METHODS FOR INCREASING CELL CULTURE TRANSFECTION EFFICIENCY AND CELLULAR REPROGRAMMING

BACKGROUND

Transfection methods can be used to introduce nucleic acids into cultured cells. Transfection methods have become a mainstay of studies related to gene regulation, gene function, molecular therapy, signal transduction, drug screening, and gene therapy. Transfection efficiency can vary based on cell culture conditions, cell type, cell viability and health, cell confluency, cell culture media, serum, and type of nucleic acid used for transfection. A method for increasing cell culture transfection efficiency could lead to improvements in genetic manipulation of cells and, in turn, future therapeutic studies.

Stem cell reprogramming is a cell culture technique that can be used in the field of regenerative medicine. Induced pluripotent stem cells (iPSCs) can be used to replace those cells lost due to damage or disease in afflicted patients. Current methods of stem cell reprogramming can be inefficient and time-consuming. Thus, a method for increasing stem cell reprogramming efficiency could lead to improvements in future therapeutic studies.

SUMMARY OF THE INVENTION

In some embodiments, the invention provides a method for increasing transfection efficiency of a nucleic acid that is introduced into a cell, the method comprising culturing the cell in a hypoxic condition and a positive pressure condition, wherein culturing the cell in the hypoxic condition and the positive pressure condition increases expression of a polypeptide encoded by the nucleic acid that is introduced into the cell as compared to expression of the polypeptide encoded by a nucleic acid that is introduced into a cell that is cultured in the absence of the hypoxic condition and the positive pressure condition.

In some embodiments, the invention provides a method for reprogramming a cell, the method comprising culturing the cell in a hypoxic condition and a positive pressure condition, wherein the cell exhibits a rate of reprogramming that is higher than the rate of reprogramming of a cell cultured in the absence of the hypoxic condition and the positive pressure condition.

INCORPORATION BY REFERENCE

Each patent, publication, and non-patent literature cited in the application is hereby incorporated by reference in its entirety as if each was incorporated by reference individually.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 depicts an illustrative transfection workflow of the invention

FIG. 2 depicts an experimental procedure for comparison of electroporation versus a method described herein.

FIG. 3 depicts the results of transfection of cells using a method described herein.

FIG. 4 depicts the results of transfection of cells using a method described herein.

FIG. 5 depicts the results of transfection of PBMCs from two different donors using a method described herein.

FIG. 6 is an illustrative computer system to be used with a method described herein.

FIG. 7 depicts a workflow that can be used for the reprogramming of stem cells.

FIG. 8 illustrates the average fold increase in stem cell colony number using a method described herein.

FIG. 9 illustrates the distribution of stem cell colony area using a method described herein.

FIG. 10 depicts the cell morphology of cells cultured using a method described herein.

FIG. 11 provides the reprogramming kinetics of stem cells cultured using a method described herein.

FIG. 12 depicts cardiomyocyte differentiation using a method described herein.

FIG. 13 depicts the effect of conditions described herein on stem cell pluripotency and differentiation.

FIG. 14 depicts the effect of conditions described herein on stem cell differentiation markers.

FIG. 15 depicts immunofluorescence of stem cell markers using a method described herein.

FIG. 16 illustrates the average colony area size of differentiated stem cells using a method described herein.

FIG. 17 shows the gene expression profile of a population of cells as a function of oxygen concentration and pressure as compared to a standard cell culture incubator.

FIG. 18 depicts the change in transfection efficiency with changes in oxygen and pressure levels.

FIG. 19 depicts the change in transfection efficiency with changes in oxygen and pressure levels.

FIG. 20 provides a workflow for measuring transfection efficiency using a method disclosed herein.

FIG. 21 shows the change in transfection efficiency via GFP expression with changes in oxygen and pressure levels.

FIG. 22 shows the quantification of transfection efficiency via GFP expression of FIG. 21 with changes in oxygen and pressure levels.

FIG. 23 shows a comparison between the transfection of CD8+ cells enriched from PBMCs and PBMCs with a GFP plasmid using a method described herein.

FIG. 24 shows the quantification of the results of FIG. 23.

FIG. 25 shows that the GFP-transfected CD8+ cells cultured under hypoxic and high pressure conditions developed more multicellular clusters than did cells grown at standard incubator conditions.

FIG. 26 shows the percent GFP in the multicellular clusters in cells grown under hypoxic and high pressure conditions compared to cells grown under standard incubator conditions.

FIG. 27 shows the quantification of the results of FIG. 26.

FIG. 28 depicts that when a CRISPR/Cas9 system was used to knockout CTLA4, and knock-in GFP using homology-directed repair, the transfection efficiency of the CRISPR/Cas9 was higher in the cells grown under hypoxic and high pressure conditions than in standard incubator conditions.

FIG. 29 shows that the cells grown under hypoxic and high pressure conditions developed a higher percentage of GFP-positive multicellular clusters than the cells grown at standard culture conditions.

FIG. 30 shows that the proliferation of the CD8+ cells grown under hypoxic and high pressure conditions was enriched over the cells grown under standard incubator conditions.

FIG. 31 depicts a limited dilution assay workflow to assess GFP-positive colonies using the CRISPR/Cas9 system.

FIG. 32 shows genome editing of the CD8-positive T-cells as indicated by the GFP signal.

FIG. 33 shows that a combination of low oxygen and high pressure enhances ectoderm commitment in defined medium, while causing changes in colony morphology to more mesoderm-like morphology.

FIG. 34 shows the change in various stem cell markers upon incubation of cells using a method disclosed herein.

FIG. 36 shows that PDL1 expression increased in ARV7-positive, 22RV1 prostate cancer cells during low oxygen and high pressure culturing conditions.

FIG. 37 (top panel) provides a western blot showing increased PDL1 protein expression under various conditions of high pressure and hypoxia in both DU145 and 22Rv1 prostate cancer cells. The bottom panel of FIG. 37 provides a quantification of the western blot results normalized to actin.

FIG. 38 shows identification of pressure and oxygen sensitive gene expression signatures in various cell lines.

FIG. 39 shows a workflow of taking a biopsy culture taken from a patient having prostate cancer.

FIG. 40 shows thst prostate cancer cells were able to form an organoid after two weeks of culture under high pressure and low oxygen conditions.

FIG. 41 shows a workflow of taking an apheresis culture taken from a patient having prostate cancer.

FIG. 42 shows the mutations found using the COSMIC database from pancreatic ductal adenocarcinoma (PDAC) and circulating tumor cells (CTC) using whole exome sequencing (top panels).

FIG. 43 shows that there was increased ex vivo expansion of primary cells under low oxygen and high pressure.

FIG. 44 shows that there was increased ex vivo expansion of primary cells under low oxygen and high pressure.

FIG. 45 shows the effect that various oxygen and pressure conditions had on the gene expression of immunotherapeutic targets in donor PBMCs.

FIG. 46 shows the results of the ex vivo culture and expansion of tumor-infiltrating lymphocytes (TILs) enriched from renal cell carcinoma tumors using high pressure and low conditions.

FIG. 47 shows that hypoxic and high pressure conditions can lead to greater enrichment of CD8+ cells from fresh blood samples than culture under standard incubator conditions.

FIG. 48 shows an expanded culture time, which indicated that the culture under hypoxic and high pressure conditions generates more CD8+ cells from whole blood than culture under standard conditions.

FIG. 49 shows induction of neural precursor markers, PAX6 and NESTIN, in iPSCs after two weeks in culture under 5% O₂and 2 PSI in stem cell maintenance media.

FIG. 50 shows ex vivo cultures of pancreatic ductal adenocarcinoma colonies from a fine-needle aspirate.

FIG. 51 shows ex vivo cultures of pancreatic ductal adenocarcinoma colonies from a fine-needle aspirate.

FIG. 52 shows ex vivo cultures of pancreatic ductal adenocarcinoma colonies from a fine-needle aspirate.

FIG. 53 shows the transfection of human dermal fibroblasts using electroporation of a GFP plasmid.

FIG. 54 shows the transfection of PBMCs using electroporation of a GFP plasmid.

FIG. 55 shows the transfection of activated CD8+ T-cells using electroporation of a GFP plasmid.

FIG. 56 shows the post-transfection effects of CD8+ T-cells using low oxygen and high pressure conditions.

FIG. 57 shows a heatmap of the effect on pressure-sensitive genes under various experimental conditions.

FIG. 58 shows a heatmap of the effect on oxygen-sensitive genes under various experimental conditions.

FIG. 59 is a molecular confirmation of a genome editing experiment.

FIG. 60 is a molecular confirmation of a genome editing experiment.

DETAILED DESCRIPTION

Transfection.

A method described herein can be used to increase, for example, transfection and transduction efficiency in cells. Transduction can be used, for example, to introduce a viral vector in a cell. Viral nucleic acid delivery systems can use recombinant viruses to deliver nucleic acids for gene therapy. Non-limiting examples of viruses that can be used to deliver nucleic acids include retrovirus, adenovirus, herpes simplex virus, adeno-associated virus, vesicular stomatitis virus, reovirus, vaccinia, pox virus, lentivirus, and measles virus.

Transfection methods that can be used with methods of the invention include, for example, lipofection, electroporation, calcium phosphate transfection, chemical transfection, polymer transfection, gene gun, magnetofection, or sonoporation. FIG. 1 depicts an illustrative transfection workflow of the invention. FIG. 1 shows the transfection of, for example, DU145 (human prostate cancer), LnCaP (androgen-sensitive human prostate adenocarcinoma), U87 (human primary glioblastoma), PANC10 (pancreatic adenocarcinoma), or PBMCs (peripheral blood mononuclear cells) with a GFP (green fluorescent protein) plasmid. The cells can be cultured in hypoxic conditions, for example, at 1% or 5% oxygen, and at conditions that are about 2 PSI greater or less than normal pressure conditions. The transfection allows introduction of the GFP-expressing plasmid into the cell.

Viral nucleic acid delivery methods can use recombinant viruses for nucleic acid transfer. Non-viral nucleic acid delivery can comprise injecting naked DNA or RNA, use of carriers including lipid carriers, polymer carriers, chemical carriers and biological carriers such as biologic membranes, bacteria, and virus-like particles, and physical/mechanical approaches. A combination of viral and non-viral nucleic acid delivery methods can be used for efficient gene therapy.

Non-viral nucleic acid transfer can include injection of naked nucleic acid, for example, nucleic acid that is not protected or devoid of a carrier. Hydrodynamic injection methods can increase the targeting ability of naked nucleic acids.

Non-viral nucleic acid delivery systems can include chemical carriers. These systems can include lipoplexes, polyplexes, dendrimers, and inorganic nanoparticles. A lipoplex is a complex of a lipid and a nucleic-acid that protects the nucleic acid from degradation and facilitates entry into cells, and can be prepared from neutral, anionic, or cationic lipids. Lipoplexes can enter cells by endocytosis, and release the nucleic acid contents into the cytoplasm. A polyplex is a complex of a polymer and a nucleic acid, and are prepared from cationic polymers that facilitate assembly by ionic interactions between nucleic acids and polymers. Uptake of polyplexes into cells can occur by endocytosis. Inside the cells, polyplexes require co-transfected endosomal rupture agents such as inactivated adenovirus, for the release of the polyplex particle from the endocytic vesicle. Examples of polymeric carriers include polyethyleneimine, chitosan, poly(beta-amino esters) and polyphosphoramidate. Dendrimers can be constructed to have a positively-charged surface and/or carry functional groups that aid temporary association of the dendrimer with nucleic acids. These dendrimer-nucleic acid complexes can be used for gene therapy. The dendrimer-nucleic acid complex can enter the cell by endocytosis. Nanoparticles prepared from inorganic material can be used for nucleic acid delivery. Examples of inorganic material can include gold, silica/silicate, silver, iron oxide, and calcium phosphate. Inorganic nanoparticles with a size of less than 100 nm can be used to encapsulate nucleic acids efficiently. The nanoparticles can be taken up by the cell via endocytosis, and the nucleic acid can be released from the endosome without degradation. Nanoparticles based on quantum dots can be prepared and offers the use of a stable fluorescence marker coupled with gene therapy. Organically modified silica or silicate can be used to target nucleic acids to specific cells in an organism.

Non-viral nucleic acid delivery systems can include biological methods including bactofection, biological liposomes, and virus-like particles (VLPs). The bactofection method comprises using attenuated bacteria to deliver nucleic acids to a cell. Biological liposomes, such as erythrocyte ghosts and secretion exosomes, are derived from the subject receiving gene therapy to avoid an immune response. Virus-like particles (VLP) or empty viral particles are produced by transfecting cells with only the structural genes of a virus and harvesting the empty particles. The empty particles are loaded with nucleic acids to be transfected for gene therapy.

Examples of physical methods of transfection include electroporation, gene gun, sonoporation, and magnetofection. The electroporation method uses short high-voltage pulses to transfer nucleic acid across the cell membrane. These pulses can lead to formation of temporary pores in the cell membrane, thereby allowing nucleic acid to enter the cell. Electroporation can be efficient for a broad range of cells. Electron-avalanche transfection is a type of electroporation method that uses very short, for example, microsecond, pulses of high-voltage plasma discharge for increasing efficiency of nucleic acid delivery. The gene gun method utilizes nucleic acid-coated gold particles that are shot into the cell using high-pressure gas. Force generated by the gene gun allows penetration of nucleic acid into the cells, while the gold is left behind on a stopping disk. The sonoporation method uses ultrasonic frequencies to modify permeability of cell membrane. Change in permeability allows uptake of nucleic acid into cells. The magnetofection method uses a magnetic field to enhance nucleic acid uptake. In this method, nucleic acid is complexed with magnetic particles. A magnetic field is used to concentrate the nucleic acid complex and bring them in contact with cells.

Non-limiting examples of viruses that can be used to deliver nucleic acids include retrovirus, adenovirus, herpes simplex virus, adeno-associated virus, vesicular stomatitis virus, reovirus, vaccinia, pox virus, and measles virus.

Non-limiting examples of retroviral vectors include Moloney murine leukemia viral (MMLV) vectors, HIV-based viral vectors, gammaretroviral vectors, C-type retroviral vectors, and lentiviral vectors. Lentivirus is a subclass of retrovirus. While some retroviruses can infect only dividing cells, lentiviruses can infect and integrate into the genome of actively dividing cells and non-dividing cells.

An adenovirus is a non-enveloped virus with a linear double-stranded genome. Adenoviruses can enter host cells using interactions between viral surface proteins and host cell receptors that lead to endocytosis of the adenovirus particle. Once inside the host cell cytoplasm, the adenovirus particle is released by the degradation of the endosome. Using cellular microtubules, the adenovirus particle gains entry into the host cell nucleus, where adenoviral DNA is released. Inside the host cell nucleus, the adenoviral DNA is transcribed and translated, without integrating into the host cell genome.

Herpes simplex virus (HSV)-based vectors can be used in the disclosure. The HSV is an enveloped virus with a linear double-stranded DNA genome. Interactions between surface proteins on the host cell and HSV lead to pore formation in the host cell membrane. These pores allow HSV to enter the host cell cytoplasm, and once inside the host cell, the HSV uses the nuclear entry pore to enter the host cell nucleus where HSV DNA is released. HSV can persist in host cells in a state of latency. Herpes simplex virus 1 and 2 (HSV-1 and HSV-2), also known as human herpes virus 1 and 2 (HHV-1 and HHV-2), are members of the herpes virus family.

Alphavirus-based vectors can be used to deliver nucleic acids. Examples of alphavirus-based vectors include vectors derived from semliki forest virus and sindbis virus.

Pox/vaccinia-based vectors such as orthopox or avipox vectors can be used in the present invention. Pox virus is a double stranded DNA virus that can infect diving and non-dividing cells. Pox viral genome can accommodate up to 25 kb transgenic sequence. Multiple genes can be delivered using a single vaccinia viral vector.

Adeno-associated virus (AAV) is a small, non-enveloped virus that belongs to the Parvoviridae family. The AAV genome is a linear single-stranded DNA molecule of about 4,800 nucleotides. The AAV DNA comprises two inverted terminal repeats (ITRs) at both ends of the genome and two sets of open reading frames. The ITRs serve as origins of replication for the viral DNA and as integration elements. The open reading frames encode for the Rep (non-structural replication) and Cap (structural capsid) proteins. AAV can infect dividing cells and quiescent cells. AAV can be engineered for use as a gene therapy vector by substituting the coding sequence for both AAV genes with a transgene (transferred nucleic acid) to be delivered to a cell. The substitution eliminates immunologic or toxic side effects due to expression of viral genes. The transgene can be placed between the two ITRs (145 bp) on the AAV DNA molecule.

A pseudotyped virus can be used for the delivery of nucleic acids. Pseudotyping involves substitution of endogenous envelope proteins of the virus by envelope proteins from other viruses or chimeric proteins. The foreign envelope proteins can confer a change in host tropism or alter stability of the virus. An example of a pseudotyped virus useful for gene therapy includes vesicular stomatitis virus G-pseudotyped lentivirus (VSV G-pseudotyped lentivirus) that is produced by coating the lentivirus with the envelope G-protein from Vesicular stomatitis virus. VSV G-pseudotyped lentivirus can transduce almost all mammalian cell types.

A hybrid vector having properties of two or more vectors can be used for nucleic acid delivery to a host cell. Hybrid vectors can be engineered to reduce toxicity or improve therapeutic transgene expression in target cells. Non-limiting examples of hybrid vectors include AAV/adenovirus hybrid vectors, AAV/phage hybrid vectors, and retrovirus/adenovirus hybrid vectors.

A viral vector can be replication-competent. A replication-competent vector contains all the genes necessary for replication, making the genome lengthier than replication-defective viral vectors. A viral vector can be replication-defective, wherein the coding region for the genes essential for replication and packaging are deleted or replaced with other genes. Replication-defective viruses can transduce host cells and transfer the genetic material, but do not replicate. A helper virus can be supplied to help a replication-defective virus replicate.

A viral vector can be derived from any source, for example, humans, non-human primates, dogs, fowl, mouse, cat, sheep, and pig.

The nucleic acid of the disclosure can be generated using any method. The nucleic acid can be synthetic, recombinant, isolated, and/or purified.

A vector of the present disclosure can comprise one or more types of nucleic acids. The nucleic acids can include DNA or RNA. RNA nucleic acids can include a transcript of a gene of interest. DNA nucleic acids can include the gene of interest, promoter sequences, untranslated regions, and termination sequences. A combination of DNA and RNA can be used. The nucleic acids can be double-stranded or single-stranded. The nucleic acid can include non-natural or altered nucleotides.

A vector of the disclosure can comprise nucleic acids encoding a selectable marker. The selectable marker can be positive, negative or bifunctional. The selectable marker can be an antibiotic-resistance gene. Examples of antibiotic resistance genes include markers conferring resistance to kanamycin, gentamicin, ampicillin, chloramphenicol, tetracycline, doxycycline, hygromycin, puromycin, zeomycin, or blasticidin. The selectable marker can allow imaging of the host cells, for example, a fluorescent protein. Examples of imaging marker genes include GFP, eGFP, RFP, CFP, YFP, dsRed, Venus, mCherry, mTomato, and mOrange.

The transfection can be a stable or transient transfection. The transfection can be used to transfect DNA plasmids, RNA, siRNA, shRNA, or any nucleic acid. The plasmids can encode, for example, green fluorescent protein (GFP), selectable markers, and other proteins of interest. The selectable markers can provide resistance to, for example, G418, hygromycin B, puromycin, and blasticidin.

A Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)—CRISPR associated (Cas) (CRISPR-Cas) system can be used to modify a target or deliver a nucleic acid of the disclosure. The CRIPSR-Cas system is a targeted genome-editing system comprising a Cas nuclease that is guided to specific DNA sequences, for example, a genomic locus in a subject, by a guide RNA molecule. The Cas nuclease can modify the genomic locus, for example, by cleaving the genomic locus, thus generating mutations that result in loss of function of the target sequence. The Cas nuclease can also modify the genomic locus, for example, by cleaving the genomic locus, and adding a transgene, for example, a therapeutic nucleic acid of the disclosure. The CRIPSR/Cas system can be used in conjunction with other nucleic acid delivery methods such as viral vectors and non-viral methods as described herein.

A CRISPR interference (CRISPRi) system can be used to modify the expression of a target of the disclosure. The CRISPRi system is a targeted gene regulatory system comprising a nuclease deficient Cas enzyme fused to a transcriptional regulatory domain that is guided to specific DNA sequences, for example, a genomic locus in a subject, by a guide RNA molecule. The Cas/regulator fusion protein can occupy the genomic locus and induce, for example, transcriptional repression of the target gene through the function of a negative regulatory domain fused to the Cas protein. The CRISPRi system can be used in conjunction with other nucleic acid delivery methods such as viral vectors and non-viral methods as described herein.

A method of the invention can increase the transfection or transduction efficiency by, for example, about 2-fold, about 3-fold, about 4-fold, about 5-fold, about 6-fold, about 7-fold, about 8-fold, about 9-fold, about 10-fold, about 12-fold, about 14-fold, about 16-fold, about 18-fold, about 20-fold, about 25-fold, about 30-fold, about 35-fold, about 40-fold, about 45-fold, about 50-fold, about 60-fold, about 70-fold, about 80-fold, about 90-fold, or about 100-fold.

In some embodiments, a hypoxic or positive pressure condition is applied to a cell prior to transfection. In some embodiments, a hypoxic or positive pressure condition is applied to a cell after transfection. A method described herein can comprise a conditioning step, where the conditioning step is for 24-48 hours and comprises culturing the cell to be transfected in a hypoxic or high pressure condition prior to the transfection. A method described herein can comprise a recovery period, where the recovery period comprises culturing a cell post-transfection in a hypoxic or positive pressure condition. In some embodiments, a transfection method described herein comprises a conditioning step, where the conditioning step comprises culturing the cell prior to transfection in a hypoxic or positive pressure condition for 24-48 hours. In some embodiments, a transfection method described herein comprises a recovery period, where the recovery period comprises culturing the cell after transfection in a hypoxic or positive pressure condition. In some embodiments, a transfection method described herein comprises both a conditioning step and a recovery period.

In some embodiments, a conditioning step prior to transfection can use moderate oxygen and moderate pressure levels to efficiently propagate cells while maintaining, for example, pluripotency. The oxygen levels can vary from about 5% to about 15%. Pressure levels can vary from about 0.1 PSI to about 2 PSI.

In some embodiments, a recovery phase after a transfection can use low oxygen and high pressure levels to increase transfection and recovery of cells by increasing cell viability. The oxygen levels can vary from about 0.1% to about 2%. Pressure levels can vary from about 2 PSI to about 5 PSI.

In some embodiments, positive pressure is used to increase transfection efficiency. In some embodiments, hypoxia is used to increase transfection efficiency. In some embodiments, hypoxia and positive pressure are used to increase transfection efficiency.

Stem Cells.

A method disclosed herein can be used to reprogram, for example, fibroblasts to pluripotent stem cells. A method disclosed herein can, for example, increase the efficiency and increase the rate of cell reprogramming. A method disclosed herein can further increase, for example, the number and size of stem cell colonies that form as a result of the reprogramming protocol. The cells can be reprogrammed into, for example, totipotent, pluripotent, multipotent, oligopotent, or unipotent stem cells.

Reprogramming of cells into pluripotent stem cells can be enhanced by, for example, culturing the cells under hypoxic and positive pressure conditions. The cells can be reprogrammed by transfecting cells with, for example, an RNA replicon vector encoding several stem cell transformation factors. The stem cell transformation factors can include, for example, Oct4, Sox2, KLF-4, GLIS1, and c-MYC. Additional stem cell transformation factors include, for example, Nanog and Lin28. After transfection of the cells with the reprogramming factors, the cells can be maintained in media designed to differentiate and maintain stem cell populations. The cells can be grown under hypoxic and high pressure conditions as disclosed herein to induce differentiation of the cells.

Adult stem cells can be found in many organs and tissues including, for example, brain, bone marrow, peripheral blood, blood vessels, skeletal muscle, skin, teeth, heart, gut, liver, ovarian epithelium, and testis. The stem cells can reside in stem cell niches within the various areas of the body. In many tissues, some types of stem cells are pericytes, which are cells that compose the outermost layer of small blood vessels. Stem cells may remain quiescent non-dividing for long periods of time until they are activated by a normal need for more cells to maintain tissues, or by disease or tissue injury.

Markers that can be used to identify iPSCs include, for example, SSEA-3, SSEA-4, TRA-1-60, TRA-1-81, TRA-2-49/6E, Nanog, Oct3/4, Sox2, GDF3, REX1, FGF4, ESG1, DPPA2, DPPA4, and hTERT.

The iPSCs can be induced to differentiate into, for example, neuronal cells, hippocampal progenitors, dentate granule cell neurons, MGE progenitors, cortical interneurons, dorsal cortical progenitors, excitatory cortical neurons, glial progenitors, astrocytes, neural crest stem cells, dopaminergic neurons, oligodendrocytes, dopaminergic neurons, hematopoietic cells, B-cells, T-cells, NK cells, granulocytes, monocytes, macrophages, erythrocytes, megakaryocytes, platelets, cardiomyocytes, hepatocytes, skeletal muscle cells, adipocytes, pancreatic beta-cells, or cells from the ectoderm, mesoderm, or endoderm.

The stem cells obtained using a method disclosed herein can be cultured on, for example, a gelatin-coated culture dish. The cells can be in cultured in medium containing inactivated mouse embryonic fibroblast (MEF) medium, basic FGF solution, pluripotent culture medium, leukemia inhibitory factor, and a collagenase solution. The stem cells can additionally be grown over a layer of feeder cells, which can be, for example, MEFs, JK1 cells, or SNL 76/7 cells.

Expression markers that can be measured to assess the differentiation or gene expression profile of an initial cell culture to iPSCS can include, for example, IGF1, CTNNB1, AXIN1, KAT2A, CD4, CXCL12, FZD9, CD44, ACTC1, JAG1, BMP1, FZD2, IL6ST, FZD7, LIFR, SMAD4, DVL1, CTNNA1, FGFR1, WNT1, PPARG, COL1A1, FGF1, GLL, DNMT3B, PSEN1, ALDH1A1, JUND, SDAD1, NCSTN, FZD6, TCF7, NOTCH1, APC, RB1, NUMB, CREBBP, GATA6, PSEN2, HDAC2, CCND1, CCNE1, EP300, Notch2, MME, GLI2, BTRC, STAT3, PPARD, Notch3, Notch4, GLI3, CDC42, CCNA2, ISL1, BMP2, PAX6, S100B, CD3D, FZD5, Nanog, CDH1, Sox1, DLL1, CCND2, SMO, COL2AI, LIFR, or COX2.

Plant Cells.

A method disclosed herein can be used to genetically engineer or to reprogram plant cells. A method disclosed herein can be used to create plant cells with a particular genotype that alters the cell's ability to produce a specific molecule or that results in a specific phenotype. Some embodiments of the invention comprise modulating local pressure and oxygen conditions during transformation of plant cells.

A method disclosed herein can be applied to any type of plant cell or tissue. Plant cells or tissues used in the invention can include roots, leaves, monocotyledons such as cotton, soybean, Brassica, and peanut, dicotyledons such as asparagus, barley, maize, oat, rice, sugarcane, tall fescue, and wheat, hypocotyl tissue, callus tissue, nodal explants, shoot meristem, cell cultures, immature embryos, scutellar tissue, and immature inflorescence.

In addition to or in conjunction with the methods described herein, the invention can include the use of Agrobacterium tumor-inducing (Ti) plasmid genes, which can contain a transfer DNA region (T-DNA), for engineering a plant cell's DNA. Agrobacterium can be used in the invention to produce Ti plasmid genes, and Agrobacterium strains used in the invention can include Agrobacterium tumefaciens strain C58, nopaline strains, octopine strains such as LBA4404, and agropine strains such as EHA101, EHA105, and EHA 109.

The invention can also include the use of promoters such as nopaline synthase (NOS) promoter, octopine synthase (OCS) promoter, caulimovirus promoters such as cauliflower mosaic virus (CaMV) 19S and 35S promoters, enhanced CaMV 35S promoter (e35S), figwort mosaic virus (FMV) 35S promoter, and promoters from the ribulose bisphosphate carboxylase (Rubisco) family such as Rubisco small subunit and Rubisco activase promoters in engineered plant cells.

Conditions Used in Methods Disclosed Herein.

The present invention can use a substrate to culture the cells during transfection. The cells can be applied to, for example, a culture dish coated with a substrate that can promote growth and enrichment of the cells. Cells that do not adhere to the substrate can be washed away with media. Once adhered, the cells can spread and begin dividing on the substrate.

The substrate can comprise, for example, 1, 2, 3, 4, or 5 layers. The distance between two substrates layers may range from about 0.1 to about 20 mm, about 1 to about 10 mm, or about 1 to about 5 mm and each layer can be about 0.1, about 1, about 2, about 3, about 4, about 5, about 6, about 7, about 8, about 9, about 10, about 12, about 15, about 17, or about 20 mm.

The cells can be plated on a material made of, for example, plastic, glass, gelatin, polyacrylamide, or any combination thereof. The dishes used to the plate the cells can be, for example, microscope slides, culture plates, culture dishes, Petri dishes, microscope coverslips, an enclosed environmental chamber, a sealed culture dish, or multi-well culture dishes.

The binding surface layer of the substrate can be the portion of the substrate that is in contact with the cells. In some instances, the binding surface layer is the only layer, adjacent to the base layer, or separated from the base layer by one or more middle layers.

The binding surface layer of the substrate can comprise, for example, cell monolayers, cell lysates, biological materials associated with the extracellular matrix (ECM), gelatin, or any combination thereof.

Biological materials associated with the ECM can include, for example, collagen type I, collagen type IV, laminin, fibronectin, elastin, reticulin, hygroscopic molecules, glycosaminoglycanse, roteoglycans, glycocalyx, bovine serum albumin, Poly-L-lysine, Poly-D-lysine, or Poly-L-ornithine. The gelatin can be from an animal source, for example, the gelatin can porcine or bovine.

The monolayer of cells used in the substrate can be, for example, mammalian cells, endothelial cells, vascular cells, venous cells, capillary cells, human umbilical vein endothelial cells (HUVEC), human lung microvascular endothelial cells (HLMVEC). The cell lines can be obtained from a primary source or from an immortalized cell line. The monolayer of cells can be irradiated by ultraviolet light or X-ray sources to cause senescence of cells. The monolayer can also contain a mixture of one or more different cell types. The different cell types may be co-cultured together. One non-limiting example of co-culture is a combination of primary human endothelial cells co-cultured with transgenic mouse embryonic fibroblasts mixed to form a monolayer.

The binding surface layer of the substrate can contain, for example, a mixture of intracellular components. One method that can be used to obtain a mixture of intracellular components is lysis of the cells and collection of the cytosolic components. The lysed cells can be primary or immortalized. The lysed cells can be from either mono- or co-cultures.

The binding surface layer of the substrate can contain biological materials associated with the extracellular matrix (ECM) or binding moieties. For example, gelatin can be mixed directly with cells, binding moieties, biological materials associated with the ECM, or any combination thereof, to make a binding surface layer for the substrate. For example, the binding surface layer can be comprised of a gelatin mixed with a collagen.

The substrate can have one or more middle layers. The middle layer of the substrate can be one or more monolayers of cells. The cells of the monolayer can be of varying origin. For example, the middle layer of the substrate can be made by growing a confluent monolayer of mouse embryonic fibroblasts on the base layer and then growing another layer of cells, for example, the binding surface layer, on top of the confluent mouse embryonic fibroblasts.

A feeder layer can be used in the substrate for growth or reprogramming of the cells. A feeder layer can sit adjacent to a base layer and can be separated from the binding surface layer of the substrate. The feeder layer can be a monolayer of feeder cells. The cells of the monolayer can be of varying origin. For example, the feeder layer can be made by growing a monolayer of human endothelial cells or mouse embryonic fibroblasts on a base layer.

Conjugation of layers of the substrate can be done by allowing cells to grow in a monolayer on top of the base layer or middle layer. Conjugation of layers can also be done by pre-treating the surface with a surface of either net positive, net negative, or net neutral charge. The conjugation procedure can be aided by chemical moieties, linkers, protein fragments, nucleotide fragments, or any combination thereof.

The media used for growing the cells can be supplemented or made with culture media that has been collected from cell cultures, blood plasma, or any combination thereof. The enrichment media can be, for example, Plating Culture Medium, Type R Long Term Growth Medium, Type DF Long Term Growth Medium, Type D Long Term Growth Medium, and MEF—Enrichment Medium, or any combination thereof. The enrichment medium can contain, for example, a primary nutrient source, animal serum, ions, elements, calcium, glutamate, magnesium, zinc, iron, potassium, sodium, amino acids, vitamins, glucose, growth factors, hormones, tissue extracts, proteins, small molecules, or any combination thereof. In some embodiments, the culture media used for transfection does not contain serum.

Non-limiting examples of amino acids include essential amino acids, phenylalanine, valine, threonine, tryptophan, isoleucine, methionine, leucine, lysine, and histidine, arginine, cysteine, glycine, glutamine, proline, serine, tyrosine, alanine, asparagine, aspartic acid, glutamic acid, or any combination thereof.

Non-limiting examples of growth factors include Epidermal Growth Factor (EGF), Nerve Growth Factor (NGF), Brain Derived Neurotrophic Factor (BDNF), Fibroblast Growth Factor (FGF), Stem Cell Factor (SCF), Insulin-like Growth Factor (IGF), Transforming Growth Factor-beta (TGF-β), or any combination thereof.

Non-limiting examples of hormones include peptide hormones, insulin, steroidal hormones, hydrocortisone, progesterone, testosterone, estrogen, dihydrotestosterone, or any combination thereof.

Non-limiting examples of tissue extracts include pituitary extract. Non-limiting examples of small molecule additives include sodium pyruvate, endothelin-1, transferrin, cholesterol, or any combination thereof.

The culturing conditions in a method of the invention can be adjusted to simulate oxygen and pressure levels found, for example, in pathological conditions. The oxygen level used during culturing conditions can be, for example, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, or about 25% oxygen in the incubator. In some embodiments, the cells can be grown under hypoxic conditions during transfection.

The culturing condition in a method of the invention can be adjusted to simulate the pressure found, for example, in pathological conditions. The pressure used during culturing conditions can be about 0 PSI, about 0.1 PSI, about 0.15 PSI about 0.2 PSI, about 0.25 PSI, about 0.3 PSI, about 0.35 PSI, about 0.4 PSI, about 0.45 PSI, 0.5 PSI, about 0.55 PSI, about 0.6 PSI, about 0.65 PSI, about 0.7 PSI, about 0.75 PSI, about 0.8 PSI, about 0.85 PSI, about 0.9 PSI, about 0.95 PSI, about 1 PSI, about 1.1 PSI, about 1.2 PSI, about 1.3 PSI, about 1.4 PSI, about 1.5 PSI, about 1.6 PSI, about 1.7 PSI, about 1.8 PSIG, about 1.9 PSI, about 2 PSI, about 2.1 PSI, about 2.2 PSI, about 2.3 PSI, about 2.4 PSI, about 2.5 PSI, about 2.6 PSI, about 2.7 PSI, about 2.8 PSI, about 2.9 PSI, about 3 PSI, about 3.5 PSI, about 4 PSI, about 4.5 PSI, about 5 PSI, about 6 PSI, about 7 PSI, about 8 PSI, about 9 PSI, or about 10 PSI. A pressure used in a method disclosed herein can be an above atmospheric pressure value. A pressure used in a method disclosed herein can be positive pressure.

The culturing condition in a method of the invention can be adjusted to simulate the pressure found, for example, in pathological conditions. The pressure used during culturing conditions can be a PSI gauge (PSIG) reading of, for example, about 0.5 PSIG, about 0.6 PSIG, about 0.7 PSIG, about 0.8 PSIG, about 0.9 PSIG, about 1 PSIG, about 1.1 PSIG, about 1.2 PSIG, about 1.3 PSIG, about 1.4 PSIG, about 1.5 PSIG, about 1.6 PSIG, about 1.7 PSIG, about 1.8 PSIG, about 1.9 PSIG, about 2 PSIG, about 2.5 PSIG, about 3 PSIG, about 3.5 PSIG, about 4 PSIG, about 4.5 PSIG, about 5 PSIG, about 6 PSIG, about 7 PSIG, about 8 PSIG, about 9 PSIG, about 10 PSIG, about 15 PSIG, about 20 PSIG, about 25 PSIG, about 30 PSIG, about 35 PSIG, about 40 PSIG, about 45 PSIG, about 50 PSIG, or about 55 PSIG.

The pressure used during culturing conditions can be, for example, about 3.45 kPa, about 4.14 kPa, about 4.83 kPa, about 5.52 kPa, about 6.21 kPa, about 6.89 kPa, about 7.58 kPa, about 8.27 kPa, about 8.96 kPa, about 9.65 kPa, about 10.3 kPa, about 11 kPa, about 11.7 kPa, about 12.4 kPa, about 13.1 kPa, about 13.8 kPa, about 17.2 kPa, about 20.7 kPa, about 24.1 kPa, about 27.6 kPa, about 31 kPa, about 34.4 kPa, about 41.4 kPa, about 48.3 kPa, about 55.2 kPa, about 62.1 kPa, about 68.9 kPa, about 103 kPa, about 138 kPa, about 172 kPa, about 207 kPa, about 241 kPa, about 276 kPa, about 310 kPa, about 345 kPa, or about 379 kPa.

The pressure used in a method of the invention can be delivered continuously or via pulses of pressure produced by repeated depressurizations and pressurizations of an incubator used in the method. The pulses of pressure can be separated by, for example, about 1 minute, about 1.5 minutes, about 2 minutes, about 2.5 minutes, about 3 minutes, about 3.5 minutes, about 4 minutes, about 4.5 minutes, about 5 minutes, about 6 minutes, about 7 minutes, about 8 minutes, about 9 minutes, about 10 minutes, about 11 minutes, about 12 minutes, about 13 minutes, about 14 minutes, about 15 minutes, about 16 minutes, about 17 minutes, about 18 minutes, about 19 minutes, or about 20 minutes, about 21 minutes, about 22 minutes, about 23 minutes, about 24 minutes, about 25 minutes, about 26 minutes, about 27 minutes, about 28 minutes, about 29 minutes, about 30 minutes, about 32 minutes, about 34 minutes, about 36 minutes, about 38 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 1 hour, about 1.5 hours, about 2 hours, about 2.5 hours, about 3 hours, about 3.5 hours, about 4 hours, about 4.5 hours, or about 5 hours.

The pH of the media used in a method of the invention can be, for example, about 2, about 2.1, about 2.2, about 2.3, about 2.4, about 2.5, about 2.6, about 2.7, about 2.8, about 2.9, about 3, about 3.1, about 3.2, about 3.3, about 3.4, about 3.5, about 3.6, about 3.7, about 3.8, about 3.9, about 4, about 4.1, about 4.2, about 4.3, about 4.4, about 4.5, about 4.55, about 4.6, about 4.7, about 4.8, about 4.9, about 5, about 5.5, about 6, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, about 7, about 7.1, about 7.2, about 7.3, about 7.4, about 7.5, about 7.6, about 7.7, about 7.8, about 7.9, about 8, about 8.5, about 9, about 9.5, about 10, about 10.5, or about 11 pH units.

The viscosity of the media can be adjusted by, for example, at least 0.001 Pascal-second (Pa·s), at least 0.001 Pa·s, at least 0.0009 Pa·s, at least 0.0008 Pa·s, at least 0.0007 Pa·s, at least 0.0006 Pa·s, at least 0.0005 Pa·s, at least 0.0004 Pa·s, at least 0.0003 Pa·s, at least 0.0002 Pa·s, at least 0.0001 Pa·s, at least 0.00005 Pa·s, or at least 0.00001 Pa·s depending on the cell types being cultured.

The oxygen solubility of the media can be, for example, about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 21%, about 22%, about 23%, about 24%, about 25%, about 26%, about 27%, about 28%, about 29%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99%.

In some embodiments, a culture media used for a method described herein can contain, for example, an L-alanine-L-glutamine dipeptide, B27 TM supplement, human bFGF, human EGF, human HGH, 1 mg/mL human insulin, 0.55 mg/mL human transferrin, 0.5 μg/mL sodium selenite, beta-mercaptoethanol, non-essential amina acids solution, and high glucose media.

In some embodiments, for PBMC or CD8+ cell culture, a culture media for a method described herein can contain, for example, PHA-P (10 μg/mL), IL-2 (100 U/mL), IL-4 (20 ng/mL), IL-15 (100 ng/mL), GM-CSF (20 ng/mL), and LPS (100 ng/mL).

The oxygen concentration used in a method disclosed herein can be used to mimic oxygen concentration found in, for example, solid tumors (about 1.1%), muscle (about 3.8%), prostate (about 3.9%), brain (about 4.4%), peripheral tissues (about 5.3%), venous blood (about 5.3%), lung (about 5.6%), bone marrow (about 6.4%), intestinal tissue (about 7.6%), kidney (about 9.5%), and arterial blood (about 13.2%).

The pressure conditions used in a method disclosed herein can be used to mimic the interstitial fluid pressure found in, for example, normal breast (about 0.02 PSI), normal skin (about 0.04 PSI), lymphoma (about 0.14 PSI), brain tumors (about 0.15 PSI), sarcoma (about 0.17 PSI), lung carcinoma (about 0.25 PSI), rectal carcinoma (about 0.33 PSI), breast carcinoma (about 0.37 PSI), head and neck carcinoma (about 0.41 PSI), metastatic melanoma (about 0.43 PSI), colorectal carcinoma liver metastases (about 0.43 PSI), cervical carcinoma (about 0.44 PSI), ovarian carcinoma (about 0.48 PSI), and renal cell carcinoma (about 0.72 PSI).

Therapeutic Uses.

Subjects can be, for example, elderly adults, adults, adolescents, pre-adolescents, children, toddlers, infants. Subjects can be non-human animals, for example, a subject can be a mouse, rat, cow, horse, donkey, pig, sheep, dog, cat, or goat. A subject can be a patient.

A method disclosed herein can be used to identify a therapeutic, a biomarker, a genetic mutation, or a therapeutic target for, for example, stem cell differentiation or differentiation of various cell types.

Genomic, proteomic, and metabolic analysis can be conducted on the transfected cells to, for example, identify biomarkers that can be used for development of cancer therapies, drug development, cancer vaccines, cancer screening, diagnostics, personalized antibody development, hematopoietic stem cell transplantation, organ transplantation, or cardiovascular disease treatment. A method described herein can be used to induce phenotypic and genotypic changes in cells to determine the effect of cancer therapies. The cancer therapies can include, for example, chemotherapeutics or gene therapy.

Non-limiting examples of cancers that can be analyzed in a method disclosed herein include: acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, AIDS-related cancers, AIDS-related lymphoma, anal cancer, appendix cancer, astrocytomas, basal cell carcinoma, bile duct cancer, bladder cancer, bone cancers, brain tumors, such as cerebellar astrocytoma, cerebral astrocytoma/malignant glioma, ependymoma, medulloblastoma, supratentorial primitive neuroectodermal tumors, visual pathway and hypothalamic glioma, breast cancer, bronchial adenomas, Burkitt lymphoma, carcinoma of unknown primary origin, central nervous system lymphoma, cerebellar astrocytoma, cervical cancer, childhood cancers, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative disorders, colon cancer, cutaneous T-cell lymphoma, desmoplastic small round cell tumor, endometrial cancer, ependymoma, esophageal cancer, Ewing's sarcoma, germ cell tumors, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gliomas, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, Hodgkin lymphoma, Hypopharyngeal cancer, intraocular melanoma, islet cell carcinoma, Kaposi sarcoma, kidney cancer, laryngeal cancer, lip and oral cavity cancer, liposarcoma, liver cancer, lung cancers, such as non-small cell and small cell lung cancer, lymphomas, leukemias, macroglobulinemia, malignant fibrous histiocytoma of bone/osteosarcoma, medulloblastoma, melanomas, mesothelioma, metastatic squamous neck cancer with occult primary, mouth cancer, multiple endocrine neoplasia syndrome, myelodysplastic syndromes, myeloid leukemia, nasal cavity and paranasal sinus cancer, nasopharyngeal carcinoma, neuroblastoma, non-Hodgkin lymphoma, non-small cell lung cancer, oral cancer, oropharyngeal cancer, osteosarcoma/malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial cancer, ovarian germ cell tumor, pancreatic cancer, pancreatic cancer islet cell, paranasal sinus and nasal cavity cancer, parathyroid cancer, penile cancer, pharyngeal cancer, pheochromocytoma, pineal astrocytoma, pineal germinoma, pituitary adenoma, pleuropulmonary blastoma, plasma cell neoplasia, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma, renal pelvis and ureter transitional cell cancer, retinoblastoma, rhabdomyo sarcoma, salivary gland cancer, sarcomas, skin cancers, skin carcinoma merkel cell, small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, stomach cancer, T-cell lymphoma, throat cancer, thymoma, thymic carcinoma, thyroid cancer, trophoblastic tumor (gestational), cancers of unknown primary site, urethral cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms tumor.

Methods that can be used to determine the presence of, for example, biological markers or transfection of desired genes can include, for example, qPCR, RT-PCR, immunofluorescence, immunohistochemistry, western blotting, high-throughput sequencing, or mRNA sequencing.

Computer Systems.

A method of the invention can be used to, for example, sequence, image, or characterize the transfected cells. Further methods can be found in PCT/US14/13048, the entirety of which is incorporated herein by reference.

The invention provides a computer system that is configured to implement the methods of the disclosure. The system can include a computer server (“server”) that is programmed to implement the methods described herein. FIG. 6 depicts a system 600 adapted to enable a user to detect, analyze, and process images of cells and sequence cells. The system 600 includes a central computer server 601 that is programmed to implement exemplary methods described herein. The server 601 includes a central processing unit (CPU, also “processor”) 605 which can be a single core processor, a multi core processor, or plurality of processors for parallel processing. The server 601 also includes memory 610 (e.g. random access memory, read-only memory, flash memory); electronic storage unit 615 (e.g. hard disk); communications interface 620 (e.g. network adaptor) for communicating with one or more other systems; and peripheral devices 625 which may include cache, other memory, data storage, and/or electronic display adaptors. The memory 610, storage unit 615, interface 620, and peripheral devices 625 are in communication with the processor 605 through a communications bus (solid lines), such as a motherboard. The storage unit 615 can be a data storage unit for storing data. The server 601 is operatively coupled to a computer network (“network”) 630 with the aid of the communications interface 620. The network 630 can be the Internet, an intranet and/or an extranet, an intranet and/or extranet that is in communication with the Internet, a telecommunication or data network. The network 630 in some cases, with the aid of the server 601, can implement a peer-to-peer network, which may enable devices coupled to the server 601 to behave as a client or a server. The microscope and micromanipulator can be peripheral devices 625 or remote computer systems 640.

The storage unit 615 can store files, such as individual images, time lapse images, data about individual cells, cell colonies, or any aspect of data associated with the invention. The data storage unit 615 may be coupled with data relating to locations of cells in a virtual grid.

The server can communicate with one or more remote computer systems through the network 630. The one or more remote computer systems may be, for example, personal computers, laptops, tablets, telephones, Smart phones, or personal digital assistants.

In some situations the system 600 includes a single server 601. In other situations, the system includes multiple servers in communication with one another through an intranet, extranet and/or the Internet.

The server 601 can be adapted to store cell profile information, such as, for example, cell size, morphology, shape, migratory ability, proliferative capacity, kinetic properties, and/or other information of potential relevance. Such information can be stored on the storage unit 615 or the server 601 and such data can be transmitted through a network.

Methods as described herein can be implemented by way of machine (e.g., computer processor) computer readable medium (or software) stored on an electronic storage location of the server 601, such as, for example, on the memory 610, or electronic storage unit 615. During use, the code can be executed by the processor 605. In some cases, the code can be retrieved from the storage unit 615 and stored on the memory 610 for ready access by the processor 605. In some situations, the electronic storage unit 615 can be precluded, and machine-executable instructions are stored on memory 610. Alternatively, the code can be executed on a second computer system 640.

Aspects of the systems and methods provided herein, such as the server 601, can be embodied in programming. Various aspects of the technology may be thought of as “products” or “articles of manufacture” typically in the form of machine (or processor) executable code and/or associated data that is carried on or embodied in a type of machine readable medium (e.g., computer readable medium). Machine-executable code can be stored on an electronic storage unit, such memory (e.g., read-only memory, random-access memory, flash memory) or a hard disk. “Storage” type media can include any or all of the tangible memory of the computers, processors or the like, or associated modules thereof, such as various semiconductor memories, tape drives, disk drives and the like, which may provide non-transitory storage at any time for the software programming. All or portions of the software may at times be communicated through the Internet or various other telecommunication networks. Such communications, for example, may enable loading of the software from one computer or processor into another, for example, from a management server or host computer into the computer platform of an application server. Thus, another type of media that may bear the software elements includes optical, electrical, and electromagnetic waves, such as used across physical interfaces between local devices, through wired and optical landline networks and over various air-links. The physical elements that carry such waves, such as wired or wireless likes, optical links, or the like, also may be considered as media bearing the software. As used herein, unless restricted to non-transitory, tangible “storage” media, terms such as computer or machine “readable medium” refer to any medium that participates in providing instructions to a processor for execution.

Hence, a machine readable medium, such as computer-executable code, may take many forms, including but not limited to, tangible storage medium, a carrier wave medium, or physical transmission medium. Non-volatile storage media can include, for example, optical or magnetic disks, such as any of the storage devices in any computer(s) or the like, such may be used to implement the system. Tangible transmission media can include: coaxial cables, copper wires, and fiber optics (including the wires that comprise a bus within a computer system). Carrier-wave transmission media may take the form of electric or electromagnetic signals, or acoustic or light waves such as those generated during radio frequency (RF) and infrared (IR) data communications. Common forms of computer-readable media therefore include, for example: a floppy disk, a flexible disk, hard disk, magnetic tape, any other magnetic medium, a CD-ROM, DVD, DVD-ROM, any other optical medium, punch cards, paper tame, any other physical storage medium with patterns of holes, a RAM, a ROM, a PROM and EPROM, a FLASH-EPROM, any other memory chip or cartridge, a carrier wave transporting data or instructions, cables, or links transporting such carrier wave, or any other medium from which a computer may read programming code and/or data. Many of these forms of computer readable media may be involved in carrying one or more sequences of one or more instructions to a processor for execution.

EXAMPLES
Example 1: Transfection Efficiency Using a Method Disclosed Herein

FIG. 2 depicts an experimental procedure for comparison of electroporation versus a method of the invention, which includes hypoxic and high pressure conditions. The cells were cultured in either a standard CO₂incubator or under hypoxic and positive pressure conditions. The cells were further cultured in media and a substrate that contained FBS or serum-free media and a serum-free substrate.

FIG. 3 depicts the results of the transfection of DU145 (human prostate cancer) cells. 5×10̂6 cells/mL were transfected with 0.5 μg GFP at 1260 V for 40 ms with 2 pulses. After transfection, the cells were evenly split across the tested conditions and cultured for 48 hours prior to imaging and assessment of transfection efficiency. FIG. 3 shows that transfection of the cells under hypoxic and high pressure conditions, along with culturing in serum-free media allowed for the greatest transfection efficiency as indicated by the brightest GFP staining in the bottom-right panel.

FIG. 4 depicts the results of the transfection of human dermal fibroblast cells. 5×10̂6 cells/mL were transfected with 0.5 μg GFP at 1000 V for 30 ms with 1 pulse. After transfection, the cells were evenly split across the tested conditions and cultured for 48 hours prior to imaging and assessment of transfection efficiency. FIG. 4 shows that transfection of the cells under hypoxic and high pressure conditions, along with culturing in serum-free media allowed for the greatest transfection efficiency as indicated by the brightest GFP staining in the bottom-right panel.

FIG. 5 depicts the results of the transfection of healthy donor peripheral blood mononuclear cells (PBMCs). 2×10̂7 cells/mL were transfected with 1 μg GFP at 1500 V for 10 ms with 1 pulse. After transfection, the cells were evenly split across the tested conditions and cultured for 48 hours prior to imaging and assessment of transfection efficiency. FIG. 5 shows that transfection of the cells under hypoxic and high pressure conditions, along with culturing in serum-free media allowed for the greatest transfection efficiency as indicated by the brightest GFP staining in the bottom panels.

TABLE 1 below provides a quantitative analysis of the change in fold expression of the GFP plasmid using different methods.

TABLE 1

Fold Expression change of GFP

Transfection Condition
DU145
PC3
Panc10
Dermal fibroblasts

Standard, FBS
1
1
1
1

Standard, Serum-free
5.56
1.88
0.574
3.81

media and substrate

1% O₂2 PSI, FBS
2.55
5.033
15.731
5.018

1% O₂2 PSI, Serum-free
14.2
4.234
36.533
6.642

media and substrate

Example 2: Transfection of Human Dermal Fibroblasts

FIG. 18 depicts the transfection of human dermal fibroblasts under 21% O₂and 0 PSI, 5% O₂and 0, 2, or 5 PSI, and 1% O₂, and 0, 2, or 5 PSI. The cells were transfected with a GFP plasmid and imaged 48 hours post-transfection. The experiments were repeated in triplicate. FIG. 18 shows that transfection efficiency increased at lower oxygen and higher pressure conditions as indicated by brighter GFP expression.

FIG. 19 provides a quantitative analysis of GFP transfection of human dermal fibroblasts grown under various hypoxic and high pressure conditions in FIG. 18. The results indicate that cells grown under the hypoxic and positive pressure conditions provided higher GFP transfection efficiency than cells grown under 21% O₂and 0 PSI.

FIG. 53 shows the transfection of human dermal fibroblasts using electroporation of a GFP plasmid. The cells were cultured under several conditions of low oxygen and high pressure. The results indicated that 1% oxygen and 2 PSI pressure provided the greatest proportion of transfected cells (52.8% GFP+ cells versus 9.4% GFP+ under standard culture conditions).

FIG. 54 shows the transfection of PBMCs using electroporation of a GFP plasmid. The cells were cultured under several conditions of low oxygen and high pressure. The results indicated that 1% oxygen and 5 PSI pressure provided the greatest proportion of transfected cells (7.8% GFP+ cells versus 3.7% GFP+ under standard culture conditions).

FIG. 55 shows the transfection of activated CD8+ T-cells using electroporation of a GFP plasmid. The cells were cultured under several conditions of low oxygen and high pressure. The results indicated that 1% oxygen and 5 PSI pressure provided the greatest proportion of transfected cells (55.6% GFP+ cells versus 3.7% GFP+ under standard culture conditions).

FIG. 56 shows the post-transfection effects of CD8+ T-cells using low oxygen and high pressure conditions. The results indicated that lower oxygen (10% O₂) and positive pressure (5 PSI) promotes cell proliferation post-transfection (1.1×10⁵cells under higher oxygen and positive pressure versus 3×10⁴cells under standard conditions 2 days after transfection).

Example 3: Transfection of Immune Cells

FIG. 20 depicts a sample workflow for transfection of immune cells using a method disclosed herein. FIG. 20 shows that after a sample is obtained from a donor, the sample can be enriched for CD8+ cells. A DNA plasmid is transfected using electroporation. The transfected cells are then subjected to decreasing oxygen levels and either 0 PSI or high pressure conditions. Then, the percent GFP-positive cells, percent viable cells, and relative expression of GFP in the cells are assessed.

FIG. 21 shows the transfection of peripheral blood mononuclear cells (PBMC) with a GFP plasmid using electroporation of cells at passage zero. The 2-5 PSI pulsed pressure condition was performed at a frequency of 30 minute pulses. The experiments were repeated four times. FIG. 22 depicts a quantification of the results from FIG. 21 indicating that there was an almost 2.5-fold increase in GFP-positive cells using the hypoxic and positive pressure conditions compared to standard incubator conditions.

FIG. 23 shows a comparison between the transfection of CD8+ cells enriched from PBMC and PBMC with a GFP plasmid using electroporation of cells at passage zero. The experiment was performed in triplicate. FIG. 24 depicts a quantification of the results from FIG. 23 indicating that enriched CD8+ cells have higher transfection efficiency than the PBMC. “ST” in FIG. 24 denotes standard culture conditions.

FIG. 25 shows that the GFP-transfected CD8+ cells cultured under hypoxic and positive pressure conditions developed more multicellular clusters than did cells grown at standard incubator conditions. In each panel, the first number indicates the oxygen level; the second number represents the pressure level in PSI. The top left panel is a control without transfection. FIG. 26 shows the percent GFP in the multicellular clusters in cells grown under hypoxic and positive pressure conditions compared to cells grown under standard incubator conditions. FIG. 27 is a quantification of the results of FIG. 26. “ST” in FIG. 27 denotes standard culture conditions.

FIG. 47 shows that hypoxic and positive pressure conditions can lead to greater enrichment of CD8+ cells from fresh blood samples than culture under standard incubator conditions. For example, FIG. 47 shows that 8.3 million CD8+ cells were obtained after one week under hypoxic and positive pressure conditions versus 3 million CD8+ cells obtained under standard conditions. An expanded culture time up to 11 days is shown in FIG. 48 indicating that the culture under hypoxic and positive pressure conditions generates more CD8+ cells from whole blood than culture under standard conditions.

Example 4: Immune Cell Genome Editing

A method disclosed herein can be used to introduce the CRISPR/Cas9 system into immune cells, for example, CD8+ T-cells, as shown in FIGS. 28-30. PBMCs were freshly isolated from healthy donors, and post-cell counting PBMCs were enriched for CD8+ cells. The CD8+ cells were cultured at 2 million cells/mL in IL-2 containing media in a standard incubator, or with PBMC media under low oxygen and positive pressure conditions. Once the cells demonstrated doubling times of 36-48 hours, the cells were transfected at 20 million cells/ml with 1 μg/μl CTLA4 CRISPR Knockout and a GFP HDR DNA plasmid. The cells were expanded under low oxygen and positive pressure conditions or under conventional culture conditions. Images were taken 1, 3, and 5 days post-transfection prior to cell splitting and counting. Quantification was performed through cell counting across entire wells and assessed as GFP+ multicellular clusters/total multicellular clusters at 5 days post-transfection as well as total cell count at 5 days post-transfection.

FIG. 28 shows that when a CRISPR/Cas9 system was used to knockout CTLA4, and knock-in GFP using homology-directed repair, the transfection efficiency of the CRISPR/Cas9 system was higher in the cells grown under hypoxic and high pressure conditions than in standard incubator conditions. This is indicated by the bright GFP signal seen in the top panels of FIG. 28. The results also indicate that the GFP expression persisted through subsequent expansion of the CD8+ cells for at least five days. The experiment was repeated four times, and the cells were transfected at passage 1+ while in the exponential growth phase.

FIGS. 29 and 30 provide a quantification of the results of from FIG. 28. FIG. 29 shows that the cells grown under hypoxic and positive pressure conditions developed a higher percentage of GFP-positive multicellular clusters than the cells grown at standard culture conditions. FIG. 30 shows that the proliferation of the CD8+ cells grown under hypoxic and positive pressure conditions was enriched over the cells grown under standard incubator conditions. The error bars represent standard deviation.

FIG. 31 depicts a limited dilution assay workflow to assess GFP-positive colonies using the CRISPR/Cas9 system. A sample is taken from a donor and then subjected to enrichment for CD8+ cells. Then, the cells are split to be cultured under either positive pressure and low oxygen conditions or under standard incubator conditions. The cells are then transfected using electroporation using a guide RNA and a homology-directed repair plasmid to effect the genomic GFP insertion. The cells are then expanded under either positive pressure and low oxygen conditions or under standard incubator conditions. After one week, the GFP-positive cells are assessed.

FIG. 32 shows that less input of T-cells still allowed for successful genome editing of the CD8-positive T-cells as indicated by the GFP signal visible in the cells even at the lowest concentration of 5000 cells. TABLE 2 below provides a quantification of the results of the FIG. 32. The results indicate that a use of hypoxic and positive pressure conditions to culture cells provided 45-times increased transfection efficiency over standard culture conditions. The p-value of the experiment was 1.68×10⁻¹⁸.

TABLE 2

GFP+ Cell

Low Oxygen and Positive

Cell Count
Standard
Pressure

5000
0/12
1/12

10000
0/12
12/12

50000
1/12
12/12

100000
4/12
12/12

FIGS. 59-60 provide molecular confirmation of the genome editing experiments performed above. FIG. 59 provides data relating to the PD1 knockout, and FIG. 60 provides data relating to the CTLA4 knockout. Both figures show that the highlighted bands in the DNA gels (upper panels) were extracted and used as a template for sequencing. For FIG. 59, genome editing was confirmed for Donors 76, 78, 82, and MOLT4. The PD1-pos-HDR was a positive control from previous MOLT4 knockout cells. For FIG. 60, genome editing was confirmed for Donors 74, 75, and 76.

Example 5: Reprogramming of Cells Using a Method of the Invention

FIG. 7 depicts a workflow of a stem cell reprogramming experiment using a method of the invention. Prior to initiation of the reprogramming process, human fibroblasts were plated in fibroblast medium until the cells reached a desired confluency. The cells were cultured either under standard conditions or under hypoxic and positive pressure conditions. The fibroblasts were then transfected with a RNA vector encoding Oct4, KLF-4, SOX2, GLIS1, and c-MYC, and a puromycin resistance gene. After 5 days of puromycin selection post-transfection, the cells were cultured in reprogramming media for the remainder of the reprogramming induction phase until the induced pluripotent stem cell (iPSC) colonies emerged. Recombinant B18R Protein was also added during the first 2 weeks after transfection to inhibit the interferon response and increase cell viability. After about 20 days, the iPSC colonies were isolated and propagated in maintenance media. The maintenance media was a complete, serum-free media designed for the feeder-free maintenance and expansion of stem cells. The maintenance media contained recombinant human basic fibroblast growth factor and recombinant human transforming growth factor β. The reprogramming media was a complete, xeno-free defined reprogramming media designed for generating iPSCs under feeder-free conditions.

The colonies formed from standard or hypoxic and high pressure conditions were assessed for various markers. FIG. 8 shows that by day 19, the average fold increase in colony number was higher in cells grown under hypoxic and either standard or high pressure conditions as compared to standard conditions (18% oxygen and 0 PSI). For example, cells grown under 5% oxygen and 0 PSI had about a 13-fold increase in colony number as compared to cells grown in 18% oxygen and 0 PSI, which increase was statistically significant. Further, cells grown under 5% oxygen and 2 PSI had about a 10-fold increase in colony number as compared to cells grown in 18% oxygen and 0 PSI, which increase was statistically significant.

FIG. 9 depicts the frequency distribution of colony area between cells cultured in 18% oxygen; 0 PSI, 5% oxygen; 0 PSI, and 5% oxygen; 2 PSI conditions. The graph illustrates that the colony area (measured in μm²), was greater in cells grown under hypoxic and either standard or positive pressure conditions.

FIG. 10 shows microscopy images of the morphology of cells grown at 18% oxygen; 0 PSI, 5% oxygen; 0 PSI, and 5% oxygen; 2 PSI conditions. The images show that cells grown under hypoxic and either standard or high pressure conditions had a greater cell area than those cells grown under standard conditions.

FIG. 11 shows the reprogramming kinetics of cells grown under 18% oxygen; 0 PSI (standard), 5% oxygen; 0 PSI, and 5% oxygen; 2 PSI conditions. The graph shows that cells grown under hypoxic and either standard or positive pressure conditions had a higher rate of reprogramming, as indicated by the increase in stem cell colony count per days post-transfection, as compared to cells grown under standard conditions.

FIG. 12 shows the effect of hypoxia and positive pressure conditions on pre-cardiomyocyte differentiation and morphology. H9c2 pre-cardiomyocytes were cultured under 20% oxygen; 0 PSI (standard conditions), 5% oxygen; 2 PSI, and 1% oxygen; 5 PSI. The cells were then stained with DAPI to identify the nuclei of the cells, F-actin to visualize the individual cells, and cardiac troponin, a marker of cardiomyocytes. The results indicated that with decreasing oxygen levels and increasing pressure levels, the cells expressed increasing levels of cardiac troponin as shown by the more intense staining in the cytoplasm of the cells.

FIG. 13 shows staining of fibroblasts with DAPI to identify the nuclei of the cells and Sox2, a stem cell marker. After removal of pluripotency supporting mouse embryonic fibroblasts (MEFs), the cells grown under standard conditions (20% oxygen and 0 PSI) differentiated, while the cells grown under 1% oxygen and 2 PSI conditions maintained their dedifferentiated state. The differentiation was indicated by the Sox2 staining and the morphology of the cells after culture in the varying oxygen and pressure conditions. The cells were assessed for other stem cells markers by qPCR as shown in FIG. 14.

FIG. 14 shows that the cells grown under 1% oxygen and 2 PSI had higher levels of Nanog, Oct4, and Sox2 as compared to cells grown under standard conditions.

FIG. 15 shows staining of fibroblasts 23 days post-transfection of the reprogramming RNA vector as described above under 20% oxygen; 0 PSI (standard conditions); 5% oxygen; 0 PSI, and 5% oxygen; 2 PSI. The cells were stained with DAPI, Sox2, and SSEA4, the latter two of which are stem cells markers. Cells of the same size from each condition were analyzed for expression of SSEA4, a human embryonic stem cell marker. The results indicated that the cells grown under hypoxic and either standard or high pressure conditions showed greater staining for SSEA4, as indicated by the more intense staining around the periphery of the cells in FIG. 15. FIG. 16 shows the average colony area over the experimental period for the aforementioned conditions.

FIG. 49 shows induction of neural precursor markers, PAX6 and NESTIN, in iPSCs after two weeks in culture under 5% O₂and 2 PSI in stem cell maintenance media.

FIG. 33 shows that a combination of low oxygen and positive pressure enhances ectoderm commitment in defined medium, while causing changes in colony morphology to more mesoderm-like morphology. The directed differentiation of iPSCs to all three germ layers was performed using defined medium under the indicated cell culture conditions. Each experiment was performed in triplicate using independent iPSC reprogrammed cells lines at passage 5. The results indicate that the brachyury (mesoderm indicator) was more prominent along the edges of the cells under culture at hypoxic and high pressure conditions. FOXA2 (endoderm indicator) was more prominent in cells under culture at hypoxic and high pressure conditions. Finally, PAX6 (ectoderm indicator) was more highly expressed in the cells under grown under low oxygen and high pressure conditions.

FIG. 34 shows that induction of PAX6 in iPSCs was accompanied by a loss of E-cadherin (indicated by CDH1 staining) under conditions of low oxygen and positive pressure (A; left panel is PAX6 staining; right panel is CDH1 staining). Additionally, SOX2, SSEA4, and NANOG staining decreased at low oxygen and positive pressure conditions, while OCT4 remained fairly constant throughout the three experimental conditions (B; left panel is SOX2 staining, right panel is SSEA4 staining. C; left panel is OCT4 staining, right panel is NANOG staining).

FIG. 17 shows the gene expression profiles as a function of oxygen concentration and pressure as compared to standard incubation conditions. The results indicated that oxygen concentration and pressure had an effect on the gene expression profile of several iPSC marker genes of interest. FIG. 45 shows the effect that various oxygen and pressure conditions had on the gene expression of immunotherapeutic targets in donor PBMCs. The cell culture conditions were created to mimic the vasculature and tumor microenvironments. The sample size for each condition was 12. The results of FIG. 45 are quantified (on a logarithmic scale) in TABLE 3 below.

TABLE 3

Donor 1
Donor 2
Donor 3
Donor 4
Donor 5
Donor 6

TNFRSF4
6.619909
6.866825
6.948752
6.77406
7.199577
7.044961

TNFRSF9
7.11055
7.182824
7.046122
7.770995
7.907799
8.281298

TNFSF4
6.959052
7.238442
6.69186
7.131686
7.270319
7.912743

IL10
7.571088
7.472796
8.289168
8.460592
10.14985
9.663655

IL20
3.923793
3.7464
3.066
3.650412
4.757204
3.658009

GNLY
11.58694
11.58746
11.92236
12.00283
12.14272
12.32933

CD8A
10.58403
10.74872
11.73939
11.03479
11.5482
11.26316

IL1B
12.26734
12.36899
13.85733
13.13851
17.2512
16.41079

CTLA4
7.218212
7.548015
7.96451
8.191502
8.123371
8.438327

ICOS
8.459099
8.514474
9.260944
9.310994
9.568987
9.989088

PDCD1
5.298568
5.887669
5.870134
5.689587
6.187069
6.070108

CX3CR1
6.848331
6.521741
6.770097
6.755467
7.244198
7.098837

BTLA
6.550678
6.491376
7.731419
7.463986
8.060028
7.759103

IL12A
5.238696
4.780255
5.055161
4.893
4.910049
5.020782

CXCL9
4.88878
4.39225
4.078183
6.442779
5.653539
4.950788

CXCL10
6.477897
6.717857
5.181365
7.381816
4.957034
4.986273

CXCL13
8.705631
8.945058
8.734548
9.69251
9.49176
10.32852

IL13
3.67695
4.39225
4.078183
4.453252
3.883496
4.668556

IL4
4.262466
4.885367
4.356257
5.157029
3.736401
4.668556

HAVCR2
9.170135
9.146919
9.582496
9.055441
8.495221
8.383747

MICA
9.5119
9.485452
8.457327
8.581001
8.713503
8.310856

VEGFA
10.82009
10.66857
11.57516
10.85657
12.58646
12.20046

IL17A
4.394877
4.019712
3.658276
4.555729
5.5696
4.950788

IL6
10.67563
10.36244
9.83626
11.30245
13.4968
12.44336

FOXP3
7.649995
7.533283
8.282547
8.390411
8.306643
8.195861

CD40LG
6.142305
6.637095
6.475081
7.027289
6.392924
7.242464

IDO1
10.16081
10.41552
9.648315
11.00966
9.463818
10.17725

IL7
5.298568
5.509989
5.591429
5.876287
6.829225
6.680652

CD274
9.902727
9.830315
8.97929
9.246269
10.94272
10.59646

PDCD1LG2
9.004413
8.817339
6.071134
7.61544
6.963616
6.700756

IL33
7.31835
7.105141
3.066
10.27267
3.883496
3.066

IL15
7.450272
7.687581
7.290701
7.714615
7.036545
7.434076

PRF1
9.608044
9.799925
10.05587
10.23814
9.88916
10.17636

CD27
7.499824
7.739823
8.341071
7.842887
7.983301
8.152978

LAG3
7.703818
7.605478
7.701923
8.135379
8.251017
8.432363

CD4
9.963468
10.03466
10.39339
10.6011
10.25358
10.45002

IFNG
3.066
4.019712
4.356257
4.339943
4.701601
5.020782

CXCL1
13.46389
13.51905
14.50338
14.0498
15.36751
14.96332

CCL7
10.178
10.03205
11.89334
9.941749
6.974265
7.373004

CCL5
11.94975
12.08067
12.31532
12.19939
11.9531
12.05798

CD40
8.757388
8.690513
9.051137
8.895786
8.983975
8.414324

CD70
5.700041
5.446335
5.546533
6.159697
5.988991
5.736881

ICAM1
10.56529
10.50489
9.923909
10.34862
10.86652
10.23831

TGFB1
11.26375
11.17533
11.36419
11.25554
11.26814
11.0061

CD33
7.582629
7.291983
9.093345
7.724167
7.278922
6.414203

TABLE 4 below shows the effect that high atmospheric pressure had on iPSCs grown under hypoxic conditions as assessed by digital PCR. The results indicate that that was a change in gene expression of various neuronal, bone, and cardiomyocyte factors. TABLE 4 shows relative gene expression changes with 1 being no change, and values above 1 indicating greater expression, and values below 1 indicating lower expression.

TABLE 4

iPSC
iPSC

5% O₂+ 0 PSI
5% O₂+ 2 PSI

PAX6
13.167
116.833

FABP7
3.083
7.488

LEFTY2
3.047
1.526

COL2A1
2.997
21.564

FOXA2
2.761
1.674

BMP2
2.682
2.071

OTX2
2.179
2.513

NCAM1
2.038
3.667

CDH2
2.025
7.926

GATA2
2.000
1.000

FGFR1
1.994
1.683

COL9A1
1.791
1.764

ACTC1
1.784
7.826

RUNX1
1.667
7.667

COMMD3
1.656
4.008

PTEN
1.574
1.902

COL1A1
1.568
2.147

NES
1.535
3.579

ALDH2
1.444
2.235

SOX2
1.438
1.472

CDC42
1.387
1.239

EP300
1.382
1.412

FGF2
1.343
0.948

REST
1.323
1.651

HSPA9
1.276
1.060

GPI
1.274
1.253

GABRB3
1.264
0.821

CCNA2
1.259
1.173

MYBL2
1.258
1.165

CDK1
1.229
1.233

LEFTY1
1.225
0.360

TCF3
1.218
1.439

ALPL
1.209
1.119

DPPA3
1.208
0.531

SFRP2
1.197
1.921

GRB7
1.189
0.533

PSMB4
1.167
0.997

NAT1
1.151
0.792

APC
1.145
1.361

GDF3
1.138
0.443

REEP5
1.133
0.996

LIN28A
1.125
1.065

BRIX1
1.111
0.957

PSMB2
1.104
1.016

NUMB
1.090
1.090

HPRT1
1.089
0.774

AFP
1.082
1.135

CCNE1
1.073
0.977

PODXL
1.068
0.737

GJA1
1.067
1.177

PARD6A
1.060
0.766

KAT7
1.052
1.037

KAT8
1.019
0.843

POU5F1
0.982
0.548

RAB7A
0.973
1.062

CDH1
0.954
0.365

KAT2A
0.946
0.996

DNMT3B
0.935
0.589

CD9
0.894
0.398

FOXD3
0.880
0.343

DPPA2
0.876
0.425

MYCN
0.867
1.018

KLF4
0.806
1.032

MYC
0.759
0.822

TDGF1
0.757
0.291

TBX3
0.750
1.250

HDAC2
0.739
0.773

ALDH1A1
0.708
1.542

RUNX2
0.684
0.947

EMX2
0.667
44.000

ZFP42
0.606
0.464

NODAL
0.583
0.313

GAL
0.485
0.175

NANOG
0.485
0.182

NR5A2
0.218
0.113

FGF5

1.500

Example 6: Change in Gene Expression in Cancer Cells

FIG. 35 show that different combinations of tumor (disease) extracellular matrix (ECM), low oxygen, and high pressure can alter the gene expression of EGFR and other metabolic regulators in DU145 (prostate cancer) and Panc10 (pancreatic cell lines). The markers measured included EGFR, ErbB2, LDHA, SLC1A5, and TRX1. The results indicate that, generally, gene expression increased with low oxygen, high pressure, and tumor ECM conditions. However, for ErbB2 in DU145 cells, the ErbB2 expression decreased compared to standard incubator conditions when the cells were cultured under hypoxic and high pressure conditions.

FIG. 36 shows that PDL1 expression increased in ARV7-positive, 22RV1 prostate cancer cells during low oxygen and positive pressure culturing conditions. The top panel of FIG. 37 provides a western blot showing increased PDL1 protein expression under various conditions of high pressure and hypoxia in both DU145 and 22Rv1 prostate cancer cells. The bottom panel of FIG. 37 provides a quantification of the western blot results normalized to actin. The results indicated that the change in PDL1 expression was more pronounced in the 22Rv1 prostate cancer cells, rather than the DU145 prostate cancer cells.

TABLES 5-10 below show the effect of varying oxygen and pressure on the gene expression profiles of various cancer targets when compared to traditional culturing approaches.

TABLE 5

LnCAP (2 PSI)

1% O₂
10% O₂
20% O₂

VEGFA (higher)
EGFR (higher)
PRPF40A (lower)

GPI (higher)
GPI (higher)
SSSCA1 (lower)

HK2 (higher)
HSP90B1 (lower)
VEGFA (higher)

HSP90B1 (lower)
VEGFA (higher)
EGFR (higher)

EPAS1 (lower)
GPX1 (lower)
NFκB1 (lower)

EGFR (higher)
SPG7 (lower)
PARP2 (lower)

TABLE 6

LnCAP (5 PSI)

1% O₂
10% O₂
20% O₂

GPI (higher)
EGFR (higher)
NFκB1 (lower)

HK2 (higher)
HDAC3 (higher)
HDAC3 (higher)

ITGAV (lower)
PK3C2A (higher)
LAMA3 (lower)

EGFR (higher)
PLOD3 (higher)
PARP2 (lower)

VEGFA (higher)
GP1 (higher)
HSP90B1 (lower)

HDAC3 (higher)
CDK5 (lower)
SSSCA1 (higher)

TABLE 7

PC-3 (2 PSI)

1% O₂
10% O₂
20% O₂

ATF2 (higher)
ITGAV (lower)
SSSCA1 (lower)

HDAC3 (higher)
ATF2 (lower)
PEA15 (higher)

GP1 (higher)
PEA15 (lower)
CDK2 (higher)

PEA15 (lower)
IGF1R (lower)
ATF2 (higher)

HK2 (higher)
PLK4 (higher)
NFκB1 (lower)

HPRT1 (higher)
RHOB (lower)
ABCC1 (lower)

TABLE 8

PC-3 (5 PSI)

1% O₂
10% O₂
20% O₂

GP1 (higher)
PI3KC2a (lower)
P992CB (higher)

HK2 (higher)
CDC25A (lower)
CDK7 (lower)

PARP2 (lower)
PLK4 (lower)
HDAC3 (lower)

PLK1 (lower)
PRPF40A (lower)
ERBB2 (higher)

SSSCA1 (lower)
MAN2B1 (higher)
PEA15 (higher)

AURKA (lower)
HSP90B1 (higher)
HK2 (lower)

TABLE 9

PANC-10.05 (2 PSI)

1% O₂
10% O₂
20% O₂

GP1 (higher)
GP1 (higher)
CTSB (lower)

ITGAV (lower)
SPG7 (lower)
ITGAL (lower)

VEGFA (higher)
PARP2 (lower)
ITGAV (lower)

CDK7 (lower)
CDC25A (lower)
CDK2 (higher)

MAN2B1 (higher)
MMP1 (higher)
GPI (higher)

HRAS (lower)
COL12A1 (lower)
ENO1 (lower)

TABLE 10

PANC-10.05 (5 PSI)

1% O₂
10% O₂
20% O₂

GP1 (higher)
GP1 (higher)
RHOB (lower)

VEGFA (higher)
CDK5 (higher)
CTSB (lower)

ADM (higher)
CDC25A (lower)
BIRC5 (lower)

DR1 (lower)
PPP2CB (higher)
ITGAL (lower)

HK2 (higher)
TXN_ALT (higher)
PIK3C3 (lower)

NAA10 (lower)
TXNRD1 (higher)
ITGAV (lower)

FIG. 38 shows identification of pressure and oxygen sensitive gene expression signatures in various cell lines. The results indicated that there was an enrichment of metabolic processes involved in cell survival. The results in the top panel of FIG. 38 were corrected for any false discovery rate. The top panel of FIG. 38 provides various gene ontology terms that were enriched under low oxygen or positive pressure conditions. The bars to the right of the 0 on the x-axis indicated upregulated genes, and the bars to the left of the 0 on the x-axis indicated downregulated genes. The bottom panel of FIG. 38 shows that over 130 genes were shown to be co-regulated by low oxygen and positive pressure conditions across various cell lines.

FIGS. 57 and 58 show the effect of various experimental conditions on various cells lines. FIG. 57 shows pressure-sensitive genes, while FIG. 58 depicts oxygen-sensitive genes. The cells lines tested for both figures was (from left to right) PANC10, LNCaP, PC3, and 22Rv1. Within each group of cell lines, the cells were exposed to high (>18%; left-most columns of cells) and low (<5%; right-most columns of cells) oxygen, low pressure (0 PSI; left-most columns of cells) and positive pressure (2 PSI; right-most columns of cells). TABLES 11-16 below provide the quantitative data for the heatmaps of FIGS. 57-58.

TABLE 11

Oxygen-Sensitive Candidates

O2
PSI
Expt
CellLine
FUT11
PFKFB4
PPFIA4
ANKZF1
PFKFB3
MIR210HG
BNIP3L
LDHA

LNCaP_25
20
0
A
LNCaP
148.8648
142.0982
8.458226
419.528
316.3376
10.14987
1283.959
11777.23

22RV1_3
20
0
A
22RV1
221.0045
97.77776
6.697107
420.5783
297.3515
18.7519
488.8888
10510.44

PC3_3
20
0
A
PC3
982.6681
450.2195
9.330973
306.7557
521.9513
20.4115
713.2363
19104.58

DU145_3
20
0
A
DU145
374.04
107.181
41.56
192.4884
1296.016
28.43579
3087.47
17411.45

PANC10_12
20
0
B
PANC10
261.2557
36.13111
2.779316
141.7451
390.4939
16.6759
922.733
18838.21

LNCaP_1
20
0
B
LNCaP
157.7928
136.224
1.1352
407.5369
294.0168
21.5688
1683.502
11943.44

22RV1_12
20
0
B
22RV1
222.767
104.2549
6.237475
440.1875
308.3095
24.9499
619.2922
9452.448

DU145_12
20
0
B
DU145
446.3176
143.8638
32.85079
190.308
1184.894
52.10815
3061.92
23495.11

PANC10_21
20
0
C
PANC10
338.7363
30.03138
1.766552
150.1569
314.0046
18.10716
1237.028
22815.9

LNCaP_10
20
0
C
LNCaP
159.8962
153.9
8.994158
376.7553
293.8092
17.98832
1323.141
11189.73

22RV1_21
20
0
C
22RV1
195.1418
84.6398
2.351106
391.4591
246.8661
28.21327
739.4227
8315.861

PC3_25
20
0
C
PC3
670.9391
423.8815
8.262797
356.1266
355.3003
13.22048
796.5337
17632.81

DU145_21
20
0
C
DU145
371.5608
119.1418
33.31931
209.0029
1529.659
36.34834
2347.497
18875.89

PANC10_8
20
0
A
PANC10
271.5097
21.81774
4.242339
199.996
283.6307
12.12097
994.5255
21212.91

LNCaP_26
20
0
A
LNCaP
193.4126
109.4788
3.649293
385.0005
328.4364
18.24647
1591.092
13212.27

22RV1_6
20
0
A
22RV1
236.1916
112.9612
4.107679
465.1947
291.6452
15.4038
626.4211
9411.72

PC3_6
20
0
A
PC3
814.0391
385.4435
16.82201
409.5794
430.7898
22.67315
815.5019
21881.78

DU145_6
20
0
A
DU145
390.234
120.8689
34.53398
188.7858
1281.211
35.68512
3075.827
18655.26

PANC10_15
20
0
B
PANC10
266.3327
11.38174
1.138174
161.6207
357.3867
15.93444
1108.582
20638.51

LNCaP_4
20
0
B
LNCaP
218.3846
168.5067
4.04416
413.8524
376.1069
22.91691
1480.163
12063.73

22RV1_15
20
0
B
22RV1
224.198
107.2251
3.899096
416.2285
296.3313
16.57116
611.1833
9461.156

PC3_15
20
0
B
PC3
1154.691
535.3124
26.03343
434.9752
612.328
71.04957
896.5263
22944.13

DU145_15
20
0
B
DU145
500.0377
221.7909
62.50471
264.1328
1630.163
87.70823
2433.651
26165.28

PANC10_24
20
0
C
PANC10
279.1962
23.19056
4.547169
155.9679
344.6754
12.27736
968.0923
18497.43

LNCaP_13
20
0
C
LNCaP
191.8114
184.7509
5.883786
398.9207
303.6034
25.88866
1423.876
11905.25

22RV1_24
20
0
C
22RV1
222.8496
119.8661
5.064763
446.5433
254.0823
27.8562
882.9571
8997.552

PC3_26
20
0
C
PC3
752.2415
421.9328
6.159603
333.3885
432.7121
12.31921
878.5134
18287.86

DU145_24
20
0
C
DU145
315.9268
100.4485
42.12358
241.4005
1213.483
42.12358
1699.524
15059.18

PANC10_9
20
0
A
PANC10
308.4363
25.97358
0
220.7754
269.4759
12.98679
1042.19
19941.22

LNCaP_27
20
0
A
LNCaP
187.4626
94.73918
0
417.2555
264.0603
20.15727
1408.993
12487.43

22RV1_9
20
0
A
22RV1
221.6903
108.7925
0
472.1183
279.1656
12.31613
665.071
9027.723

PC3_9
20
0
A
PC3
1047.944
516.1348
17.91357
383.4624
615.779
34.14775
1031.15
25294.52

DU145_9
20
0
A
DU145
525.8409
352.0274
81.40633
323.4251
1627.027
82.50642
3651.184
22824.57

PANC10_18
20
0
B
PANC10
294.2244
34.51794
9.862269
198.8891
295.8681
32.87423
1290.313
22765.4

LNCaP_7
20
0
B
LNCaP
208.0811
123.3848
4.182534
367.0174
324.1464
13.59324
1846.589
13925.75

22RV1_18
20
0
B
22RV1
251.6665
100.153
3.852038
376.2157
290.1869
19.26019
735.7393
8831.439

PC3_18
20
0
B
PC3
876.1958
531.8069
48.03854
466.1768
508.1259
40.59595
883.6384
21119.36

DU145_18
20
0
B
DU145
358.0772
119.3591
30.744
233.2927
888.8633
55.15835
2062.561
18450.92

PANC10_27
20
0
C
PANC10
267.4496
25.71631
0
161.1555
330.8832
22.28747
865.7824
20482.18

LNCaP_16
20
0
C
LNCaP
166.4081
147.7803
12.41851
409.811
294.3188
33.52999
1435.58
11798.83

22RV1_27
20
0
C
22RV1
220.7981
118.5017
4.051341
430.455
238.0163
16.20536
751.5237
8691.139

PC3_27
20
0
C
PC3
699.4571
410.7506
11.15457
398.9399
359.5708
14.43532
812.3151
17792.19

DU145_27
20
0
C
DU145
996.1885
0
110.6876
0
1217.564
221.3752
1771.002
19591.71

PC3_PSI_1
20
0
A
PC3
1664.168
254.2092
11.80753
548.0084
587.5984
77.7908
2127.44
18376.69

PC3_PSI_2
20
0
B
PC3
1500.024
210.9101
5.913367
488.1813
424.4483
96.585
2270.733
21171.83

PC3_PSI_3
20
0
C
PC3
1481.33
225.2641
12.73578
491.1234
602.5615
61.29093
2450.841
18856.11

PANC10_4
10
2
A
PANC10
388.3242
57.908
13.62541
170.3176
530.2556
40.87623
1196.765
12772.69

PC3_2
10
2
A
PC3
1127.084
485.2885
36.79082
395.9393
637.1236
50.80638
873.0521
21860.76

DU145_2
10
2
A
DU145
414.3253
80.87098
69.79276
168.3889
1087.881
86.41008
4175.38
18361.04

PANC10_11
10
5
B
PANC10
246.0487
33.96941
4.590461
169.8471
377.3359
6.426645
943.7988
18659.31

LNCaP_2
10
5
B
LNCaP
247.6243
405.2033
14.06956
419.2729
478.3651
66.12694
2207.514
15593.29

22RV1_11
10
5
B
22RV1
211.1379
131.6928
6.441494
396.5097
334.242
19.32448
704.9857
10325.71

DU145_11
10
5
B
DU145
424.3177
102.2594
22.33251
202.168
983.8058
75.22529
3838.841
24570.46

PANC10_19
20
2
C
PANC10
234.8746
38.70092
6.672573
146.7966
334.9632
33.36287
914.1425
20149.84

PANC10_20
20
5
C
PANC10
253.3356
45.50203
5.53403
153.1082
348.029
33.20418
1054.54
21815.15

LNCaP_11
20
2
C
LNCaP
161.5547
163.0369
4.446461
339.4132
250.484
34.08953
1551.815
10816.76

LNCaP_12
20
5
C
LNCaP
160.2608
163.7447
0
369.2966
280.4564
27.87144
1477.186
10847.22

22RV1_19
20
2
C
22RV1
197.8738
119.4952
6.424474
409.8815
267.2581
23.12811
1029.201
8685.889

PC3_19
20
5
C
PC3
670.3506
441.7176
16.96196
342.0661
474.228
29.68342
804.9861
18424.92

PC3_22
20
2
C
PC3
616.4342
412.4344
10.13664
311.7016
456.1486
35.47823
866.0488
20432.29

DU145_19
20
2
C
DU145
283.305
110.0012
31.13242
176.4171
1270.203
53.96286
2028.796
17877.27

DU145_20
20
5
C
DU145
272.9211
81.08527
29.66534
288.7427
812.8303
55.3753
2099.317
18893.86

LNCaP_23
10
2
A
LNCaP
210.4404
134.5439
3.449843
322.5603
260.4631
36.22335
1540.355
12221.07

22RV1_5
10
2
A
22RV1
231.5576
109.639
8.771119
511.3563
292.0783
16.66513
735.0198
9873.649

PC3_5
10
2
A
PC3
932.2809
406.043
18.49152
375.2238
530.8608
29.27824
976.1983
24877.26

DU145_5
10
2
A
DU145
531.7783
186.5714
121.7204
222.4889
1420.736
130.6997
4202.346
19979.1

PANC10_14
10
5
B
PANC10
244.5717
46.2249
8.404527
179.0164
401.7364
42.02264
1121.164
21281.94

LNCaP_5
10
5
B
LNCaP
236.4201
149.1802
3.489596
343.7252
305.3396
28.78916
2145.229
14346.6

22RV1_14
10
5
B
22RV1
212.7266
123.0226
4.271619
423.7446
325.4974
22.21242
609.1329
10182.69

PC3_14
10
5
B
PC3
1372.075
660.5132
42.33261
462.546
750.1588
115.1696
1169.127
29175.88

DU145_14
10
5
B
DU145
718.0632
286.0285
113.6933
319.5381
1571.362
183.1061
3811.719
26951.31

PANC10_22
20
2
C
PANC10
241.9556
31.37496
1.898129
153.1902
385.2086
11.50043
835.9584
16977.98

PANC10_23
20
5
C
PANC10
224.4627
34.78357
1.086987
107.6117
385.3367
7.608906
980.4619
18153.76

LNCaP_14
20
2
C
LNCaP
185.8022
182.617
0
425.7525
285.6046
35.03699
1472.615
12031.49

LNCaP_15
20
5
C
LNCaP
210.3339
161.9813
3.626446
388.0297
233.3014
20.54986
1646.407
13451.7

22RV1_22
20
2
C
22RV1
185.5875
131.6668
7.523819
500.334
259.5718
35.11116
852.6995
8938.298

22RV1_23
20
5
C
22RV1
195.4212
109.9976
5.850934
439.9902
320.6312
19.89317
788.7058
9155.541

PC3_20
20
5
C
PC3
961.2017
488.5887
13.31304
398.7256
536.5156
34.61391
896.6334
22240.1

PC3_23
20
2
C
PC3
975.2113
431.7469
17.56259
385.4012
456.1394
34.63732
887.8863
22027.38

DU145_22
20
2
C
DU145
352.0325
123.6235
72.99672
243.7148
1377.519
65.93252
2081.584
15409.37

DU145_23
20
5
C
DU145
321.1689
97.3239
30.08193
139.7925
1235.129
32.73622
2099.542
16100.91

PANC10_6
10
2
A
PANC10
275.0814
24.77833
4.619688
152.4497
294.4001
27.71813
1201.959
24722.47

LNCaP_24
10
2
A
LNCaP
191.2567
137.5476
7.859865
328.8044
331.4243
32.74944
1923.047
15162.99

22RV1_8
10
2
A
22RV1
210.2406
129.0285
4.553949
465.2618
245.1542
15.93882
881.9481
10532.52

PC3_8
10
2
A
PC3
1268.069
676.1431
25.66617
421.0856
741.9127
52.13441
1305.766
32049.02

DU145_8
10
2
A
DU145
484.3028
162.0973
97.45725
226.7373
1511.582
72.5957
3500.505
20059.29

PANC10_17
10
5
B
PANC10
322.1212
23.81882
3.402689
168.4331
326.6581
23.25171
1291.32
21974

LNCaP_8
10
5
B
LNCaP
177.4626
216.7482
10.83741
356.2799
376.6001
40.64029
1574.134
14511.29

22RV1_17
10
5
B
22RV1
239.3598
120.3411
5.28972
435.0795
323.9953
15.86916
756.4299
10143.04

PC3_17
10
5
B
PC3
972.7494
524.2362
29.85234
401.9144
527.1486
72.81059
1108.905
25722.52

DU145_17
10
5
B
DU145
321.8763
162.586
56.02626
221.9079
1329.25
71.40601
2352.004
19793.75

PANC10_26
20
5
C
PANC10
210.1558
32.4252
2.634548
134.3619
362.9596
7.29567
992.0085
20848.8

LNCaP_17
20
2
C
LNCaP
144.6707
201.7776
6.345206
418.7836
253.8082
31.72603
1463.205
10600.3

LNCaP_18
20
5
C
LNCaP
200.3051
120.9572
0.967658
311.5857
244.8174
20.32081
1815.326
11864.45

22RV1_25
20
2
C
22RV1
208.1508
121.137
5.118463
380.4724
226.9185
23.88616
759.2387
9999.771

22RV1_26
20
5
C
22RV1
192.6104
118.7764
1.605087
393.2463
287.3105
16.05087
828.2248
10298.24

PC3_21
20
5
C
PC3
675.8017
388.9932
10.86022
359.8681
427.004
23.69502
776.9992
21501.26

PC3_24
20
2
C
PC3
710.7665
408.105
10.25144
335.8567
405.6641
20.01472
809.8638
21619.31

DU145_25
20
2
C
DU145
327.3233
93.92135
39.62393
168.0582
1223.67
43.63578
1988.066
17555.27

DU145_26
20
5
C
DU145
0
0
0
241.1813
482.3626
0
1688.269
6994.258

PC3_PSI_4
20
0.5
A
PC3
1470.105
299.8807
7.468294
589.4208
498.0778
88.47056
2114.676
20392.47

PC3_PSI_7
20
2
A
PC3
913.9944
249.5563
6.27251
402.3367
398.7524
49.28401
1836.053
21830.13

PC3_PSI_10
20
5
A
PC3
1082.442
231.3616
11.47627
386.0616
405.8008
42.23267
1590.611
23326.66

PC3_PSI_5
20
0.5
B
PC3
1393.246
294.2318
10.64655
573.4617
486.8376
80.33304
2149.635
19581.9

PC3_PSI_8
20
2
B
PC3
929.2346
237.8557
8.875211
387.8467
434.8853
56.80135
1786.58
20405

PC3_PSI_11
20
5
B
PC3
955.0823
239.8718
12.81452
364.4129
430.8883
49.25581
1538.143
23672.02

PC3_PSI_6
20
0.5
C
PC3
1275.758
348.6979
1.400393
639.9797
494.3388
113.4318
1960.55
20218.88

PC3_PSI_9
20
2
C
PC3
867.4324
285.8406
11.47535
366.6897
484.0513
45.90142
1520.484
22023.81

PC3_PSI_12
20
5
C
PC3
1123.57
261.2875
9.009913
433.8106
426.8029
52.05727
1841.359
19512.13

PANC10_1
1
2
A
PANC10
731.0598
187.9868
26.10928
313.3113
1096.59
219.3179
1921.643
20991.86

LNCaP_19
1
2
A
LNCaP
702.2483
1298.139
193.7739
971.7834
1169.928
273.906
5826.33
49116.59

22RV1_1
1
2
A
22RV1
214.5837
163.0836
11.44447
504.9871
266.0838
58.65289
826.8627
10547.51

PC3_1
1
2
A
PC3
2573.914
1346.416
88.59052
662.4336
1702.375
122.9094
1581.86
31345.88

DU145_1
1
2
A
DU145
1051.173
659.8602
338.699
599.574
3639.096
358.429
6371.707
26577.46

PANC10_10
1
5
B
PANC10
804.5866
418.4743
147.303
571.3569
1063.483
446.3726
2510.846
31510.56

LNCaP_3
1
5
B
LNCaP
604.2898
627.438
92.59279
855.265
741.9607
159.6007
4498.061
44259.36

22RV1_10
1
5
B
22RV1
580.0314
467.6222
151.7524
784.6161
753.1415
229.3147
2421.294
24631.1

DU145_10
1
5
B
DU145
1203.968
624.6599
214.7803
558.7712
2454.143
245.5855
6173.865
31692.51

PANC10_2
1
2
A
PANC10
1183.027
307.4562
149.4509
695.9279
1512.121
346.7059
5590.067
78193.51

22RV1_4
1
2
A
22RV1
532.976
494.5483
162.0648
826.1964
652.4362
311.5988
2659.868
25173.51

PC3_4
1
2
A
PC3
4049.774
3173.175
378.6605
1172.901
2545.545
256.5425
3821.631
65465.66

DU145_4
1
2
A
DU145
1195.26
788.3113
406.9489
642.1021
3689.346
383.7992
8898.048
36787.45

PANC10_13
1
5
B
PANC10
772.2797
374.3285
86.31362
516.9731
1433.715
536.9616
3733.291
68947.32

LNCaP_6
1
5
B
LNCaP
526.9173
551.0878
56.80072
831.4659
807.2953
123.2696
4291.476
43798.19

22RV1_13
1
5
B
22RV1
526.6807
412.5491
108.8962
703.637
692.1191
216.7453
2371.634
24520.49

PC3_13
1
5
B
PC3
4524.925
3367.721
270.9948
1122.156
2841.377
296.029
4216.379
64623.19

DU145_13
1
5
B
DU145
1366.53
885.2882
312.0682
808.0923
2534.322
594.572
6794.874
49986.75

PANC10_3
1
2
A
PANC10
867.7696
348.604
80.79235
647.8349
1524.581
323.1694
3840.629
65821.82

LNCaP_21
1
2
A
LNCaP
818.9263
785.0397
141.1942
1016.598
1157.792
200.4958
5475.511
50954.16

22RV1_7
1
2
A
22RV1
719.2968
771.1298
254.6183
940.2691
1096.678
405.5706
4000.236
34090.67

PC3_7
1
2
A
PC3
4536.24
3476.616
242.0746
1253.534
2829.208
184.5271
4467.433
75690.53

DU145_7
1
2
A
DU145
1027.857
677.8808
337.3639
457.1754
3470.329
259.5916
6680.016
32923.99

PANC10_16
1
5
B
PANC10
758.2926
364.7887
74.44668
503.0468
1655.907
450.9342
3356.482
61379.16

LNCaP_9
1
5
B
LNCaP
570.9571
438.3332
47.20511
745.1664
690.0938
122.5085
4372.093
45783.34

22RV1_16
1
5
B
22RV1
550.355
384.2356
128.3036
709.0463
815.7409
237.0241
1979.927
29000.67

PC3_16
1
5
B
PC3
3915.276
2814.212
252.5061
1071.863
2282.289
302.3202
3744.648
70023.21

DU145_16
1
5
B
DU145
961.737
931.1199
90.05028
1010.364
1732.567
781.6364
3632.628
30377.56

TABLE 12

Oxygen-Sensitive Candidates

O2
PSI
Expt
CellLine
P4HA1
PDK1
HK2
SLC2A1

LNCaP_25
20
0
A
LNCaP
649.5917
517.6434
827.2145
779.8484

22RV1_3
20
0
A
22RV1
630.8675
163.4094
1745.266
1078.234

PC3_3
20
0
A
PC3
2446.465
314.3372
1463.796
1660.33

DU145_3
20
0
A
DU145
970.0978
260.2968
407.9442
4884.393

PANC10_12
20
0
B
PANC10
380.7663
162.59
1438.296
993.6055

LNCaP_1
20
0
B
LNCaP
743.5561
615.2785
860.4817
648.1993

22RV1_12
20
0
B
22RV1
691.4687
140.7887
1638.674
762.7541

DU145_12
20
0
B
DU145
1446.568
546.0028
391.9439
3900.182

PANC10_21
20
0
C
PANC10
647.8829
346.2442
1475.071
673.9396

LNCaP_10
20
0
C
LNCaP
632.5891
510.6683
658.5723
1055.315

22RV1_21
20
0
C
22RV1
615.9897
192.7907
1689.269
945.1445

PC3_25
20
0
C
PC3
1517.876
222.2692
1004.756
1670.738

DU145_21
20
0
C
DU145
1021.792
319.0576
492.7219
3892.301

PANC10_8
20
0
A
PANC10
610.8968
233.3287
1392.699
760.5908

LNCaP_26
20
0
A
LNCaP
806.4939
585.7116
718.9108
689.7165

22RV1_6
20
0
A
22RV1
688.0363
169.4418
1500.33
909.8509

PC3_6
20
0
A
PC3
2284.137
313.7671
1392.57
1420.363

DU145_6
20
0
A
DU145
1002.637
309.6547
439.7327
4949.871

PANC10_15
20
0
B
PANC10
557.7053
237.8784
1526.291
630.5484

LNCaP_4
20
0
B
LNCaP
676.7228
544.6136
812.8762
1005.648

22RV1_15
20
0
B
22RV1
695.9886
139.3927
1692.208
884.12

PC3_15
20
0
B
PC3
3292.687
381.8236
1419.364
2126.063

DU145_15
20
0
B
DU145
1396.275
404.2644
595.8111
4661.642

PANC10_24
20
0
C
PANC10
442.4396
237.8169
1573.321
888.9716

LNCaP_13
20
0
C
LNCaP
701.3473
571.904
890.8053
1093.208

22RV1_24
20
0
C
22RV1
732.7024
187.3962
1635.074
869.451

PC3_26
20
0
C
PC3
1839.411
267.9427
989.3862
2629.381

DU145_24
20
0
C
DU145
889.4556
210.6179
708.0002
3149.548

PANC10_9
20
0
A
PANC10
454.5376
347.3966
1451.274
811.6744

LNCaP_27
20
0
A
LNCaP
683.3315
522.0734
905.0615
776.055

22RV1_9
20
0
A
22RV1
734.8624
125.214
1239.824
927.8151

PC3_9
20
0
A
PC3
2725.102
467.9921
1644.13
1780.161

DU145_9
20
0
A
DU145
1434.512
503.8392
614.9478
4513.651

PANC10_18
20
0
B
PANC10
734.739
325.4549
1568.101
761.0384

LNCaP_7
20
0
B
LNCaP
860.5564
654.5666
808.2747
734.0347

22RV1_18
20
0
B
22RV1
647.1424
179.7618
1580.62
837.1763

PC3_18
20
0
B
PC3
2106.253
341.0059
1355.905
2117.755

DU145_18
20
0
B
DU145
1187.261
348.1306
602.2207
3117.803

PANC10_27
20
0
C
PANC10
454.3215
276.0217
1273.815
1016.651

LNCaP_16
20
0
C
LNCaP
706.6135
599.8143
702.8879
1046.881

22RV1_27
20
0
C
22RV1
679.6124
180.2847
1556.728
803.1783

PC3_27
20
0
C
PC3
1478.965
232.9337
1005.224
1622.662

DU145_27
20
0
C
DU145
332.0628
110.6876
553.4381
2324.44

PC3_PSI_1
20
0
A
PC3
5714.151
404.2344
1986.444
2360.812

PC3_PSI_2
20
0
B
PC3
6109.165
376.4844
1645.887
2259.563

PC3_PSI_3
20
0
C
PC3
5512.204
439.3843
2096.627
2465.965

PANC10_4
10
2
A
PANC10
465.5349
346.3125
2496.856
1017.364

PC3_2
10
2
A
PC3
3003.416
527.9191
1531.199
1958.673

DU145_2
10
2
A
DU145
653.6147
435.3739
301.3275
5786.152

PANC10_11
10
5
B
PANC10
370.9092
186.3727
1402.845
747.3271

LNCaP_2
10
5
B
LNCaP
938.4397
1060.845
896.231
1066.473

22RV1_11
10
5
B
22RV1
587.6074
191.8134
1524.487
815.2069

DU145_11
10
5
B
DU145
1067.259
684.08
218.6235
4109.182

PANC10_19
20
2
C
PANC10
358.9844
322.9525
1034.249
899.4629

PANC10_20
20
5
C
PANC10
426.7352
347.4141
1170.755
843.0173

LNCaP_11
20
2
C
LNCaP
641.7725
672.8978
542.4682
1050.847

LNCaP_12
20
5
C
LNCaP
646.2691
630.5914
674.1405
921.4995

22RV1_19
20
2
C
22RV1
481.8356
168.3212
1372.268
898.1415

PC3_19
20
5
C
PC3
1454.841
293.3005
1160.127
1879.95

PC3_22
20
2
C
PC3
1389.986
332.6084
1096.657
2104.619

DU145_19
20
2
C
DU145
771.0463
292.6448
372.5513
4544.296

DU145_20
20
5
C
DU145
777.2319
282.8096
354.0064
3331.418

LNCaP_23
10
2
A
LNCaP
562.3244
757.2405
726.1919
867.6354

22RV1_5
10
2
A
22RV1
526.2672
200.8586
1475.302
942.0182

PC3_5
10
2
A
PC3
2330.702
503.894
1410.749
1630.336

DU145_5
10
2
A
DU145
895.9417
565.7004
529.7829
4733.126

PANC10_14
10
5
B
PANC10
469.8131
350.4688
1268.243
837.9314

LNCaP_5
10
5
B
LNCaP
1092.243
1116.671
734.5599
789.521

22RV1_14
10
5
B
22RV1
574.9599
165.7388
1411.343
768.8914

PC3_14
10
5
B
PC3
3483.102
719.0319
1566.307
2744.149

DU145_14
10
5
B
DU145
1287.727
807.8211
539.7442
4438.828

PANC10_22
20
2
C
PANC10
305.3755
231.9067
1287.936
823.788

PANC10_23
20
5
C
PANC10
376.0974
271.2032
1108.183
748.3903

LNCaP_14
20
2
C
LNCaP
678.4435
713.4805
748.5175
1030.937

LNCaP_15
20
5
C
LNCaP
724.0804
889.6881
620.1223
956.173

22RV1_22
20
2
C
22RV1
605.6675
225.7146
1423.256
872.7631

22RV1_23
20
5
C
22RV1
576.902
196.5914
1425.287
903.3841

PC3_20
20
5
C
PC3
2397.013
392.7347
1262.742
2501.521

PC3_23
20
2
C
PC3
2171.906
408.3301
1249.383
2355.338

DU145_22
20
2
C
DU145
704.0651
276.6811
521.5733
3496.778

DU145_23
20
5
C
DU145
755.5873
307.8974
391.9499
3486.85

PANC10_6
10
2
A
PANC10
496.4065
395.1933
1267.474
753.0091

LNCaP_24
10
2
A
LNCaP
818.736
872.445
791.2264
755.857

22RV1_8
10
2
A
22RV1
557.8587
231.4924
1137.728
997.3148

PC3_8
10
2
A
PC3
3033.42
677.7473
1817.486
2119.063

DU145_8
10
2
A
DU145
782.6413
521.0979
348.0616
3922.157

PANC10_17
10
5
B
PANC10
537.6248
352.7454
1374.119
722.5042

LNCaP_8
10
5
B
LNCaP
732.88
872.4116
803.3231
1007.879

22RV1_17
10
5
B
22RV1
677.0841
207.6215
1496.991
763.0421

PC3_17
10
5
B
PC3
2249.119
491.4714
1362.286
2311.736

DU145_17
10
5
B
DU145
949.1507
449.3086
342.7489
3878.994

PANC10_26
20
5
C
PANC10
395.9928
313.5112
1105.091
765.0321

LNCaP_17
20
2
C
LNCaP
540.6116
719.5464
697.9727
1271.579

LNCaP_18
20
5
C
LNCaP
748.967
979.2695
737.3551
769.2878

22RV1_25
20
2
C
22RV1
544.2632
220.0939
1337.625
897.4372

22RV1_26
20
5
C
22RV1
598.6974
218.2918
1362.719
882.7977

PC3_21
20
5
C
PC3
1519.443
293.2259
1094.414
1690.245

PC3_24
20
2
C
PC3
1659.757
289.4812
1111.061
1648.041

DU145_25
20
2
C
DU145
677.9484
283.5227
335.7317
3924.363

DU145_26
20
5
C
DU145
723.5439
0
1447.088
2411.813

PC3_PSI_4
20
0.5
A
PC3
5684.521
567.5904
2330.108
2964.338

PC3_PSI_7
20
2
A
PC3
3168.962
676.5351
2363.84
1947.166

PC3_PSI_10
20
5
A
PC3
2911.759
659.1968
2217.215
2038.185

PC3_PSI_5
20
0.5
B
PC3
5978.036
587.9798
2240.13
2297.235

PC3_PSI_8
20
2
B
PC3
2980.296
670.966
2292.467
1868.232

PC3_PSI_11
20
5
B
PC3
2440.766
653.941
2114.396
1842.888

PC3_PSI_6
20
0.5
C
PC3
5698.2
504.1415
2181.813
3509.385

PC3_PSI_9
20
2
C
PC3
2633.072
619.6691
2340.451
2198.052

PC3_PSI_12
20
5
C
PC3
3495.846
623.3525
2334.235
2044.583

PANC10_1
1
2
A
PANC10
1154.03
1091.368
4140.931
1968.64

LNCaP_19
1
2
A
LNCaP
2533.63
3489.387
2233.499
2788.596

22RV1_1
1
2
A
22RV1
570.7928
193.1254
1821.101
1194.516

PC3_1
1
2
A
PC3
5150.222
1217.122
2708.795
3858.077

DU145_1
1
2
A
DU145
1531.27
959.0991
1274.78
8591.336

PANC10_10
1
5
B
PANC10
1489.769
1259.887
3536.387
2274.268

LNCaP_3
1
5
B
LNCaP
2708.339
2442.744
1751.953
1772.665

22RV1_10
1
5
B
22RV1
1570.356
460.8777
3189.049
1848.007

DU145_10
1
5
B
DU145
1849.165
1211.669
1052.509
7289.696

PANC10_2
1
2
A
PANC10
3117.334
2465.185
3551.597
2408.323

22RV1_4
1
2
A
22RV1
1187.918
595.63
2880.41
1901.338

PC3_4
1
2
A
PC3
7777.686
2626.01
3321.799
6459.001

DU145_4
1
2
A
DU145
1791.063
1147.742
1363.401
8994.303

PANC10_13
1
5
B
PANC10
2011.562
1618.153
2430.41
2528.535

LNCaP_6
1
5
B
LNCaP
2837.619
2337.289
1760.822
1827.291

22RV1_13
1
5
B
22RV1
1331.884
471.1855
2938.103
1777.94

PC3_13
1
5
B
PC3
8870.855
2186.108
3833.356
8164.266

DU145_13
1
5
B
DU145
2156.555
1241.703
998.6182
8236.958

PANC10_3
1
2
A
PANC10
2281.636
1934.528
2818.755
2720.009

LNCaP_21
1
2
A
LNCaP
3128.863
3035.675
2180.038
1843.996

22RV1_7
1
2
A
22RV1
1699.577
959.3655
3535.557
2450.701

PC3_7
1
2
A
PC3
8564.561
2882.376
3866.938
7705.728

DU145_7
1
2
A
DU145
2227.022
982.6645
873.3627
7695.261

PANC10_16
1
5
B
PANC10
2214.257
1636.763
2495.027
2823.656

LNCaP_9
1
5
B
LNCaP
2806.456
2419.824
1773.563
1452.119

22RV1_16
1
5
B
22RV1
1160.135
524.6943
2679.52
1509.931

PC3_16
1
5
B
PC3
7363.903
1915.84
3168.637
7527.66

DU145_16
1
5
B
DU145
3322.855
633.954
520.4906
5664.163

TCAF2
PGK1
NDRG1
RP11.61L23.2

LNCaP_25
5.074935
6913.754
590.3842
1.691645

22RV1_3
13.39421
5498.325
375.038
17.41248

PC3_3
48.40442
11756.44
1036.321
15.74602

DU145_3
5.468421
6583.978
928.5378
5.468421

PANC10_12
8.337949
8091.979
3051.689
15.28624

LNCaP_1
13.6224
6934.938
532.4089
1.1352

22RV1_12
9.801746
5673.429
393.852
18.71243

DU145_12
4.531143
12527.48
1383.132
3.398358

PANC10_21
24.73173
8389.797
2002.828
15.89897

LNCaP_10
9.993509
8129.72
811.473
7.994808

22RV1_21
24.68661
5088.968
517.2432
17.63329

PC3_25
45.44538
10514.41
714.732
23.96211

DU145_21
7.067732
6815.313
1210.601
6.058056

PANC10_8
12.12097
8984.668
2512.071
23.63589

LNCaP_26
27.3697
7249.322
607.6074
0

22RV1_6
9.242278
5890.412
443.6294
18.48456

PC3_6
46.07768
12791.31
1051.741
32.91263

DU145_6
3.453398
7278.613
935.871
13.81359

PANC10_15
13.65809
7513.087
1914.409
25.03983

LNCaP_4
9.436374
8479.256
825.0087
4.04416

22RV1_15
11.69729
5622.496
439.6231
6.823418

PC3_15
75.93084
14487.6
1961.185
35.2536

DU145_15
9.073265
9858.607
2695.768
7.056984

PANC10_24
27.28301
7942.54
1993.479
25.46415

LNCaP_13
10.59082
8366.744
839.0279
4.707029

22RV1_24
24.47969
5327.287
538.5532
15.19429

PC3_26
50.04677
11911.9
878.5134
23.09851

DU145_24
16.20138
5913.503
959.1215
1.620138

PANC10_9
45.45376
8035.576
1733.736
6.493395

LNCaP_27
16.12582
7077.218
713.5674
6.047182

22RV1_9
10.26344
5603.839
504.9613
16.42151

PC3_9
82.85027
13963.07
1870.289
48.70252

DU145_9
14.30111
9117.509
1654.529
5.500428

PANC10_18
24.65567
8032.818
2118.744
31.23052

LNCaP_7
7.319434
7679.132
725.6696
2.091267

22RV1_18
20.5442
5183.559
493.0609
11.55611

PC3_18
74.4259
13456.2
2266.607
43.97894

DU145_18
12.65929
7606.427
939.5005
4.521177

PANC10_27
15.42979
8119.496
2350.471
24.00189

LNCaP_16
14.90222
7503.267
909.0353
1.241851

22RV1_27
31.39789
5252.563
513.5074
17.2182

PC3_27
38.71292
10959.04
728.3278
22.30914

DU145_27
0
7858.821
1106.876
0

PC3_PSI_1
136.1339
11451.22
8597.273
67.37239

PC3_PSI_2
101.8413
12176.28
8531.675
62.41887

PC3_PSI_3
99.49826
10847.7
8207.413
55.71903

PANC10_4
20.43812
6899
9683.125
10.21906

PC3_2
74.74961
13316.53
1232.785
29.19907

DU145_2
22.15643
7367.014
1418.012
5.539108

PANC10_11
27.54277
7995.665
2921.369
19.27994

LNCaP_2
28.13912
11285.19
1315.504
5.627824

22RV1_11
5.725772
5800.923
302.7502
15.03015

DU145_11
7.052371
13331.33
1395.194
4.701581

PANC10_19
21.35223
8454.15
2243.319
16.01418

PANC10_20
14.75741
8604.187
2191.476
24.59569

LNCaP_11
25.19661
8225.953
867.0599
13.33938

LNCaP_12
10.45179
8051.363
918.0156
5.225895

22RV1_19
20.55832
5171.702
395.7476
25.6979

PC3_19
50.17912
10696.63
830.4291
22.26257

PC3_22
43.0807
11520.29
807.7632
21.54035

DU145_19
7.264232
6830.453
906.9912
0

DU145_20
13.84383
6998.054
810.8527
4.944224

LNCaP_23
12.07445
7374.039
721.0171
5.174764

22RV1_5
11.40246
5530.191
318.3916
7.016895

PC3_5
63.17937
13791.59
1123.36
30.04872

DU145_5
22.94728
6909.127
2174.005
21.94957

PANC10_14
26.05403
8333.929
2506.23
17.64951

LNCaP_5
10.46879
8439.587
819.1825
2.617197

22RV1_14
12.81486
5604.364
309.2652
11.96053

PC3_14
132.6007
19719.53
3896.468
67.85669

DU145_14
21.5419
12404.54
3389.258
13.16449

PANC10_22
21.661
7066.846
1907.062
21.88431

PANC10_23
12.50035
7958.916
1831.572
10.32637

LNCaP_14
9.555543
8537.347
745.3323
2.123454

LNCaP_15
8.461708
9301.835
704.7394
3.626446

22RV1_22
13.79367
5359.467
392.4926
21.31749

22RV1_23
14.04224
5195.629
359.2473
12.87205

PC3_20
81.87521
13993.34
1294.028
43.26739

PC3_23
81.47088
13702.72
1202.549
34.63732

DU145_22
12.95103
5573.652
898.3305
1.177366

DU145_23
6.193339
5824.393
844.9484
1.769525

PANC10_6
20.99858
7886.647
1809.238
29.39801

LNCaP_24
7.859865
7931.914
858.0353
11.7898

22RV1_8
13.66185
6635.862
321.8124
9.107898

PC3_8
135.5495
15911.42
2852.153
60.95715

DU145_8
13.92246
7322.221
1228.16
6.961232

PANC10_17
10.20807
7970.231
1772.234
30.6242

LNCaP_8
21.67482
8740.373
957.7563
4.064029

22RV1_17
14.54673
5682.482
416.5654
10.57944

PC3_17
117.9531
15416.19
2191.599
45.87067

DU145_17
12.08409
7421.831
1130.412
5.49277

PANC10_26
7.498328
8108.327
1471.699
14.794

LNCaP_17
10.15233
7925.162
719.5464
3.807124

LNCaP_18
13.54721
7599.983
600.9154
6.773603

22RV1_25
22.18001
5497.229
406.0647
15.35539

22RV1_26
20.86613
5348.149
378.8005
16.05087

PC3_21
47.88369
11973.88
763.1771
24.18867

PC3_24
60.04415
12247.05
845.4998
28.80167

DU145_25
7.364225
6414.625
902.9969
3.792026

DU145_26
0
4582.445
1205.906
0

PC3_PSI_4
116.0458
10641.75
6466.968
55.15048

PC3_PSI_7
65.41332
11098.76
5544.003
27.77826

PC3_PSI_10
69.31666
12269.51
4685.072
45.90507

PC3_PSI_5
109.853
10928.2
7134.154
59.52388

PC3_PSI_8
65.67656
11224.48
5054.433
49.70118

PC3_PSI_11
75.28531
11772.94
4058.599
29.63358

PC3_PSI_6
138.6389
11600.86
6114.117
60.21691

PC3_PSI_9
72.50337
10328.86
5277.098
31.81803

PC3_PSI_12
64.40419
10479.2
6131.746
27.02974

PANC10_1
104.4371
9649.989
16746.49
36.55299

LNCaP_19
167.5489
25812.72
4163.954
37.88061

22RV1_1
14.30558
5440.413
343.334
17.1667

PC3_1
228.2603
19178.65
3507.706
102.1584

DU145_1
134.8219
11298.74
4204.691
44.94065

PANC10_10
249.9687
14071.9
14980.26
214.2588

LNCaP_3
65.78962
19872.12
3352.834
45.07807

22RV1_10
24.73002
10677.75
1065.639
31.47457

DU145_10
103.5395
15795.34
4245.976
51.34191

PANC10_2
311.985
30121.65
32468.58
255.6264

22RV1_4
37.59235
12027.88
614.0085
33.41543

PC3_4
642.7761
54808.26
21785.28
307.6616

DU145_4
197.3824
18925.56
10973
73.1046

PANC10_13
191.7071
29308.47
24094.22
162.633

LNCaP_6
65.2604
20293.57
3592.948
26.58757

22RV1_13
34.5536
9575.536
962.2655
33.50652

PC3_13
699.7048
56612.26
37270.24
392.4105

DU145_13
93.62045
27383.16
14038.14
70.62596

PANC10_3
204.9732
30376.43
24958.85
187.0193

LNCaP_21
163.7853
26070.1
3826.363
14.11942

22RV1_7
47.28626
16204.64
1003.924
42.7395

PC3_7
636.1495
71921.8
46350.09
299.622

DU145_7
91.43509
15639.6
9443.038
52.5489

PANC10_16
173.3544
26166.94
30138.14
189.3073

LNCaP_9
73.05553
19577.76
3049.225
32.59401

22RV1_16
23.63488
11998.41
1079.101
41.19222

PC3_16
550.2457
61562.82
36378.06
275.4092

DU145_16
34.21911
17995.65
11369.75
36.02011

TABLE 13

Oxygen-Sensitive Candidates

O2
PSI
Expt
CellLine
BNIP3
PPP1R3G
KDM3A
BHLHE40

LNCaP_25
20
0
A
LNCaP
1884.493
0
336.6374
201.3058

22RV1_3
20
0
A
22RV1
377.7168
1.339421
310.7458
2.678843

PC3_3
20
0
A
PC3
535.9478
26.82655
506.7885
2151.956

DU145_3
20
0
A
DU145
1537.72
43.74737
534.8115
313.8873

PANC10_12
20
0
B
PANC10
6.94829
13.89658
821.2879
187.6038

LNCaP_1
20
0
B
LNCaP
2310.132
1.1352
353.0473
188.4432

22RV1_12
20
0
B
22RV1
455.3357
1.782136
336.8237
5.346407

DU145_12
20
0
B
DU145
2355.062
58.90487
412.3341
404.4046

PANC10_21
20
0
C
PANC10
4.41638
14.57405
1080.247
197.4122

LNCaP_10
20
0
C
LNCaP
2106.632
0
274.8215
273.8222

22RV1_21
20
0
C
22RV1
384.4058
2.351106
355.0169
8.22887

PC3_25
20
0
C
PC3
473.4583
21.48327
481.7211
1764.933

DU145_21
20
0
C
DU145
1772.991
56.54185
465.4606
379.6382

PANC10_8
20
0
A
PANC10
12.12097
7.272581
898.7699
173.3299

LNCaP_26
20
0
A
LNCaP
1737.064
0
346.6829
209.8344

22RV1_6
20
0
A
22RV1
375.8526
0
370.718
1.02692

PC3_6
20
0
A
PC3
652.4015
31.44985
544.1555
1584.195

DU145_6
20
0
A
DU145
1697.921
55.25438
534.1256
370.6648

PANC10_15
20
0
B
PANC10
11.38174
3.414522
1059.64
175.2788

LNCaP_4
20
0
B
LNCaP
2423.8
0
315.4445
227.821

22RV1_15
20
0
B
22RV1
447.4213
0.974774
380.1618
5.848644

PC3_15
20
0
B
PC3
859.6456
43.93141
709.411
1654.75

DU145_15
20
0
B
DU145
2327.797
101.8222
502.054
634.1204

PANC10_24
20
0
C
PANC10
9.549055
6.820754
841.681
201.4396

LNCaP_13
20
0
C
LNCaP
2220.541
0
262.4169
267.1239

22RV1_24
20
0
C
22RV1
497.1909
4.220636
406.8693
11.81778

PC3_26
20
0
C
PC3
486.6086
29.25811
480.449
1790.905

DU145_24
20
0
C
DU145
1195.662
46.98399
460.1191
272.1831

PANC10_9
20
0
A
PANC10
9.740092
6.493395
986.996
181.8151

LNCaP_27
20
0
A
LNCaP
1826.249
0
368.8781
288.249

22RV1_9
20
0
A
22RV1
418.7484
2.052688
336.6409
2.052688

PC3_9
20
0
A
PC3
882.2434
50.94172
802.1921
2231.359

DU145_9
20
0
A
DU145
3016.435
74.80582
741.4577
861.367

PANC10_18
20
0
B
PANC10
8.218557
8.218557
1145.667
261.3501

LNCaP_7
20
0
B
LNCaP
2469.786
2.091267
397.3407
248.8608

22RV1_18
20
0
B
22RV1
360.8076
0
382.6358
1.284013

PC3_18
20
0
B
PC3
726.6674
42.62574
606.9094
1504.756

DU145_18
20
0
B
DU145
1682.782
56.06259
432.2245
348.1306

PANC10_27
20
0
C
PANC10
3.428841
15.42979
917.215
231.4468

LNCaP_16
20
0
C
LNCaP
2055.264
0
289.3514
295.5606

22RV1_27
20
0
C
22RV1
369.6848
3.038506
383.8645
10.12835

PC3_27
20
0
C
PC3
432.4036
14.43532
459.9619
1728.958

DU145_27
20
0
C
DU145
996.1885
0
0
110.6876

PC3_PSI_1
20
0
A
PC3
1212.009
49.31381
1193.255
2956.051

PC3_PSI_2
20
0
B
PC3
1408.695
53.87734
1015.128
3076.922

PC3_PSI_3
20
0
C
PC3
1089.705
60.49494
1467.798
3081.262

PANC10_4
10
2
A
PANC10
6.812705
44.28258
1556.703
436.0131

PC3_2
10
2
A
PC3
756.8398
47.30249
706.0334
1985.537

DU145_2
10
2
A
DU145
1657.301
74.22405
546.156
310.19

PANC10_11
10
5
B
PANC10
7.344738
8.26283
893.3037
181.7823

LNCaP_2
10
5
B
LNCaP
3307.754
0
434.7494
406.6103

22RV1_11
10
5
B
22RV1
422.9914
2.862886
388.6368
5.010051

DU145_11
10
5
B
DU145
2160.376
45.84041
443.124
266.8147

PANC10_19
20
2
C
PANC10
6.672573
14.67966
898.1284
218.8604

PANC10_20
20
5
C
PANC10
6.763815
15.9872
863.3087
270.5526

LNCaP_11
20
2
C
LNCaP
2048.336
1.482154
385.3599
183.7871

LNCaP_12
20
5
C
LNCaP
1867.387
1.741965
341.4252
209.0358

22RV1_19
20
2
C
22RV1
388.0382
6.424474
489.5449
3.854685

PC3_19
20
5
C
PC3
448.7851
26.50305
519.8133
1988.082

PC3_22
20
2
C
PC3
454.248
28.50929
527.1051
2062.805

DU145_19
20
2
C
DU145
1517.187
52.92512
459.7221
214.8137

DU145_20
20
5
C
DU145
1555.453
59.33068
324.3411
163.1594

LNCaP_23
10
2
A
LNCaP
1740.446
0
395.007
222.5149

22RV1_5
10
2
A
22RV1
405.2257
3.508448
440.3102
0.877112

PC3_5
10
2
A
PC3
759.6934
52.39265
621.007
1768.252

DU145_5
10
2
A
DU145
2189.969
97.77537
631.5491
451.9617

PANC10_14
10
5
B
PANC10
12.60679
15.9686
1015.267
196.6659

LNCaP_5
10
5
B
LNCaP
2971.391
2.617197
423.1135
247.7613

22RV1_14
10
5
B
22RV1
448.52
3.417295
387.0087
2.562971

PC3_14
10
5
B
PC3
1152.941
83.42015
827.3535
1848.939

DU145_14
10
5
B
DU145
2995.52
122.0707
499.0539
581.6312

PANC10_22
20
2
C
PANC10
8.039135
13.84518
803.5786
181.3272

PANC10_23
20
5
C
PANC10
12.50035
11.41336
705.9978
197.8316

LNCaP_14
20
2
C
LNCaP
2168.046
0
295.1601
237.8268

LNCaP_15
20
5
C
LNCaP
2407.96
0
291.3245
273.1923

22RV1_22
20
2
C
22RV1
427.6037
2.50794
408.7942
0

22RV1_23
20
5
C
22RV1
399.0337
0
342.8647
7.02112

PC3_20
20
5
C
PC3
667.6491
40.60478
574.4578
1722.042

PC3_23
20
2
C
PC3
632.2531
49.76066
530.2925
1614.782

DU145_22
20
2
C
DU145
1321.005
34.14363
492.1391
250.779

DU145_23
20
5
C
DU145
1298.832
57.50957
452.1137
222.0754

PANC10_6
10
2
A
PANC10
15.11898
16.79887
1069.248
212.9256

LNCaP_24
10
2
A
LNCaP
2202.072
1.309978
421.8128
273.7853

22RV1_8
10
2
A
22RV1
425.7942
1.517983
412.8914
3.035966

PC3_8
10
2
A
PC3
1170.217
52.93647
982.533
2756.707

DU145_8
10
2
A
DU145
1988.923
83.53478
681.2063
594.6881

PANC10_17
10
5
B
PANC10
5.104033
15.3121
1093.397
250.0976

LNCaP_8
10
5
B
LNCaP
2407.26
0
306.1569
268.2259

22RV1_17
10
5
B
22RV1
427.1449
2.64486
407.3084
3.96729

PC3_17
10
5
B
PC3
858.4368
53.15173
736.115
1716.145

DU145_17
10
5
B
DU145
1856.556
97.77131
515.2219
582.2337

PANC10_26
20
5
C
PANC10
7.498328
11.75414
959.3807
143.6842

LNCaP_17
20
2
C
LNCaP
2044.425
1.269041
280.4581
291.8795

LNCaP_18
20
5
C
LNCaP
2672.67
0
345.4538
241.9144

22RV1_25
20
2
C
22RV1
390.7094
3.412309
412.8894
3.412309

22RV1_26
20
5
C
22RV1
420.5327
1.605087
418.9277
3.210174

PC3_21
20
5
C
PC3
500.5573
35.04888
484.267
1771.696

PC3_24
20
2
C
PC3
491.581
24.89636
471.0781
1757.878

DU145_25
20
2
C
DU145
1469.877
60.67242
375.8503
206.1983

DU145_26
20
5
C
DU145
964.7252
0
241.1813
0

PC3_PSI_4
20
0.5
A
PC3
1100.137
90.7685
1469.531
3095.321

PC3_PSI_7
20
2
A
PC3
918.0267
43.90757
1006.738
2725.854

PC3_PSI_10
20
5
A
PC3
907.5433
41.77361
820.3236
2921.858

PC3_PSI_5
20
0.5
B
PC3
1087.4
70.17042
1380.18
3091.854

PC3_PSI_8
20
2
B
PC3
913.2592
46.1511
1042.837
2678.539

PC3_PSI_11
20
5
B
PC3
872.1883
46.45264
863.7788
3215.243

PC3_PSI_6
20
0.5
C
PC3
1179.131
120.4338
1625.856
2820.392

PC3_PSI_9
20
2
C
PC3
888.8183
68.85212
973.8403
2787.989

PC3_PSI_12
20
5
C
PC3
916.3415
54.72688
1090.533
2917.877

PANC10_1
1
2
A
PANC10
10.44371
130.5464
2637.037
877.2717

LNCaP_19
1
2
A
LNCaP
11168.95
2.913893
1041.717
926.6181

22RV1_1
1
2
A
22RV1
467.7926
1.430558
702.4041
0

PC3_1
1
2
A
PC3
1877.161
114.1301
1523.597
3456.626

DU145_1
1
2
A
DU145
4441.451
204.9732
1043.5
1381.103

PANC10_10
1
5
B
PANC10
10.04338
92.62231
2322.253
790.0795

LNCaP_3
1
5
B
LNCaP
9018.051
6.091631
877.1949
565.3034

22RV1_10
1
5
B
22RV1
2023.365
16.86138
919.5071
21.35775

DU145_10
1
5
B
DU145
4712.332
162.5827
842.8631
1135.512

PANC10_2
1
2
A
PANC10
11.57364
82.02187
3135.449
946.5223

22RV1_4
1
2
A
22RV1
1756.816
22.55541
1064.281
11.6954

PC3_4
1
2
A
PC3
5423.365
169.4506
1770.238
5201.848

DU145_4
1
2
A
DU145
4238.848
313.1314
1129.466
1706.992

PANC10_13
1
5
B
PANC10
6.359951
131.7418
2111.504
914.9243

LNCaP_6
1
5
B
LNCaP
8195.015
6.04263
861.679
576.4669

22RV1_13
1
5
B
22RV1
1859.612
20.94158
894.2053
32.45944

PC3_13
1
5
B
PC3
4191.345
268.4914
2037.781
5547.57

DU145_13
1
5
B
DU145
5865.239
364.627
652.0583
1364.888

PANC10_3
1
2
A
PANC10
5.984618
133.1578
2558.424
951.5543

LNCaP_21
1
2
A
LNCaP
8482.947
2.823884
1042.013
883.8756

22RV1_7
1
2
A
22RV1
3168.179
34.55534
1164.879
24.55248

PC3_7
1
2
A
PC3
5787.271
200.7905
2149.898
6611.701

DU145_7
1
2
A
DU145
4169.23
240.674
1067.794
1692.075

PANC10_16
1
5
B
PANC10
18.07991
142.5122
2173.843
1168.813

LNCaP_9
1
5
B
LNCaP
7559.562
6.743587
987.9356
560.8417

22RV1_16
1
5
B
22RV1
1931.307
14.18093
937.2917
31.06298

PC3_16
1
5
B
PC3
4419.143
254.2238
1826.518
4920.72

DU145_16
1
5
B
DU145
8228.795
590.7299
304.37
633.954

TABLE 14

Pressure-Sensitive Candidates

O2
PSI
Expt
CellLine
RBM3
CBX3P2
TMEM260
TBCK
COX11
FBXO36
ACBD6
EXOSC9

LNCaP_25
20
0
A
LNCaP
2341.237
20.29974
228.3721
221.6055
676.6581
42.29113
338.329
605.609

22RV1_3
20
0
A
22RV1
3323.104
4.018264
225.0228
117.8691
409.8629
121.8873
543.8051
692.4809

PC3_3
20
0
A
PC3
4555.265
7.581416
397.1495
82.2292
964.0062
51.32035
285.7611
415.8115

DU145_3
20
0
A
DU145
4080.535
15.31158
320.4494
79.83894
926.3505
29.52947
530.4368
692.3021

PANC10_8
20
0
A
PANC10
6270.177
0.606048
235.7528
93.33146
307.2666
54.54436
1032.101
522.4138

LNCaP_26
20
0
A
LNCaP
2751.567
25.54505
242.678
215.3083
824.7403
58.3887
326.6118
571.1144

22RV1_6
20
0
A
22RV1
2628.915
6.161519
216.6801
138.6342
417.9564
137.6073
546.3213
668.5248

PC3_6
20
0
A
PC3
4268.403
5.851134
368.6215
136.7703
1071.489
65.09387
277.9289
398.6085

DU145_6
20
0
A
DU145
4347.829
12.66246
279.7253
90.93949
920.9063
18.41813
569.8107
626.2163

PANC10_9
20
0
A
PANC10
5259.65
0
249.9957
126.6212
288.9561
25.97358
922.0621
461.031

LNCaP_27
20
0
A
LNCaP
2539.816
34.26736
255.9974
203.5885
691.3944
28.22018
336.6264
554.325

22RV1_9
20
0
A
22RV1
2625.388
6.158065
221.6903
131.3721
359.2205
143.6882
582.9635
689.7033

PC3_9
20
0
A
PC3
3764.649
4.478393
378.4242
124.2754
1153.746
75.01308
274.8614
372.8262

DU145_9
20
0
A
DU145
3828.298
1.100086
361.9281
116.6091
1103.386
19.80154
561.0436
597.3464

PC3_PSI_1
20
0
A
PC3
3345.699
18.75314
585.5147
181.9749
1030.728
113.908
508.4185
203.5063

PANC10_12
20
0
B
PANC10
6561.966
4.168974
244.5798
75.04154
286.2696
66.70359
926.9019
397.4422

LNCaP_1
20
0
B
LNCaP
2565.552
24.9744
296.2872
228.1752
751.5025
44.27281
333.7488
643.6585

22RV1_12
20
0
B
22RV1
2731.123
7.128543
272.6668
145.2441
412.5644
127.4227
539.9871
676.3205

DU145_12
20
0
B
DU145
6787.653
10.19507
317.18
138.1999
1218.878
44.17865
456.5127
872.2451

PANC10_15
20
0
B
PANC10
5285.68
5.69087
227.6348
138.8572
293.6489
28.45435
1040.291
452.9933

LNCaP_4
20
0
B
LNCaP
2600.395
16.17664
203.5561
188.7275
644.3695
52.57408
358.5822
474.5148

22RV1_15
20
0
B
22RV1
2673.805
3.899096
251.4917
147.1909
423.0519
125.7458
530.277
669.6697

PC3_15
20
0
B
PC3
2805.644
10.84726
374.7729
129.6248
997.9482
71.59193
258.1648
391.0438

DU145_15
20
0
B
DU145
4734.228
9.073265
275.2224
104.8466
858.9358
31.25236
499.0296
725.8612

PANC10_18
20
0
B
PANC10
4834.155
1.643711
259.7064
128.2095
386.2722
75.61073
943.4904
460.2392

LNCaP_7
20
0
B
LNCaP
3271.787
40.77971
240.4957
174.6208
737.1716
37.64281
388.9757
673.388

22RV1_18
20
0
B
22RV1
2480.712
0
296.6069
118.1292
403.18
116.8452
463.5286
680.5267

PC3_18
20
0
B
PC3
3387.732
10.82559
347.0953
169.1498
1035.873
58.18752
288.2312
374.1593

DU145_18
20
0
B
DU145
3878.265
9.042353
272.1748
134.7311
993.7546
41.59482
646.5282
697.1654

PC3_PSI_2
20
0
B
PC3
4171.552
19.71122
517.7481
95.27091
949.4239
122.2096
739.8279
224.0509

PANC10_21
20
0
C
PANC10
6169.241
5.299656
268.0743
135.1412
317.9793
61.82932
1016.651
535.7069

LNCaP_10
20
0
C
LNCaP
3535.704
18.98767
196.8721
163.8936
670.5645
58.96171
354.7696
599.6106

22RV1_21
20
0
C
22RV1
3391.47
12.93108
228.0572
124.6086
410.2679
136.3641
557.212
618.3408

PC3_25
20
0
C
PC3
3779.403
14.04676
329.6856
147.0778
1022.934
69.4075
303.2447
444.5385

DU145_21
20
0
C
DU145
4444.594
2.019352
268.5738
122.1708
1058.14
33.31931
698.6958
657.2991

PANC10_24
20
0
C
PANC10
6779.829
1.364151
243.7283
105.949
321.4849
50.92829
866.2357
544.7509

LNCaP_13
20
0
C
LNCaP
3172.538
24.7119
182.3974
167.0995
728.4128
48.24705
358.911
487.1775

22RV1_24
20
0
C
22RV1
2659.845
5.064763
244.7969
152.787
405.1811
157.8518
522.5148
608.6157

PC3_26
20
0
C
PC3
3815.104
10.00935
309.52
130.8916
942.4193
62.36598
322.6092
448.8811

DU145_24
20
0
C
DU145
3565.923
8.100688
251.1213
132.8513
852.1924
43.74372
665.8766
638.3342

PANC10_27
20
0
C
PANC10
5134.69
3.428841
183.443
132.0104
294.8803
39.43167
872.6401
375.4581

LNCaP_16
20
0
C
LNCaP
3575.29
19.86962
194.9707
166.4081
613.4746
45.9485
356.4114
500.4661

22RV1_27
20
0
C
22RV1
2834.926
5.064176
231.9393
131.6686
408.1726
127.6172
508.4433
645.176

PC3_27
20
0
C
PC3
3813.55
12.46687
361.5393
154.8517
1049.186
84.6435
299.2049
374.0061

DU145_27
20
0
C
DU145
2988.566
0
110.6876
442.7505
1217.564
0
885.5009
221.3752

PC3_PSI_3
20
0
C
PC3
3076.486
13.53176
567.5381
179.8929
992.5947
144.0735
473.6117
220.4881

PANC10_4
10
2
A
PANC10
6386.911
1.135451
172.5885
70.39795
373.5633
44.28258
1051.428
463.264

PC3_2
10
2
A
PC3
4264.816
16.93546
345.7169
85.26127
1086.205
85.26127
321.7737
476.5288

DU145_2
10
2
A
DU145
5171.311
40.9894
187.2218
62.03801
1350.435
73.11622
820.8958
1113.361

LNCaP_23
10
2
A
LNCaP
6032.05
53.47256
196.641
134.5439
705.4928
60.37225
464.0038
679.619

22RV1_5
10
2
A
22RV1
5267.057
18.41935
207.8755
102.6221
545.5636
175.4224
621.8724
892.8999

PC3_5
10
2
A
PC3
5504.31
7.704801
338.2408
91.68713
1168.048
85.52329
336.6998
506.2054

DU145_5
10
2
A
DU145
6664.689
49.88539
160.631
51.88081
1336.929
83.80746
832.0884
1152.353

PANC10_6
10
2
A
PANC10
7171.436
6.719546
202.8463
88.19404
308.6792
103.733
1004.992
487.5871

LNCaP_24
10
2
A
LNCaP
6097.945
69.42881
182.0869
142.7876
865.8952
94.31838
427.0527
685.1183

22RV1_8
10
2
A
22RV1
6350.482
13.66185
184.4349
98.66889
553.3048
172.2911
617.0601
900.9229

PC3_8
10
2
A
PC3
4895.822
13.63515
320.025
119.5081
1160.592
82.61298
361.7326
405.8463

DU145_8
10
2
A
DU145
6087.1
52.70647
264.5268
81.54586
1264.955
48.72862
782.6413
959.6555

PC3_PSI_4
20
0.5
A
PC3
4525.786
36.1925
521.6316
144.1955
994.4321
145.919
503.8226
238.9854

PC3_PSI_7
20
2
A
PC3
8340.647
52.42027
316.7618
69.44565
1302.89
161.7412
813.1862
346.7802

PC3_PSI_10
20
5
A
PC3
7532.104
33.96975
339.2385
64.72615
1331.247
147.3553
691.3304
316.2859

PANC10_11
10
5
B
PANC10
6243.027
2.754277
179.9461
80.79211
329.5951
76.20165
1043.871
448.9471

LNCaP_2
10
5
B
LNCaP
5325.329
26.73217
175.8695
112.5565
699.2572
68.94085
448.819
583.8868

22RV1_11
10
5
B
22RV1
3995.158
9.30438
193.2448
106.6425
491.7007
130.977
642.0022
775.8422

DU145_11
10
5
B
DU145
9470.159
52.89278
228.0267
106.961
1691.394
128.1181
584.1714
1290.584

PANC10_14
10
5
B
PANC10
6439.549
6.723622
181.5378
82.36437
305.0843
90.76889
1093.429
451.3231

LNCaP_5
10
5
B
LNCaP
6890.206
63.68512
225.0789
134.3494
1019.834
81.1331
418.7515
727.5807

22RV1_14
10
5
B
22RV1
4622.746
11.10621
210.1637
109.3534
473.2954
134.9832
633.054
762.9112

PC3_14
10
5
B
PC3
4628.573
14.94092
275.162
75.32715
1221.42
89.64553
369.1653
520.4421

DU145_14
10
5
B
DU145
12797.08
55.05151
192.6803
71.80632
1505.539
106.5127
578.0409
1055.553

PANC10_17
10
5
B
PANC10
5897.427
2.835574
199.0573
86.76856
395.8461
67.48666
1159.183
498.4939

LNCaP_8
10
5
B
LNCaP
5078.682
37.93094
188.3
125.9849
800.6138
75.86188
466.0087
606.8951

22RV1_17
10
5
B
22RV1
3329.879
9.25701
259.1963
100.5047
403.3411
148.1122
536.9066
720.7243

PC3_17
10
5
B
PC3
5029.755
11.64969
297.7953
112.8564
1199.19
74.9949
363.3248
450.6975

DU145_17
10
5
B
DU145
6037.653
24.16819
253.766
110.954
1286.407
48.33638
781.0719
853.5765

PC3_PSI_5
20
0.5
B
PC3
4611.407
27.1003
517.8093
139.373
1029.328
146.1481
630.082
248.2581

PC3_PSI_8
20
2
B
PC3
8268.147
33.7258
360.3336
72.77673
1231.879
170.4041
657.6531
305.3073

PC3_PSI_11
20
5
B
PC3
7663.484
32.0363
309.5508
67.67669
1285.457
151.3715
676.3664
354.0011

PANC10_19
20
2
C
PANC10
6286.898
8.007088
149.4656
72.06379
344.3048
109.4302
1024.907
464.4111

PANC10_20
20
5
C
PANC10
5962.61
7.378707
135.8912
77.47642
336.9609
91.61894
988.7467
509.7457

LNCaP_11
20
2
C
LNCaP
5936.025
74.10768
123.0188
105.2329
812.2202
111.1615
545.4325
690.6836

LNCaP_12
20
5
C
LNCaP
4783.436
38.32323
184.6483
146.3251
769.9486
73.16253
426.7815
661.9467

22RV1_19
20
2
C
22RV1
4621.767
16.70363
156.7572
104.0765
535.8011
179.8853
719.5411
846.7457

PC3_19
20
5
C
PC3
4385.726
15.90183
302.1348
111.3128
1166.488
111.3128
318.39
461.5065

PC3_22
20
2
C
PC3
5214.032
23.44097
259.1178
104.5341
1242.371
128.6086
333.8754
611.9994

DU145_19
20
2
C
DU145
5413.928
16.60396
189.9078
64.34034
1230.768
47.73638
777.2728
801.141

DU145_20
20
5
C
DU145
6244.554
21.75458
173.0478
78.11873
1342.851
51.41992
997.7443
972.0343

PANC10_22
20
2
C
PANC10
7881.479
8.709063
133.204
64.31308
340.3234
104.0621
1066.749
517.1844

PANC10_23
20
5
C
PANC10
10103.54
11.41336
109.7856
52.71885
382.0758
135.8733
1322.863
652.192

LNCaP_14
20
2
C
LNCaP
4922.166
37.16044
170.938
128.469
864.2458
57.33326
455.4809
587.135

LNCaP_15
20
5
C
LNCaP
5734.62
37.47328
198.2457
161.9813
934.4143
59.23196
442.4264
685.3983

22RV1_22
20
2
C
22RV1
4016.466
11.28573
208.159
116.6192
448.9212
152.9843
560.5245
753.6359

22RV1_23
20
5
C
22RV1
4193.949
7.02112
208.2932
106.487
463.3939
161.4858
599.1356
753.6002

PC3_20
20
5
C
PC3
4357.359
9.31913
295.5495
114.4922
1065.043
102.5104
340.8139
451.3121

PC3_23
20
2
C
PC3
4707.749
18.05044
255.6332
97.56992
1064.976
103.912
384.4255
515.657

DU145_22
20
2
C
DU145
4967.309
18.83786
289.6321
83.59301
1186.785
28.25679
676.9857
766.4655

DU145_23
20
5
C
DU145
5566.042
17.69525
282.2393
107.0563
1304.14
45.1229
820.175
728.1597

PANC10_26
20
5
C
PANC10
8129.403
9.119588
163.7473
72.34873
346.5443
113.4882
1121.507
516.1687

LNCaP_17
20
2
C
LNCaP
6488.608
32.99507
156.0921
110.4066
736.0439
72.33535
445.4335
588.8351

LNCaP_18
20
5
C
LNCaP
7181.955
52.25351
197.4021
138.375
1079.906
64.83306
566.0797
755.7406

22RV1_25
20
2
C
22RV1
5585.949
20.47385
206.4447
98.95695
440.1878
170.6154
590.3294
766.0633

22RV1_26
20
5
C
22RV1
5535.944
9.630521
195.8206
104.3306
455.8446
216.6867
563.3855
781.6773

PC3_21
20
5
C
PC3
4587.455
12.34116
311.4908
131.3099
1080.592
81.94528
348.5143
459.091

PC3_24
20
2
C
PC3
4373.948
14.64492
308.0314
144.4965
1114.478
87.86949
330.9751
424.2144

DU145_25
20
2
C
DU145
6117.638
22.03772
206.3632
86.722
1293.521
36.49138
824.7932
820.0669

DU145_26
20
5
C
DU145
2894.176
0
241.1813
0
241.1813
0
0
2652.994

PC3_PSI_6
20
0.5
C
PC3
4629.7
26.60747
476.1337
166.6468
981.6756
166.6468
674.9895
253.4712

PC3_PSI_9
20
2
C
PC3
7738.04
31.81803
320.7883
68.85212
1257.594
162.7414
730.2498
336.4365

PC3_PSI_12
20
5
C
PC3
6790.805
28.69824
326.3591
79.42071
1058.832
142.8238
721.7941
265.9593

PANC10_1
1
2
A
PANC10
6542.985
15.66557
177.5431
31.33113
344.6425
20.88742
1206.249
386.4173

LNCaP_19
1
2
A
LNCaP
6474.671
48.07924
179.2044
88.87375
687.6788
67.01955
543.4411
619.2023

22RV1_1
1
2
A
22RV1
4530.578
25.75005
167.3753
90.12517
497.8343
175.9587
716.7097
1032.863

PC3_1
1
2
A
PC3
5083.18
13.56792
287.3206
63.84902
1140.503
91.78297
339.996
620.1336

DU145_1
1
2
A
DU145
4611.349
23.01838
203.8771
76.72793
1121.324
52.61344
643.4185
842.9111

PANC10_2
1
2
A
PANC10
9006.806
11.57364
198.2614
81.51866
371.3628
150.9605
1150.822
517.7945

22RV1_4
1
2
A
22RV1
6490.111
11.6954
183.7848
99.41089
521.2806
182.9495
675.827
860.4472

PC3_4
1
2
A
PC3
6749.623
17.98637
247.076
81.412
1196.567
88.03856
360.6741
476.1655

DU145_4
1
2
A
DU145
5878.828
43.86276
194.9456
86.50711
1046.614
71.88619
661.5966
952.7966

PANC10_3
1
2
A
PANC10
7205.48
7.480773
187.0193
71.81542
275.2924
112.2116
975.4928
409.9463

LNCaP_21
1
2
A
LNCaP
6624.831
31.06272
189.2002
121.427
796.3352
50.82991
454.6453
810.4547

22RV1_7
1
2
A
22RV1
6886.516
12.73092
185.5076
92.75382
509.2367
176.4141
649.2767
845.6966

PC3_7
1
2
A
PC3
6622.96
18.76547
282.7331
78.18946
1148.447
76.31292
363.4246
402.8321

DU145_7
1
2
A
DU145
6479.28
55.70184
243.8269
102.9958
1239.103
58.85477
715.716
903.8411

PANC10_10
1
5
B
PANC10
6992.427
6.695589
189.7084
70.30368
302.4174
104.8976
1035.584
464.2275

LNCaP_3
1
5
B
LNCaP
4050.935
20.71155
221.7354
194.9322
670.0794
70.66292
397.1744
556.7751

22RV1_10
1
5
B
22RV1
3566.743
13.4891
256.2929
130.3947
465.374
145.0078
537.3159
657.5937

DU145_10
1
5
B
DU145
7010.738
26.52666
258.421
85.56986
1233.917
72.73438
604.9789
1029.405

PANC10_13
1
5
B
PANC10
7888.156
3.634258
158.9988
53.6053
268.9351
99.94208
1051.209
480.6306

LNCaP_6
1
5
B
LNCaP
4510.219
31.42167
209.075
196.9897
685.2342
51.96662
404.8562
663.4808

22RV1_13
1
5
B
22RV1
4500.345
13.61203
276.4288
110.9904
468.0443
127.7436
639.7652
774.8384

PC3_13
1
5
B
PC3
5296.603
20.02733
262.2329
83.23859
1182.238
103.2659
369.2539
473.7715

DU145_13
1
5
B
DU145
10515.06
27.92189
192.1683
54.20132
1005.188
93.62045
796.5951
850.7964

PANC10_16
1
5
B
PANC10
5799.396
17.01638
226.5306
57.43029
307.3584
82.95487
916.7577
398.8215

LNCaP_9
1
5
B
LNCaP
4866.622
33.71794
204.5555
176.4572
904.7646
60.69229
400.1195
714.8203

22RV1_16
1
5
B
22RV1
3998.346
9.453951
249.8544
94.53951
292.3972
145.1857
644.8945
759.0172

PC3_16
1
5
B
PC3
5563.723
20.61274
300.0299
92.18477
1156.031
75.58006
368.7391
382.4809

DU145_16
1
5
B
DU145
8976.212
7.204023
198.1106
59.43319
1197.669
5.403017
1298.525
617.7449

TABLE 15

Pressure-Sensitive Candidates

O2
PSI
Expt
CellLine
SMYD3
EIF4A2
DDHD2
PDIA5
COPG2
TNFRSF13C
SLC6A6
NRAV

LNCaP_25
20
0
A
LNCaP
216.5306
4092.09
707.1077
466.8941
776.4651
16.91645
159.0146
209.764

22RV1_3
20
0
A
22RV1
226.3622
2118.965
530.4109
324.14
358.9649
57.59512
349.589
322.8006

PC3_3
20
0
A
PC3
301.5071
2661.077
534.7814
355.1602
315.5035
2.915929
1007.162
145.2133

DU145_3
20
0
A
DU145
398.101
4582.536
999.6273
230.7674
550.1231
4.374737
1817.703
283.2642

PANC10_8
20
0
A
PANC10
247.8738
3734.471
274.54
177.5722
453.3242
0.606048
451.5061
223.0258

LNCaP_26
20
0
A
LNCaP
191.5879
4698.465
738.9819
479.8821
773.6502
7.298587
120.4267
193.4126

22RV1_6
20
0
A
22RV1
234.1377
1957.309
602.8019
281.376
379.9603
83.1805
337.8566
255.703

PC3_6
20
0
A
PC3
310.1101
3292.726
553.6636
399.3399
372.2784
3.656959
895.955
130.9191

DU145_6
20
0
A
DU145
377.5716
4293.725
1016.45
212.9596
572.113
6.906797
1722.095
310.8059

PANC10_9
20
0
A
PANC10
149.3481
3389.552
334.4098
185.0618
444.7976
0
389.6037
204.5419

LNCaP_27
20
0
A
LNCaP
268.0917
3942.762
608.7496
544.2464
741.7876
8.062909
133.038
237.8558

22RV1_9
20
0
A
22RV1
254.5334
2052.688
578.8581
328.4301
387.9581
59.52796
398.2215
240.1645

PC3_9
20
0
A
PC3
346.5156
3502.103
538.5267
361.6302
401.376
3.918594
870.4876
130.4332

DU145_9
20
0
A
DU145
375.1292
3681.986
1170.491
178.2139
694.154
7.700599
2293.678
276.1215

PC3_PSI_1
20
0
A
PC3
557.0377
5043.206
582.7365
510.5022
357.0042
4.861925
977.247
120.159

PANC10_12
20
0
B
PANC10
198.7211
2358.25
244.5798
177.8762
484.9907
2.779316
518.3425
201.5004

LNCaP_1
20
0
B
LNCaP
211.1472
4754.218
711.7705
463.1617
779.8825
14.7576
148.7112
174.8208

22RV1_12
20
0
B
22RV1
210.292
2571.622
549.7889
320.7844
372.4664
65.93902
400.0895
326.1308

DU145_12
20
0
B
DU145
449.716
6546.37
835.996
164.254
408.9357
5.663929
1144.114
306.985

PANC10_15
20
0
B
PANC10
201.4568
3136.808
349.4194
188.9369
503.0729
2.276348
413.1572
180.9697

LNCaP_4
20
0
B
LNCaP
243.9977
3823.079
562.1383
571.5746
757.606
13.48053
136.1534
186.0314

22RV1_15
20
0
B
22RV1
211.526
2394.045
582.9148
311.9277
357.742
57.51166
386.9853
297.3061

PC3_15
20
0
B
PC3
319.4519
2975.404
522.8381
355.7902
319.4519
3.796542
984.9314
167.5902

DU145_15
20
0
B
DU145
377.0446
4683.821
805.5043
188.5223
523.225
3.024422
1185.573
243.97

PANC10_18
20
0
B
PANC10
190.6705
3178.938
317.2363
184.0957
567.0805
1.643711
427.365
198.8891

LNCaP_7
20
0
B
LNCaP
220.6287
4329.968
680.7074
508.1779
775.8601
3.1369
93.06138
181.9402

22RV1_18
20
0
B
22RV1
225.9862
1863.102
626.5982
306.879
340.2634
48.79248
430.1442
273.4947

PC3_18
20
0
B
PC3
349.1251
3015.602
569.6964
374.1593
349.1251
4.736194
847.1021
135.9964

DU145_18
20
0
B
DU145
406.9059
3251.63
768.6
226.0588
503.6591
15.372
1249.653
245.952

PC3_PSI_2
20
0
B
PC3
629.4451
5833.865
511.8348
660.983
308.1521
2.628163
599.2212
164.2602

PANC10_21
20
0
C
PANC10
219.9357
3093.674
298.9889
173.1221
557.7888
2.649828
340.9445
158.1064

LNCaP_10
20
0
C
LNCaP
286.8137
3509.721
533.6534
576.6255
826.4632
9.993509
147.9039
196.8721

22RV1_21
20
0
C
22RV1
186.9129
1934.96
625.3941
345.6125
403.2146
70.53317
237.4617
303.2926

PC3_25
20
0
C
PC3
334.6433
2751.511
627.9726
356.9528
349.5163
0.82628
581.7009
140.4676

DU145_21
20
0
C
DU145
350.3576
4212.368
971.3083
209.0029
442.2381
5.04838
1489.272
284.7286

PANC10_24
20
0
C
PANC10
200.9849
2964.754
299.2037
185.5245
438.8018
3.637735
426.0697
188.7075

LNCaP_13
20
0
C
LNCaP
218.8769
3543.216
601.323
557.783
776.6598
11.76757
85.90328
245.9423

22RV1_24
20
0
C
22RV1
178.955
2172.783
612.8364
324.989
344.4039
54.02414
277.7179
266.7442

PC3_26
20
0
C
PC3
307.9801
2208.218
486.6086
331.0787
298.7407
2.309851
722.2134
145.5206

DU145_24
20
0
C
DU145
325.6477
3784.642
821.4098
223.579
429.3365
12.9611
1370.636
262.4623

PANC10_27
20
0
C
PANC10
197.1584
2083.021
291.4515
269.164
444.0349
3.428841
402.8888
207.4449

LNCaP_16
20
0
C
LNCaP
264.5144
3108.354
661.9068
571.2517
746.3527
8.69296
136.6037
212.3566

22RV1_27
20
0
C
22RV1
222.8237
2059.094
600.6113
346.3896
342.3383
53.68026
260.2986
325.1201

PC3_27
20
0
C
PC3
347.1039
2788.642
591.8483
362.8516
372.0377
3.936907
576.1007
133.8548

DU145_27
20
0
C
DU145
0
3984.754
664.1257
110.6876
0
0
1549.627
110.6876

PC3_PSI_3
20
0
C
PC3
514.207
5309.227
709.2236
547.6384
350.2339
0
949.6114
183.8728

PANC10_4
10
2
A
PANC10
172.5885
2530.92
493.9211
229.3611
468.9412
6.812705
420.1168
158.9631

PC3_2
10
2
A
PC3
331.7014
2527.471
597.4129
463.6812
364.4044
5.255832
1190.738
99.86081

DU145_2
10
2
A
DU145
488.5493
2515.863
1301.69
334.5621
672.4477
27.69554
2384.032
151.7716

LNCaP_23
10
2
A
LNCaP
303.5862
2847.845
907.3086
488.1527
1010.804
20.69906
222.5149
196.641

22RV1_5
10
2
A
22RV1
235.9431
1506.878
785.8923
355.2303
416.6282
275.4131
532.4069
209.6298

PC3_5
10
2
A
PC3
339.7817
2495.585
703.4483
463.0585
459.2061
3.08192
1162.654
124.8178

DU145_5
10
2
A
DU145
426.0213
2630.956
1326.951
343.2115
786.1938
26.93811
2799.568
157.6378

PANC10_6
10
2
A
PANC10
254.5028
2378.719
462.3888
212.5056
626.5977
6.719546
646.3363
150.3498

LNCaP_24
10
2
A
LNCaP
313.0846
2703.794
1015.233
466.352
994.273
26.19955
256.7556
192.5667

22RV1_8
10
2
A
22RV1
283.8628
1546.825
713.452
379.4957
428.8302
207.2047
531.294
222.3845

PC3_8
10
2
A
PC3
404.2422
2866.59
692.9866
372.1595
535.7813
1.604136
1111.666
117.904

DU145_8
10
2
A
DU145
435.5742
3022.169
1509.593
266.5157
802.5306
21.87816
2961.507
160.1083

PC3_PSI_4
20
0.5
A
PC3
595.1656
5259.403
772.1067
695.1259
476.8219
4.021389
2031.376
129.8334

PC3_PSI_7
20
2
A
PC3
921.611
2887.595
943.1167
745.0846
798.849
4.032328
1392.945
105.7366

PC3_PSI_10
20
5
A
PC3
863.9334
3103.642
885.5088
649.5568
779.9272
2.754304
1422.598
105.5817

PANC10_11
10
5
B
PANC10
248.803
2225.455
291.9533
206.5707
500.3602
1.836184
610.5313
154.2395

LNCaP_2
10
5
B
LNCaP
315.1582
2425.592
704.885
606.3981
945.4745
15.47652
219.4851
215.2643

22RV1_11
10
5
B
22RV1
275.5528
1928.87
726.4574
335.6734
404.3827
73.0036
539.6541
249.7868

DU145_11
10
5
B
DU145
550.085
5159.985
964.9995
230.3775
530.1032
18.80632
1224.762
258.5869

PANC10_14
10
5
B
PANC10
227.7627
2120.462
394.1723
242.8908
531.1661
10.08543
562.2629
193.3041

LNCaP_5
10
5
B
LNCaP
318.4256
3359.608
896.826
488.5434
977.0867
8.723989
189.3106
153.5422

22RV1_14
10
5
B
22RV1
253.7342
1765.887
685.1677
353.6901
411.7841
80.30644
498.0708
263.1317

PC3_14
10
5
B
PC3
479.9771
2038.191
673.5865
437.6445
359.2047
6.847922
1572.532
115.1696

DU145_14
10
5
B
DU145
485.8895
5380.687
964.5983
271.6673
579.2377
11.96772
1443.307
305.1769

PANC10_17
10
5
B
PANC10
218.3392
2764.117
390.7421
242.7251
570.5175
4.536918
538.1919
156.5237

LNCaP_8
10
5
B
LNCaP
323.7677
2504.797
732.88
551.3533
842.6088
13.54676
199.1374
224.8763

22RV1_17
10
5
B
22RV1
223.4907
1539.308
752.4626
312.0935
374.2477
75.37851
518.3925
260.5187

PC3_17
10
5
B
PC3
427.3981
2114.419
707.7189
485.6466
407.0112
4.368635
1372.48
105.5753

DU145_17
10
5
B
DU145
429.5346
3315.436
1151.285
237.2877
703.0746
9.886987
2092.746
240.5833

PC3_PSI_5
20
0.5
B
PC3
617.4997
4780.3
776.2301
741.3868
489.2572
0.967868
1652.634
138.4051

PC3_PSI_8
20
2
B
PC3
831.6073
2816.104
970.0606
634.5776
678.9537
1.775042
1510.561
102.9524

PC3_PSI_11
20
5
B
PC3
789.2944
2774.344
937.4623
469.7323
840.9529
4.004538
1509.31
109.3239

PANC10_19
20
2
C
PANC10
205.5153
1706.844
345.6393
294.9277
596.528
2.669029
591.19
134.786

PANC10_20
20
5
C
PANC10
215.2123
1885.26
327.1227
261.3292
539.2605
4.304246
493.7585
142.655

LNCaP_11
20
2
C
LNCaP
352.7526
2647.126
668.4513
628.4331
967.8463
40.01815
198.6086
154.144

LNCaP_12
20
5
C
LNCaP
350.135
3013.6
632.3333
578.3324
989.4362
17.41965
162.0028
158.5188

22RV1_19
20
2
C
22RV1
227.4264
1484.054
728.5354
409.8815
459.9924
138.7686
398.3174
187.5946

PC3_19
20
5
C
PC3
372.8096
2153.108
664.3432
437.1237
400.3728
5.300611
855.872
114.1398

PC3_22
20
2
C
PC3
391.5276
1848.669
741.2415
496.0616
390.894
7.602477
947.1419
77.92539

DU145_19
20
2
C
DU145
479.4393
3235.696
935.0104
265.6633
516.7982
8.301979
1805.68
216.8892

DU145_20
20
5
C
DU145
537.9315
2679.769
654.6152
354.9952
415.3148
17.7992
962.1459
261.055

PANC10_22
20
2
C
PANC10
257.9223
1890.76
367.6788
237.1545
542.3067
9.490646
633.8635
165.8072

PANC10_23
20
5
C
PANC10
285.8775
2162.016
347.8357
238.0501
548.9282
7.608906
436.4251
174.4613

LNCaP_14
20
2
C
LNCaP
278.1725
2702.095
706.0484
679.5053
829.2088
16.98763
149.7035
198.5429

LNCaP_15
20
5
C
LNCaP
268.357
3457.212
646.7162
633.4193
796.6094
9.670523
151.1019
217.5868

22RV1_22
20
2
C
22RV1
209.413
1636.431
756.1439
403.7783
453.9371
125.397
412.5561
263.3337

22RV1_23
20
5
C
22RV1
212.974
1630.07
704.4524
362.7579
394.3529
97.1255
370.9492
270.3131

PC3_20
20
5
C
PC3
362.7804
2094.142
571.1295
421.3578
350.133
7.987825
983.1682
110.4983

PC3_23
20
2
C
PC3
420.0385
1821.143
588.8345
466.3842
356.1302
7.317744
1072.293
112.2054

DU145_22
20
2
C
DU145
402.6593
4565.827
1045.501
275.5037
487.4297
25.90206
2083.938
209.5712

DU145_23
20
5
C
DU145
444.1509
3486.85
1136.92
291.9717
518.4709
10.61715
1947.363
224.7297

PANC10_26
20
5
C
PANC10
220.8967
2337.046
395.3848
232.0429
567.441
10.13288
452.7369
172.6642

LNCaP_17
20
2
C
LNCaP
300.7628
2307.117
713.2012
626.9064
930.2072
24.11178
214.468
189.0871

LNCaP_18
20
5
C
LNCaP
315.4564
3161.337
919.2747
575.7563
941.5308
15.48252
158.6958
165.4694

22RV1_25
20
2
C
22RV1
255.9232
1339.331
759.2387
397.534
414.5955
112.6062
433.3632
288.3401

22RV1_26
20
5
C
22RV1
235.9478
1407.661
669.3212
390.0361
513.6278
99.51538
431.7683
258.419

PC3_21
20
5
C
PC3
352.4634
2255.47
633.3481
413.6756
390.9678
8.391986
894.9806
108.1085

PC3_24
20
2
C
PC3
386.1376
2445.213
680.5004
411.5221
436.9066
3.905311
845.4998
122.5291

DU145_25
20
2
C
DU145
436.3578
3645.017
950.5346
314.0787
550.5033
11.26617
1721.8
234.3912

DU145_26
20
5
C
DU145
0
964.7252
964.7252
241.1813
723.5439
0
1688.269
241.1813

PC3_PSI_6
20
0.5
C
PC3
714.2005
5237.471
603.5695
701.597
579.7628
0
1992.76
145.6409

PC3_PSI_9
20
2
C
PC3
977.4915
2647.156
907.0746
696.3453
780.8457
1.564821
1726.519
103.2782

PC3_PSI_12
20
5
C
PC3
808.5563
2683.619
886.9759
740.1477
674.075
1.668502
1572.063
123.4692

PANC10_1
1
2
A
PANC10
172.3212
2480.381
511.7418
224.5398
443.8577
5.221856
454.3014
208.8742

LNCaP_19
1
2
A
LNCaP
333.6408
2291.777
799.8637
627.944
872.7111
39.33756
335.0977
237.4823

22RV1_1
1
2
A
22RV1
204.5698
1570.753
726.7236
340.4729
436.3203
178.8198
535.0288
213.1532

PC3_1
1
2
A
PC3
359.1507
2068.708
656.0487
511.5903
374.3149
8.779241
1169.235
104.5528

DU145_1
1
2
A
DU145
428.5803
2791.801
1272.588
303.6234
601.7662
29.59506
2236.071
213.7421

PANC10_2
1
2
A
PANC10
360.7956
2703.702
559.057
205.8095
464.4551
7.548024
968.6631
170.5854

22RV1_4
1
2
A
22RV1
275.6773
1360.843
883.0026
392.6313
466.1452
405.9974
636.5639
230.5664

PC3_4
1
2
A
PC3
458.1792
2178.244
964.6375
547.1644
436.4062
9.466512
1539.255
96.55842

DU145_4
1
2
A
DU145
425.2251
2922.966
1519.357
318.005
674.9992
13.40251
2932.713
191.2904

PANC10_3
1
2
A
PANC10
291.7501
2341.482
504.2041
230.4078
529.6387
10.47308
782.4888
227.4155

LNCaP_21
1
2
A
LNCaP
324.7466
3035.675
943.1772
570.4245
827.398
45.18214
299.3317
242.854

22RV1_7
1
2
A
22RV1
311.9075
1321.287
918.4447
410.1174
428.3044
321.9103
773.8578
233.7033

PC3_7
1
2
A
PC3
497.9105
2513.322
1117.171
449.1203
439.7376
8.75722
1715.164
113.2183

DU145_7
1
2
A
DU145
463.4813
3105.64
1746.725
318.4463
838.6805
34.68227
2785.092
194.4309

PANC10_10
1
5
B
PANC10
290.1422
2337.876
388.3442
206.4473
508.8648
6.695589
594.7915
208.6792

LNCaP_3
1
5
B
LNCaP
307.0182
3542.893
738.3057
575.05
833.3352
20.71155
213.2071
202.2422

22RV1_10
1
5
B
22RV1
256.2929
2413.425
754.2656
301.2566
372.0744
68.5696
548.5568
291.1398

DU145_10
1
5
B
DU145
515.1305
4297.318
1125.244
215.636
508.2849
12.83548
1396.5
266.1222

PANC10_13
1
5
B
PANC10
285.2892
2433.135
415.2139
297.1006
530.6016
9.994208
551.4986
233.501

LNCaP_6
1
5
B
LNCaP
315.4253
3247.309
814.5465
559.5475
821.7977
18.12789
154.6913
212.7006

22RV1_13
1
5
B
22RV1
274.3347
1939.19
723.5315
313.0766
395.7958
56.54226
475.3738
288.9938

PC3_13
1
5
B
PC3
501.935
2271.85
918.1279
490.6696
371.1315
5.632687
2335.062
105.7693

DU145_13
1
5
B
DU145
502.594
4769.716
878.7183
292.3586
512.4488
16.42464
962.484
275.934

PANC10_16
1
5
B
PANC10
252.0552
2705.605
456.2518
292.4691
539.2067
13.82581
727.4504
201.006

LNCaP_9
1
5
B
LNCaP
374.2691
3716.841
777.7604
548.4784
873.2946
10.11538
149.4829
173.0854

22RV1_16
1
5
B
22RV1
283.6185
1800.302
791.4308
341.0175
415.2986
94.53951
576.691
269.4376

PC3_16
1
5
B
PC3
479.2463
2269.692
984.8311
472.3754
391.0696
5.725762
1983.977
113.3701

DU145_16
1
5
B
DU145
671.7751
1577.681
628.551
297.1659
515.0876
9.005028
394.4202
300.7679

TABLE 16

Pressure-Sensitive Candidates

O2
PSI
Expt
CellLine
CXorf38
PPID
HIST1H2AG
AC112229.1

LNCaP_25
20
0
A
LNCaP
157.323
796.7649
159.0146
16.91645

22RV1_3
20
0
A
22RV1
300.0304
661.6742
45.54033
8.036528

PC3_3
20
0
A
PC3
409.9796
470.0478
39.65664
2.915929

DU145_3
20
0
A
DU145
150.9284
185.9263
76.55789
12.03053

PANC10_8
20
0
A
PANC10
441.2033
994.5255
42.42339
67.87743

LNCaP_26
20
0
A
LNCaP
162.3936
915.9727
149.621
12.77253

22RV1_6
20
0
A
22RV1
344.0181
634.6364
44.15755
17.45764

PC3_6
20
0
A
PC3
330.5891
599.0099
70.945
1.462784

DU145_6
20
0
A
DU145
146.1939
177.2745
65.61457
11.51133

PANC10_9
20
0
A
PANC10
370.1235
1038.943
35.71367
81.16744

LNCaP_27
20
0
A
LNCaP
183.4312
896.9986
165.2896
18.14155

22RV1_9
20
0
A
22RV1
283.271
607.5958
45.15914
14.36882

PC3_9
20
0
A
PC3
358.2714
525.0916
66.61609
2.798996

DU145_9
20
0
A
DU145
127.6099
254.1198
111.1086
9.90077

PC3_PSI_1
20
0
A
PC3
346.5858
366.7281
66.67783
0

PANC10_12
20
0
B
PANC10
362.7008
715.6739
119.5106
31.96214

LNCaP_1
20
0
B
LNCaP
121.4664
863.8873
160.0632
18.1632

22RV1_12
20
0
B
22RV1
310.9827
687.9044
60.59261
5.346407

DU145_12
20
0
B
DU145
159.7228
258.2752
147.2622
10.19507

PANC10_15
20
0
B
PANC10
363.0775
1083.542
50.07966
71.70497

LNCaP_4
20
0
B
LNCaP
128.0651
699.6397
163.1145
16.17664

22RV1_15
20
0
B
22RV1
297.3061
620.931
59.46121
9.74774

PC3_15
20
0
B
PC3
318.9095
532.0582
42.84669
2.711816

DU145_15
20
0
B
DU145
151.2211
228.8479
112.9117
14.11397

PANC10_18
20
0
B
PANC10
350.1105
1150.598
93.69155
37.80536

LNCaP_7
20
0
B
LNCaP
146.3887
928.5225
170.4383
9.410701

22RV1_18
20
0
B
22RV1
294.0389
608.622
60.34859
10.2721

PC3_18
20
0
B
PC3
255.0779
515.5685
71.7195
2.706396

DU145_18
20
0
B
DU145
183.5598
332.7586
141.0607
11.75506

PC3_PSI_2
20
0
B
PC3
334.4338
421.1631
76.21673
0

PANC10_21
20
0
C
PANC10
352.8687
1048.89
70.22044
51.67164

LNCaP_10
20
0
C
LNCaP
141.9078
635.5872
101.9338
4.996755

22RV1_21
20
0
C
22RV1
353.8414
587.7764
62.3043
4.702211

PC3_25
20
0
C
PC3
279.2825
552.7811
58.66586
1.652559

DU145_21
20
0
C
DU145
148.4224
277.6609
101.9773
15.14514

PANC10_24
20
0
C
PANC10
404.2433
807.1225
49.56414
69.11697

LNCaP_13
20
0
C
LNCaP
131.7968
669.5749
147.0947
9.414058

22RV1_24
20
0
C
22RV1
324.989
683.743
112.2689
10.97365

PC3_26
20
0
C
PC3
354.1772
461.9702
37.72757
3.849752

DU145_24
20
0
C
DU145
183.0756
371.0115
98.8284
11.34096

PANC10_27
20
0
C
PANC10
298.3092
756.0595
30.85957
39.43167

LNCaP_16
20
0
C
LNCaP
136.6037
710.339
103.0737
3.725554

22RV1_27
20
0
C
22RV1
294.735
653.2787
100.2707
10.12835

PC3_27
20
0
C
PC3
259.1797
490.1449
72.17662
4.593058

DU145_27
20
0
C
DU145
110.6876
221.3752
221.3752
0

PC3_PSI_3
20
0
C
PC3
331.9262
322.3744
66.86283
0

PANC10_4
10
2
A
PANC10
600.6535
778.9193
39.74078
38.60533

PC3_2
10
2
A
PC3
472.4409
450.8336
30.36703
4.087869

DU145_2
10
2
A
DU145
275.8476
124.076
42.09722
2.215643

LNCaP_23
10
2
A
LNCaP
162.1426
550.2499
81.0713
10.34953

22RV1_5
10
2
A
22RV1
380.6666
448.2042
29.82181
3.508448

PC3_5
10
2
A
PC3
372.9124
481.5501
50.85169
0

DU145_5
10
2
A
DU145
246.4338
149.6562
30.92894
0.997708

PANC10_6
10
2
A
PANC10
419.9716
833.6437
47.4568
29.81799

LNCaP_24
10
2
A
LNCaP
157.1973
672.0185
58.94899
2.619955

22RV1_8
10
2
A
22RV1
375.7008
509.2833
34.91361
6.830923

PC3_8
10
2
A
PC3
401.836
542.9999
49.7282
0

DU145_8
10
2
A
DU145
201.8757
157.1249
94.47386
1.988923

PC3_PSI_4
20
0.5
A
PC3
328.605
267.7096
61.46981
0

PC3_PSI_7
20
2
A
PC3
481.1912
353.0527
24.19397
0

PC3_PSI_10
20
5
A
PC3
460.4279
359.4367
28.92019
0

PANC10_11
10
5
B
PANC10
454.4556
695.9139
33.05132
43.15033

LNCaP_2
10
5
B
LNCaP
182.9043
536.0503
63.31302
14.06956

22RV1_11
10
5
B
22RV1
371.4595
611.9419
46.5219
5.725772

DU145_11
10
5
B
DU145
228.0267
211.5711
117.5395
9.403162

PANC10_14
10
5
B
PANC10
415.1836
770.6951
51.26762
39.50128

LNCaP_5
10
5
B
LNCaP
153.5422
827.0341
89.85708
5.234393

22RV1_14
10
5
B
22RV1
334.8949
598.0267
33.31863
5.980267

PC3_14
10
5
B
PC3
423.3261
421.4585
32.99454
3.112692

DU145_14
10
5
B
DU145
209.4351
163.9578
111.2998
9.574176

PANC10_17
10
5
B
PANC10
421.9334
943.679
47.07053
26.08728

LNCaP_8
10
5
B
LNCaP
146.3051
621.7965
86.69929
10.83741

22RV1_17
10
5
B
22RV1
363.6682
566
31.73832
6.61215

PC3_17
10
5
B
PC3
284.6894
405.555
58.97657
0.728106

DU145_17
10
5
B
DU145
147.2062
210.9224
147.2062
6.591324

PC3_PSI_5
20
0.5
B
PC3
335.8502
326.6554
55.16847
0

PC3_PSI_8
20
2
B
PC3
469.4987
316.845
31.06324
0

PC3_PSI_11
20
5
B
PC3
539.0108
343.1889
28.03176
0.400454

PANC10_19
20
2
C
PANC10
483.0943
533.8059
40.03544
20.01772

PANC10_20
20
5
C
PANC10
452.5607
662.2389
43.04246
30.74461

LNCaP_11
20
2
C
LNCaP
240.1089
422.4138
59.28615
4.446461

LNCaP_12
20
5
C
LNCaP
158.5188
505.1699
74.9045
5.225895

22RV1_19
20
2
C
22RV1
442.0038
444.5736
50.1109
2.56979

PC3_19
20
5
C
PC3
365.3888
425.8157
43.11164
2.826993

PC3_22
20
2
C
PC3
442.2107
392.7946
48.14902
3.167699

DU145_19
20
2
C
DU145
178.4925
199.2475
75.75556
4.150989

DU145_20
20
5
C
DU145
176.0144
205.6797
74.16335
0.988845

PANC10_22
20
2
C
PANC10
467.6097
684.5547
33.94302
30.48172

PANC10_23
20
5
C
PANC10
519.5796
717.4112
30.97912
13.58733

LNCaP_14
20
2
C
LNCaP
160.3208
605.1844
91.30852
6.370362

LNCaP_15
20
5
C
LNCaP
130.5521
777.2683
126.9256
6.044077

22RV1_22
20
2
C
22RV1
321.0163
484.0324
68.96835
3.76191

22RV1_23
20
5
C
22RV1
349.8858
559.3492
65.53046
1.170187

PC3_20
20
5
C
PC3
364.1117
422.6891
29.95435
1.331304

PC3_23
20
2
C
PC3
389.304
441.5039
35.12517
2.439248

DU145_22
20
2
C
DU145
191.9107
206.0391
70.64198
9.418931

DU145_23
20
5
C
DU145
167.2201
223.845
98.20866
11.50191

PANC10_26
20
5
C
PANC10
404.7071
856.228
55.93347
14.99666

LNCaP_17
20
2
C
LNCaP
148.4778
553.302
60.91398
7.614247

LNCaP_18
20
5
C
LNCaP
188.6932
736.3874
87.08918
7.741261

22RV1_25
20
2
C
22RV1
365.117
479.4294
54.59694
3.412309

22RV1_26
20
5
C
22RV1
346.6987
537.7041
64.20347
1.605087

PC3_21
20
5
C
PC3
274.961
448.2308
56.76932
0.493646

PC3_24
20
2
C
PC3
313.4012
483.7704
50.76904
0.976328

DU145_25
20
2
C
DU145
164.7058
204.7694
73.14764
4.451509

DU145_26
20
5
C
DU145
0
241.1813
0
0

PC3_PSI_6
20
0.5
C
PC3
270.2759
310.8873
47.61337
0

PC3_PSI_9
20
2
C
PC3
498.1347
301.4888
28.16678
0

PC3_PSI_12
20
5
C
PC3
464.5111
288.9846
37.37445
1.001101

PANC10_1
1
2
A
PANC10
579.626
704.9505
46.9967
62.66227

LNCaP_19
1
2
A
LNCaP
166.0919
463.309
75.76123
8.74168

22RV1_1
1
2
A
22RV1
376.2368
414.8619
38.62507
2.861117

PC3_1
1
2
A
PC3
580.228
418.2111
35.11696
3.990564

DU145_1
1
2
A
DU145
178.6665
160.0325
46.03676
2.192227

PANC10_2
1
2
A
PANC10
587.7395
823.2379
30.1921
19.12166

22RV1_4
1
2
A
22RV1
435.2359
399.3143
36.75697
0.835386

PC3_4
1
2
A
PC3
383.3937
441.1394
46.38591
0.946651

DU145_4
1
2
A
DU145
218.0954
155.9565
42.64435
3.65523

PANC10_3
1
2
A
PANC10
408.4502
715.1619
91.26543
19.45001

LNCaP_21
1
2
A
LNCaP
200.4958
553.4812
70.5971
2.823884

22RV1_7
1
2
A
22RV1
361.0124
435.5792
35.4647
1.818702

PC3_7
1
2
A
PC3
437.861
455.3754
41.28404
1.251031

DU145_7
1
2
A
DU145
223.8583
146.0859
98.79193
3.152934

PANC10_10
1
5
B
PANC10
398.3875
639.4287
20.08677
49.10099

LNCaP_3
1
5
B
LNCaP
170.5657
650.5862
102.3394
13.40159

22RV1_10
1
5
B
22RV1
338.3516
611.506
69.69369
6.744551

DU145_10
1
5
B
DU145
158.3042
209.6461
89.84835
8.556986

PANC10_13
1
5
B
PANC10
421.5739
652.3492
69.05089
19.98842

LNCaP_6
1
5
B
LNCaP
160.734
729.9497
102.7247
14.50231

22RV1_13
1
5
B
22RV1
362.2893
560.1872
41.88315
3.141237

PC3_13
1
5
B
PC3
443.7305
362.9954
26.91172
1.251708

DU145_13
1
5
B
DU145
225.0176
197.0957
80.48074
4.927392

PANC10_16
1
5
B
PANC10
424.3461
656.1943
72.31963
22.334

LNCaP_9
1
5
B
LNCaP
206.8033
877.7903
107.8974
8.99145

22RV1_16
1
5
B
22RV1
360.6007
596.2742
47.26976
2.701129

PC3_16
1
5
B
PC3
352.1344
346.4086
57.25762
2.290305

DU145_16
1
5
B
DU145
203.5136
72.04023
102.6573
0

Example 7: Primary Tumor Culture

To culture primary tumors using a method disclosed herein, target cells are isolated from a patient tumor. The cells are enriched for, for example, T-cells, dendritic cells, macrophages, B-cells, neutrophils, cancer cells, cancer stem cells, fibroblasts, and endothelial cells. The isolated cells are then co-cultured to re-establish tumor heterogeneity. To replicate the metastatic microenvironment, the cells are grown under low oxygen and high pressure conditions in an ex vivo setting. The cells are then subcutaneously injected into mice and downstream molecular assays are performed to determine gene expression changes.

FIGS. 50-52 shows ex vivo cultures of pancreatic ductal adenocarcinoma colonies from a fine-needle aspirate. The cells were stained for DAPI. The cancerous cells were further stained for EpCAM (around periphery of cell) and CK7. The cells that did not get labeled with either CK7 or EpCAM represent the stromal cells derived from the biopsy.

FIG. 42 shows the mutations found using the COSMIC database from pancreatic ductal adenocarcinoma (PDAC) and circulating tumor cells (CTC) cultured under low oxygen (1% O₂) and positive pressure (2 PSI) using whole exome sequencing (top panels). The bottom panels of FIG. 42 show that the NANOG and Wnt signaling pathways exhibited increased gene expression in both PDAC and CTC colonies as determined by mRNA sequencing of the cells.

FIG. 43 shows that there was increased ex vivo expansion of primary cells. The individual lines represent patient PBMC populations from cryopreserved blood samples. The viability of individual patient PBMC populations was tracked following transfection and subsequent recovery and expansion under low oxygen and positive pressure conditions. FIG. 44 shows that there was increased ex vivo expansion of primary cells. The individual lines represent patient PBMC populations from cryopreserved blood samples. The viability of individual patient PBMC populations was tracked following transfection and subsequent recovery and expansion under low oxygen and positive pressure conditions.

FIG. 46 shows the results of the ex vivo culture and expansion of tumor-infiltrating lymphocytes (TILs) enriched from renal cell carcinoma tumors using positive pressure and low oxygen conditions. The experiments were performed in duplicate; RCC1 and RCC2 indicate the two different cell populations analyzed. For each data point, the left bar is RCC1 and the right bar is RCC2. The results indicate that 1% O₂and 2 PSI maintained the immune cell viability of CD3+, CD4+, CD8+, and CD11b+ cell types, as indicated by FACS analysis. The two different tumors were additionally cultured in two different culture media formulations: Media A and Media B. Media A was supplemented with 10% fetal calf serum, while Media B was animal-component free, chemically defined, and composed of recombinant human growth factors.

Example 8: Three-Dimensional Cell Culturing Using a Method Disclosed Herein

FIG. 39 shows a biopsy culture taken from a patient having prostate cancer. The cells were cultured for 48 hours under either 21% oxygen and 0 PSI or 1% oxygen and 2 PSI. The results indicated that the cells had a two-fold increase in viable cell adherence under positive pressure and low oxygen conditions. The bottom panels of FIG. 39 show that only cells grown under high pressure and low oxygen yielded enough cells for passaging at day 6, and were then able to form organoids by day 10. The tumor cell cultures were then subcutaneously injected into mice.

FIG. 40 shows prostate cancer cells obtained from another patient could form organoids after two weeks of culture under high pressure and low oxygen conditions.

FIG. 41 shows an apheresis culture taken from a patient having prostate cancer. The cells were cultured for 10 days under high pressure and low oxygen conditions to form organoids FIG. 41 shows the results of culturing under both 2D and 3D conditions. The left panels of FIG. 41 show viable and proliferating tumor cells that were positively selected for EpCAM (prostate cancer marker), and the right panels show viable and proliferating tumor cells that are EpCAM negative. After ten days in culture, the cells were subcutaneously injected into mice.

EMBODIMENTS

The following non-limiting embodiments provide illustrative examples of the invention, but do not limit the scope of the invention.

Embodiment 1

A method for increasing transfection efficiency of a nucleic acid that is introduced into a cell, the method comprising culturing the cell in a hypoxic condition and a positive pressure condition, wherein culturing the cell in the hypoxic condition and the positive pressure condition increases expression of a polypeptide encoded by the nucleic acid that is introduced into the cell as compared to expression of the polypeptide encoded by a nucleic acid that is introduced into a cell that is cultured in the absence of the hypoxic condition and the positive pressure condition.

Embodiment 2

The method of embodiment 1, wherein the cell is cultured in a culture medium that does not contain serum.

Embodiment 3

The method of any one of embodiments 1-2, wherein the cell is contacted with a substrate.

Embodiment 4

The method of any one of embodiments 1-3, wherein the substrate does not contain serum.

Embodiment 5

The method of any one of embodiments 1-4, wherein the hypoxic condition comprises an oxygen level of about 2%.

Embodiment 6

The method of any one of embodiments 1-4, wherein the hypoxic condition comprises an oxygen level of about 5%.

Embodiment 7

The method of any one of embodiments 1-6, wherein the positive pressure condition comprises a pressure level from about 2 PSI to about 10 PSI.

Embodiment 8

The method of any one of embodiments 1-7, wherein the nucleic acid is DNA.

Embodiment 9

The method of any one of embodiments 1-7, wherein the nucleic acid is RNA.

Embodiment 10

The method of any one of embodiments 1-7, wherein the nucleic acid is circular DNA.

Embodiment 11

The method of any one of embodiments 1-7, wherein the nucleic acid is supercoiled DNA.

Embodiment 12

The method of any one of embodiments 1-11, wherein the nucleic acid that is introduced into the cell is introduced via electroporation of the cell.

Embodiment 13

The method of any one of embodiments 1-11, wherein the nucleic acid that is introduced into the cell is introduced via encapsulation of the nucleic acid in a cationic liposome.

Embodiment 14

The method of any one of embodiments 1-13, wherein culturing the cell in the hypoxic condition and the positive pressure condition increases an entry rate of the nucleic acid into the cell as compared to the entry rate of the nucleic acid that is introduced into the cell that is cultured in the absence of the hypoxic condition and the positive pressure condition.

Embodiment 15

The method of any one of embodiments 1-14, wherein the positive pressure condition is applied continuously to the cell.

Embodiment 16

The method of any one of embodiments 1-14, wherein the positive pressure condition is applied in pulses of positive pressure to the cell.

Embodiment 17

The method of any one of embodiments 1-16, wherein the culturing of the cell in the hypoxic condition and the positive pressure condition occurs after the nucleic acid is introduced into the cell.

Embodiment 18

The method of any one of embodiments 1-16, wherein the culturing of the cell in the hypoxic condition and the positive pressure condition occurs before the nucleic acid is introduced into the cell.

Embodiment 19

The method of any one of embodiments 1-16, wherein the culturing of the cell in the hypoxic condition and the positive pressure condition occurs before the nucleic acid is introduced into the cell and after the nucleic acid is introduced into the cell.

Embodiment 20

The method of any one of embodiments 1-19, wherein the nucleic acid is introduced into the cell in the absence of the hypoxic condition and the positive pressure condition.

Embodiment 21

The method of any one of embodiments 1-20, wherein the cell is a mammalian cell.

Embodiment 22

A method for reprogramming a cell, the method comprising culturing the cell in a hypoxic condition and a positive pressure condition, wherein the cell exhibits a rate of reprogramming that is higher than the rate of reprogramming of a cell cultured in the absence of the hypoxic condition and the positive pressure condition.

Embodiment 23

The method of embodiment 22, wherein the hypoxic condition comprises an oxygen level of about 2%.

Embodiment 24

The method of embodiment 22, wherein the hypoxic condition comprises an oxygen level of about 5%.

Embodiment 25

The method of any one of embodiments 22-24, wherein the positive pressure condition comprises a pressure level of about 2 PSI to about 10 PSI.

Embodiment 26

The method of any one of embodiments 22-25, wherein the rate of reprogramming of the cell cultured in the hypoxic condition and the positive pressure condition is about 10% higher than the rate of reprogramming of the cell cultured in the absence of the hypoxic condition and the positive pressure condition.

Embodiment 27

The method of any one of embodiments 22-25, wherein the rate of reprogramming of the cell cultured in the hypoxic condition and the positive pressure condition is about 20% higher than the rate of reprogramming of the cell cultured in the absence of the hypoxic condition and the positive pressure condition.

Embodiment 28

The method of any one of embodiments 22-27, wherein the cell is a somatic cell.

Embodiment 29

The method of any one of embodiments 22-27, wherein the cell is a fibroblast.

Embodiment 30

The method of any one of embodiments 22-29, wherein the cell is reprogrammed into a stem cell.

Embodiment 31

The method of any one of embodiments 22-30, wherein the cell is reprogrammed into a pluripotent stem cell.

Embodiment 32

The method of any one of embodiments 22-29, wherein the cell is reprogrammed into an immune cell.

Embodiment 33

The method of any one of embodiments 22-32, wherein the cell cultured in the hypoxic condition and the positive pressure condition exhibits a greater expression level of a stem cell marker as compared to the expression level of the stem cell marker for a cell cultured in the absence of the hypoxic condition and the positive pressure condition.

Embodiment 34

The method of embodiment 33, wherein the stem cell marker is Oct4.

Embodiment 35

The method of embodiment 33, wherein the stem cell marker is Nanog.

Embodiment 36

The method of embodiment 33, wherein the stem cell marker is Sox2.

Embodiment 37

The method of any one of embodiments 22-36, wherein the cell is contacted with a substrate.

Embodiment 38

The method of any one of embodiments 22-37, wherein a nucleic acid encoding a reprogramming factor polypeptide is introduced into the cell.

Number	Date	Country
62405725	Oct 2016	US
62362214	Jul 2016	US
62353435	Jun 2016	US

METHODS FOR INCREASING CELL CULTURE TRANSFECTION EFFICIENCY AND CELLULAR REPROGRAMMING

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Provisional Applications (3)