Patent Application
20210172934
Described are clonally derived barcoded cell populations and methods of generating the same, as well as methods of use thereof, e.g., to evaluate heterogeneity of a starting population of cells.
The relevance and pervasiveness of genetic and functional heterogeneity within most cancer types has become particularly appreciated over the past decade [8-10]. It is now known that even supposedly clonal cancer cell lines are composed of subpopulations with widely differing phenotypes and functional characteristics [11-13]. Genetic and phenotypic heterogeneity has also been observed in other disease models, including bacterial antibiotic resistance and in the evolution of antiviral resistance [14-17].
CRISPR/Cas9 is a useful tool that has expanded our ability to define the role of particular factors in biological processes, including cancer biology [1, 2]. Oftentimes, studies employ the CRISPR/Cas9 system to generate loss- or gain-of-function mutations in a gene of interest and then look for a corresponding phenotypic change, indicating whether or not the targeted gene is necessary and/or sufficient for a particular behavior. Widely used protocols that employ CRISPR/Cas9 to generate genetically modified cell lines often require a subcloning and/or selection step in order to isolate a particular subpopulation in which the gene of interest was efficiently edited [3-7]. In order to correctly define the role that particular factors play, for example in cancer models, it is essential to use appropriately matched controls to compare to the edited subclone(s); however, such comparisons can be complicated by the widespread heterogeneity present in tumors and cancer cell lines derived from them.
Gene editing protocols often require the use of a subcloning step to isolate successfully edited cells, the behavior of which is then compared to the aggregate parental population and/or other non-edited subclones. The results herein demonstrate that the inherent functional heterogeneity present in many cell lines can render these populations inappropriate controls, resulting in erroneous interpretations of experimental findings. The present protocol incorporates a single-cell cloning step prior to gene editing, allowing for the generation of appropriately matched, functionally equivalent control and edited cell lines. The results demonstrate that heterogeneity should be considered during experimental design when utilizing gene editing protocols and provide a solution to account for it.
Thus provided herein are methods for creating a plurality of barcoded clonal populations (BCPs). The methods include (a) providing an initial heterogeneous population of cells; (b) dividing the initial heterogeneous population of cells into separate cultures, each culture comprising a single cell from the initial heterogeneous population; (c) maintaining the single cells in culture to provide a plurality of stable single cell-derived monoclonal populations; and (d) introducing individual identifying nucleic acid sequences into each cell of the plurality of stable single cell-derived monoclonal populations; to thereby create a plurality of barcoded clonal populations (BCPs).
In some embodiments, the methods include (e) mixing equal numbers of each BCP to create a barcoded polyclonal population of cells (BPP). In some embodiments, the methods include exposing the BPP to a test condition. In some embodiments, the methods include determining one or both of identity and relative abundance of each BCP in the BPP, e.g., using a method comprising PCR, a hybridization assay, or next-generation sequencing.
In some embodiments, the initial heterogeneous population of cells comprises cells from cancer cell lines (e.g., from a single cancer cell line) or patient-derived cells, e.g., from a single patient, optionally including cells from normal tissues, e.g., affected and/or normal cells.
In some embodiments, the identifying nucleic acid sequences comprise unique sequences of 10-40 nucleotides.
In some embodiments, the identifying nucleic acid sequences comprise unique sequences of 20-30 nucleotides, e.g., 24 nucleotides.
In some embodiments, the identifying nucleic acid sequences are flanked by uniform sequences comprising PCR primer binding sites. The sites allow for PCR amplification of the identifying nucleic acid sequences from genomic DNA preparations.
In some embodiments, the identifying nucleic acid sequences are integrated into the genomes of the cells of the plurality of stable single cell-derived monoclonal populations.
In some embodiments, the identifying nucleic acid sequences are introduced into the cells of the plurality of stable single cell-derived monoclonal populations using a viral vector. In some embodiments, the viral vectors are lentiviral vectors.
Also provided herein are methods for selecting a therapy for a subject who has a cancer. The methods comprising: creating a plurality of barcoded clonal populations (BCPs) by a method comprising (a) providing an initial heterogeneous population of cells from the cancer in the subject; (b) dividing the initial heterogeneous population of cells into separate cultures, each culture comprising a single cell from the initial heterogeneous population; (c) maintaining the single cells in culture to provide a plurality of stable single cell-derived monoclonal populations; and (d) introducing individual identifying nucleic acid sequences into each cell of the plurality of stable single cell-derived monoclonal populations; to thereby create a plurality of barcoded clonal populations (BCPs); (e) mixing equal numbers of each BCP to create a barcoded polyclonal population of cells (BPP); (f) exposing the BPP to a candidate therapeutic compound; and determining one or both of identity and relative abundance of each BCP in the BPP.
In some embodiments, identity and/or relative abundance of each BCP is determined using a method comprising PCR, a hybridization assay, or next-generation sequencing.
In some embodiments, the initial heterogeneous population of cells comprises cells from cancer cell lines (e.g., from a single cancer cell line) or patient-derived cells, e.g., from a single patient, optionally including cells from normal tissues, e.g., affected and/or normal cells.
In some embodiments, the identifying nucleic acid sequences comprise unique sequences of 10-40 nucleotides.
In some embodiments, the identifying nucleic acid sequences comprise unique sequences of 20-30 nucleotides, e.g., 24 nucleotides.
In some embodiments, the identifying nucleic acid sequences are flanked by uniform sequences comprising PCR primer binding sites. The sites allow for PCR amplification of the identifying nucleic acid sequences from genomic DNA preparations.
In some embodiments, the identifying nucleic acid sequences are integrated into the genomes of the cells of the plurality of stable single cell-derived monoclonal populations.
In some embodiments, the identifying nucleic acid sequences are introduced into the cells of the plurality of stable single cell-derived monoclonal populations using a viral vector. In some embodiments, the viral vectors are lentiviral vectors. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.
Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
(7A) Experimental schema. 2.5×104 MT-2 WT or OPN-KO tumor cells were injected into the mammary fat pads of 6-8-week-old female FVB mice. A single dose of AC-T was initiated at 14 days, when tumors reached ˜60-80 mm3 in volume, and tumor growth was monitored periodically until the end point of 44 days. Error bars represent SD. (7B) Tumor growth kinetics for MT-2 WT vehicle (blue; n=5) and AC-T treated (green; n=4) and MT-2 OPN-KO vehicle (red; n=3) and AC-T treated mice (purple; n=2). Representative of 3 biological repetitions. Error bars represent SD. (7C) Endpoint tumor mass for MT-2 WT and MT-2 OPN-KO AC-T treated mice from 2 separate experiments (Mann-Whitney, p=0.0037; endpoint tumor mass was not measured during the first of the three experimental repetitions). Data points from individual repetitions are represented with different colors. Error bars represent SD.
Due to the inherent functional heterogeneity observed in most cancer cell lines, subcloning and selection steps employed in genetic editing protocols can render the parental population an inappropriate control, as its behavior may differ from that of the selected subclonal population prior to gene editing. For example, if the aim of a study is to evaluate whether a particular gene product (protein) is relevant for primary tumor formation, it is common practice to compare the tumorigenicity of a knockout cell line with that of the parental cell line. However, if the selected subclonal population has an inherently different tumorigenic potential than the bulk parental population, it would be possible to incorrectly conclude that the knockdown of the gene of interest was responsible for any functional differences that are observed in any given biological assay.
The ability to genetically edit a cell line to either suppress, knockdown, induce, overexpress, knock-in, or mutate a protein of interest provides an indispensible tool for biological research. However, our work demonstrates that studies designed to test necessity or sufficiency of genes/gene products without choosing appropriately matched unedited controls run the risk of detecting false positive or false negative results due to inherent phenotypic differences in subclonal cellular populations that result from heterogeneity. Our alternative approach to generate subclones and screen for desired phenotypes prior to genetic manipulation provides one solution to this problem.
As demonstrated herein, our approach worked well for hypothesis-testing experimentation, when biological phenotypes to be tested are defined. Another benefit to our modified approach is that characterization of subclone phenotypes may enable one to select a range of biological properties that could be tested. Moreover, this approach enables discovery of novel properties for which mechanistic insight could be obtained in a straightforward manner. For example, one of our subclones (MC-22 KO) stimulated elevated host plasma OPN while another clone (MC-50 KO) did not, thereby enabling one to compare properties (e.g., gene expression) of related clones to yield mechanistic insights. Our approach ensures that the real function of a specific protein of interest is uncovered during experimentation.
The present disclosure provides methods of evaluating variation across cell line strains, which should be considered in experimental design and data interpretation.
The barcoding approaches described herein can be used to track and study individual subclonal populations within a heterogeneous populations of cells or tissues. Traditional cell tagging approaches currently do not enable one to enumerate cells at the end point of a study or know anything about their identity or the ability to isolate them for further study. Therefore, the present inventors developed a molecular barcoding approach that enables the analysis of intratumoral subclonal composition, tracking of cells over time, and retrieval of barcoded cells for further study.
Importantly, the present approach is different from others that have been reported in that we generate single cell subclones prior to introducing the barcode tags. Other reported approaches infect heterogeneous parental populations of cells with an entire library of barcodes at low MOI, without the ability to identify which cells are tagged with which barcode. Hence, one advantage of the present approach is that by introducing single barcodes into monoclonal populations and then generating the pooled barcoded polyclonal population rather than infecting the bulk parental population with a library of barcodes, we gain the ability to retroactively characterize barcoded monoclonal populations in any given experiment. This approach also allows us to be confident that the same barcode is not unwittingly introduced into multiple unique clonal populations, thus confounding subsequent analyses.
The general methods are as follows. First stable single cell-derived monoclonal populations (CPs) are generated from heterogeneous populations of cells, e.g., cultured cells (e.g., cancer cell lines, e.g., any of the NCI-60 cancer cell lines, see, e.g., dtp.cancer.gov/discovery development/nci-60/cell list.htm) or patient-derived cells, e.g., affected and/or normal cells; affected cells are cells that are affected by a disease, e.g., tumor cells. Methods for obtaining and culturing the cells are known in the art. For example, cells are separated (e.g., by dilution or cell sorting) into individual cells that are placed into individual culture environments, e.g., individual vials or wells of a culture dish. Where the cells are obtained from a patient tumor or other tissue, they can first be dissociated, e.g., enzymatically, chemically, or mechanically. The individual cells are then maintained in culture to produce individual clonal populations. Any number of individual clonal populations can be produced, e.g., 10, 100, 1000, 104, 105, 106, or more.
Each individual clonal population is then tagged with a unique molecular “barcode” sequence (also referred to herein as individual identifying nucleic acid sequence), e.g., using a viral vector, e.g., recombinant retroviruses, adenovirus, adeno-associated virus, alphavirus, and lentivirus vectors (Yu, et al. Nat Biotech 2016) to create barcoded clonal populations (BCPs). The individual identifying nucleic acid sequences preferably are integrated into the genome of the cells. In preferred embodiments, upon integration, each individual identifying nucleic acid sequence introduces a unique heritable DNA barcode tag of 10-50 base pairs, 20-30 base pairs, e.g., 24-base pairs, into each cell clone genome; these individual identifying nucleic acid sequences can be used to precisely follow the progeny of each cell over time. Each individual identifying nucleic acid is flanked by uniform sequences that are common to all of the cells and allow for PCR amplification of the individual identifying nucleic acid sequences from genomic DNA preparations made from the cells.
In some embodiments, substantially (i.e., within about plus or minus 10%, given difficulties in exactly determining numbers of cells) equal numbers of each BCP, or known ratios of each BCP, are then mixed together to create a barcoded polyclonal population of cells (BPP). The BPP can be exposed to a number of conditions. Then the identity and relative abundance of each clonal population (BCP) within a polyclonal mixture of cells (BPP), e.g., optionally including tumor and non-tumor stromal cells, is determined, e.g., using a Luminex-based PRISM detection (Yu, et al., Nat Biotech, 2016) or next-generation sequencing. These methods allow the identification and quantification of the representation of each individual barcode in a given tissue sample.
In some embodiments, one caveat of our approach is that isolating particular subclonal populations removes the inherent heterogeneity of a cell line, which could have important biological consequences. This is particularly relevant in circumstances in which the biology is not well understood. If heterogeneity is desirable, then one could employ a clonal pooling approach, thus ensuring that a given experiment is both properly controlled and that the heterogeneous nature of the parental cell line is not lost.
It has been reported that functional heterogeneity can arise even within a ‘clonal’ cellular population as a result of cell plasticity or epigenetic alteration [13]. Hence, although we did not test clonal plasticity in our system, it is reasonable to hypothesize that a high degree of cellular plasticity could cause differences between matched control and edited populations that may not be due to the target gene. Limiting the in vitro passage of the cell lines to minimize chances for additional selection and monitoring for unexpected functional changes in control cell lines may help to prevent this added complication.
While other gene editing, barcoding, and other cell manipulation technologies exist, the present methods derive individual clonal populations of cells from a parental cell line or tissue prior to any type of modulation so that individual clonally related cells can be tracked and studied in any given experiment. The barcode detection system can also be optimized for applicability with typical DNA sequencing technologies.
Provided herein are collections of single cell clonal populations derived from cell lines or tissues; collections of single cell clonal populations tagged with unique molecular barcodes; and collections of mixed populations of cells comprised of uniquely barcoded clonal populations.
The presence of an individual identifying nucleic acid sequences as described herein can be evaluated using methods known in the art, e.g., using polymerase chain reaction (PCR), reverse transcriptase polymerase chain reaction (RT-PCR), quantitative or semi-quantitative real-time RT-PCR, multiplex PCR, digital PCR, e.g., BEAMing ((Beads, Emulsion, Amplification, Magnetics), Diehl (2006) Nat Methods 3:551-559); various types of nucleic acid sequencing (Sanger, pyrosequencing, NextGeneration Sequencing); multiplexed gene analysis methods, e.g., oligo hybridization assays including DNA microarrays; hybridization based digital barcode quantification assays such as the nCounter® System (NanoString Technologies, Inc., Seattle, Wash.; Kulkarni, Curr Protoc Mol Biol. 2011 April; Chapter 25:Unit25B.10) and hybridization assays, e.g., utilizing branched DNA signal amplification such as the QuantiGene 2.0 Single Plex and Multiplex Assays (Affymetrix, Inc., Santa Clara, Calif.; see, e.g., Linton et a., J Mol Diagn. 2012 May-June; 14(3):223-32); SAGE; MLPA; or luminex/XMAP. See, e.g., WO2012/048113, which is incorporated herein by reference in its entirety.
The methods described herein can include exposing the barcoded polyclonal population of cells (BPPs) to test conditions, e.g., the presence or absence of one or more environmental factors (e.g., temperature, light, atmosphere (e.g., levels of oxygen or nitrogen) or test compounds (e.g., polypeptides, polynucleotides, inorganic or organic large or small molecule test compounds) to determine whether different BCPs within the BPP react differently to the test conditions.
As used herein, “small molecules” refers to small organic or inorganic molecules of molecular weight below about 3,000 Daltons. In general, small molecules useful for the invention have a molecular weight of less than 3,000 Daltons (Da). The test compounds can be, e.g., natural products or members of a combinatorial chemistry library.
The methods can include comparing genetic, genomic, epigenomic, transcriptomic, proteomic, and other profiles across and within the BCPs, e.g., preferably before being combined in a BPP, to determine heterogeneity of a starting population of cells. The methods can alternatively or in addition include determining effects on viability, proliferation, motility, cell cycle, or other cellular characteristics. In some embodiments, one or more characteristics of each BCP is determined before they are mixed together to form a BPP, e.g., genetic, genomic, epigenomic, transcriptomic, proteomic, or other profiles, or viability, proliferation, motility, cell cycle, or other cellular characteristics can be determined; such characteristics can be determined using methods known in the art. For example, the presence of therapy-resistant BCPs can be identified. In some embodiments, therapy-resistant cells are present naturally in the starting sample, and make up some proportion of the BCPs in a BPP; in other embodiments, BCPs consisting of therapy-resistant cells are intentionally spiked in (added) to the BPP.
In some embodiments, e.g., where the initial heterogeneous populations of cells comprises patient-derived cells, e.g., tumor cells and optionally non-tumor cells (e.g., from a biopsy that includes all or part of a tumor and some surrounding non-cancerous tissue), the methods can include exposing one or more populations of BCPs or BPPs generated from those patient-derived cells to one or more test conditions to determine the effect on the patient cells. Thus, for example, BCPs or BPPs generated from tumor cells can be exposed to test conditions that comprise one or more potential therapeutics (e.g., cancer therapeutic agents), and identity and/or relative abundance of each BCP is determined, e.g., using a method comprising PCR, a hybridization assay, or next-generation sequencing. Thus, an effect on the different kinds of cells in the BPP can be evaluated, e.g., an effect on viability or growth of cells having known genetic, genomic, epigenomic, transcriptomic, proteomic, or other profiles, and/or an effect on viability, proliferation, invasiveness, motility, cell cycle, or other cellular characteristics. For example, the methods can be used to determine responses to medication and potential drug resistance (e.g., to monitor the development or overgrowth of resistant cells, and optionally to identify those populations that later develop resistance). The methods can be used to identify and select therapeutics that provide the most complete response (greatest reduction in affected/tumor cells and/or that overcome resistance (e.g., reduce numbers or don't elicit development of drug-resistant populations of cells) and/or that selectively affect resistant or tumor cells and not normal cells.
These methods can also be used to evaluate drug responses and resistance mechanisms across BCPs and BPPs generated from various cell populations, e.g., primary or non-primary tumor cells, or cells from other disease tissues or models, to screen for new candidate therapeutics or to identify new targets.
The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.
Gene editing protocols often require the use of a subcloning step to isolate successfully edited cells, the behavior of which is then compared to the aggregate parental population and/or other non-edited subclones. Here we demonstrate that the inherent functional heterogeneity present in many cell lines can render these populations inappropriate controls, resulting in erroneous interpretations of experimental findings. We describe a novel CRISPR/Cas9 protocol that incorporates a single-cell cloning step prior to gene editing, allowing for the generation of appropriately matched, functionally equivalent control and edited cell lines. As a proof of concept, we generated matched control and osteopontin-knockout Her2+ and Estrogen receptor-negative murine mammary carcinoma cell lines and demonstrated that the osteopontin-knockout cell lines exhibit the expected biological phenotypes, including unaffected primary tumor growth kinetics and reduced metastatic outgrowth in female FVB mice. Using these matched cell lines, we discovered that osteopontin-knockout mammary tumors were more sensitive than control tumors to chemotherapy in vivo. Our results demonstrate that heterogeneity must be considered during experimental design when utilizing gene editing protocols and provide a solution to account for it.
Materials and Methods
Cell Lines
McNeu and Met-1 murine mammary carcinoma cells (kind gifts from Drs. Michael Campbell and Johanna Joyce, respectively) were cultured as previously described [30, 31]. Briefly, cells were cultured in DMEM (Gibco) media, supplemented with 10% fetal bovine serum (FBS) and 100 U/ml penicillin-streptomycin at 37° C. under 5% CO2. Human MDA-MB-435 cells were a generous gift from Dr. Robert Weinberg and were cultured in DMEM:F12 (1:1; Gibco), supplemented with 10% fetal bovine serum and 100 U/ml penicillin-streptomycin at 37° C. under 5% CO2. All cell lines were validated as Mycoplasma-negative. Human cells were validated using short tandem repeat (STR) profiling (Molecular Diagnostics Laboratory at Dana-Farber Cancer Institute, Boston, Mass.). For mouse cells, the murine strain of origin was confirmed by short tandem repeat analysis (Bioassay Methods Group, NIST).
New Gene Editing Protocol
Clonal subpopulations are generated from parental cell lines by sorting one single cell per well into 96-well plates using a FACSAria II cell sorter (BD Bioscience). Single cell-derived populations are subsequently allowed to proliferate for expansion. A single expanded clone is used for both control and co-transfection with the Cas9/GFP and sgRNA vectors. Select cell populations were seeded into 12-well plates overnight before transfection with 500 ng pCas9_GFP and 500 ng sgRNA expressing plasmids using FugeneHD (Roche). 48 hours after transfection, successfully transfected single cells are isolated by FACS sorting for GFP-positivity using a FACSAria II cell sorter (BD Bioscience) followed by recovery and expansion in 12-well plates for 2-3 days. At confluency, cells were collected for a second round of FACS sorting and single GFP-negative cells were sorted into individual wells in a 96-well plate to ensure that random Cas9/GFP integration did not occur. Following clonal expansion editing was validated using Sanger sequencing and phenotype verification is performed.
To generate luciferase/GFP-positive populations, cells were infected with lentivirus generated from pLV-Luc-IRES-GFP viral plasmids (a generous gift from Dr. Robert Weinberg's lab) and then sorted for GFP-positive populations.
Vector Construction
The human codon-optimized Cas9 expression plasmid pCas9 GFP was a gift from Kiran Musunuru (Addgene plasmid #44719). The sgRNA targeting mouse OPN exon 2 (5′-GTGATTTGCTTTTGCCTATT-3′ (SEQ ID NO:1)) driven by human U6 promoter was synthesized at Eurofin.
Evaluating Target Site Modification by Sanger Sequencing
OPN gene fragments were amplified with the primers OPN-F (5′-GACTTGGTGGTGATCTAGTGG-3′ (SEQ ID NO:2)) and OPN-R (5′-GCCAGAATCAGTCACTTTCAC-3′ (SEQ ID NO:3)) using Phire Animal Tissue Direct PCR Kit (Thermo Scientific). The resulting PCR products were then submitted for Sanger sequencing (Macrogen USA).
Animals and Tumor Studies
Female FVB/NJ mice 7 weeks of age were purchased from Jackson Labs (stock no. 001800). NOD/SCID mice were maintained in-house under aseptic sterile conditions. All experiments were conducted in accordance with regulations of the Children's Hospital Institutional Animal Care and Use Committee (protocol 12-11-2308R), the MIT committee on animal care (protocol 1005-076-08), and Brigham and Women's Hospital animal care protocol committee (2017N000056). Mice were 8-9 weeks of age at the time of study initiation. All efforts were made to minimize animal suffering. Animal facility personnel monitored the animals daily, checking for levels of food, water, and bedding in each cage. Mice were also physically checked three times a week by the investigators. The basic animal maintenance included housing the mice in cages (five per cage) with sufficient diet, water and bedding and cages were cleaned and sanitized on a regular basis. Investigators strictly adhered to approved protocols for humane endpoints; if any animal became severely ill prior to an experimental endpoint, that animal would be euthanized. Humane endpoints were defined as follows: ≥20% weight loss, rough hair coat, jaundice and/or anemia, coughing, labored breathing, nasal discharge, neurological signs (frequent seizure activity, paralysis, ataxia), prolapse, self-induced trauma, any condition interfering with eating or drinking, excessive or prolonged hyperthermia or hypothermia, tumor size ≥1.5 cm3 in volume. Animals were randomly assigned to treatment groups and no animals were excluded from analysis.
For tumor studies, murine mammary carcinoma cells were injected orthotopically, using a total of 105 or 106 McNeu cells, or 2.5×104 or 2.5×105 Met-1 cells implanted into the fourth mammary fat pad of 7-10 week old female FVB mice. Where indicated, either 1×105 or 1×106 cells of the McNeuA parental cell line were implanted subcutaneously. 2.5×105 human MDA-MB-435 cells were injected subcutaneously into 8-10 week old female NOD-SCID mice. Thereafter, tumors were monitored and measured using calipers with volume calculated as 0.5(length×width2).
For the Met-1 metastasis assay, mice received tail vein injections with 106 cells of luciferase-labeled Met-1 cells suspended in 100 μl of sterile phosphate-buffered saline. Pulmonary metastases were monitored weekly by bioluminescent imaging using the Spectrum Imaging System and Living Image software (Caliper Life Sciences, Inc.). Prior to imaging, mice were intraperitoneally administered 150 mg/kg D-luciferin (Perkin-Elmer) and were anesthetized using isoflurane inhalation. Luminescent signal was detected for the regions of interest as radiance (p/sec/cm2/sr) and analyzed using the Living Image Software Version 4.1 (Caliper Life Sciences). Lungs were fixed and stained using Hematoxylin/Eoisin and metastases were classified as multi- or single-focal and were counted manually on 3 separate sections spaced 50 microns apart per mouse. Total lung area was quantified using Cell Profiler and metastases counts were normalized total lung area.
Chemotherapy
For AC-T chemotherapy trials, 2.5×105 Met-1 Luc/GFP cells were injected into the mammary fat pad of 6-8-week-old female FVB mice. Doxorubicin (Teva), paclitaxel (Hospira), and cyclophosphamide (Sigma) were diluted in PBS for in vivo experiments. Mice were treated with two to four doses of 5 mg/kg doxorubicin, 10 mg/kg paclitaxel, and 120 mg/kg cyclophosphamide administered every two weeks. Doxorubicin was administered via retro-orbital injection, and paclitaxel and cyclophosphamide were administered via intraperitoneal injection.
For studies investigating the role of OPN in chemotherapeutic response, 2.5×104 WT or OPN KO tumor cells were injected into the mammary fat pad of 6-8-week-old female FVB mice. When established tumors reached 60-80 mm3 in volume, treatment was initiated. Four treatment arms were included: vehicle control (PBS) on WT or OPN KO cohorts or one dose of paclitaxel (10 mg/kg), doxorubicin (5 mg/kg) and cyclophosphamide (120 mg/kg) by intraperitoneal injection (paclitaxel and cyclophosphamide) and retro-orbital injection (doxorubicin) on WT or OPN KO cohorts. Tumor growth was monitored using caliper measurements. Average tumor mass at sacrifice was measured and is presented as the average±standard error of mean.
Osteopontin ELISAs and Western Blotting
To assess circulating secreted murine osteopontin (mOPN) or human osteopontin (hOPN) protein levels, whole blood was collected in EDTA-coated tubes (VWR) and centrifuged at 1.5×g for 8 minutes to isolate plasma. mOPN and hOPN concentrations were determined by ELISA according to manufacturer's instructions (R&D) and analyzed using a plate reader (Molecular Device).
To quantify secreted mOPN levels in conditioned medium, cells were grown to 80-90% confluence in growth medium containing 10% FBS. Then the medium was replaced with serum-free medium and was collected 24 hours later. mOPN levels in conditioned media were quantified by ELISA or western blotting.
Whole cell lysates were prepared following culture in the presence or absence of brefeldin A (used to prevent the secretion of OPN and ensure detection of protein expression). Cell lysates or concentrated conditioned medium were subjected to SDS-PAGE on 12% gels and then transferred onto a polyvinylidenedifluoride membrane, which was incubated with mouse anti-OPN (final dilution: 1:200, Clone AKm2A1, Santa Cruz Catalog #sc-21742, mouse monoclonal antibody raised against recombinant OPN of mouse origin, references with validation available on manufacturer's datasheet) antibody at 4 C overnight. After being washed, membranes were incubated with horseradish peroxidase-conjugated anti-mouse IgG for 1 hour. The enzyme bound to OPN was visualized using the SuperSignal™ West Pico Chemiluminescent kit (ThermoFisher). The blot was then stripped and incubated with rabbit anti-mouse (3-actin antibody as a loading control (final dilution: 1:1000, Rockland Catalog #600-401-886, rabbit polyclonal antibody raised against human beta-actin, references with validation available on manufacturer's datasheet).
Immunohistochemistry, Immunofluorescence and Microscopy
Formalin-fixed, paraffin embedded tissues were sectioned onto ProbeOn Plus microscope slides (Fisher Scientific) and immunohistochemistry was performed as described [21]. For the OPN immunohistochemistry studies, anti-OPN (final dilution: 1:200, Maine Biotechnology Services Catalog #, MAB197P, mouse monoclonal antibody raised against recombinant OPN of human origin, [44]) was used and was detected using the Vector ABC kit (Vector Laboratories, Burlingame, Calif., USA). For immunofluorescence, anti-OPN (final dilution: 1:50, Clone AKm2A1, Santa Cruz Catalog #sc-21742, mouse monoclonal antibody raised against recombinant OPN of mouse origin, references with validation available on manufacturer's datasheet) was used and was detected using a goat anti-mouse IgG AF549 conjugated secondary antibody (final dilution: 1:1000, Invitrogen Catalog #A11032, polyclonal, references with validation available on manufacturer's datasheet). Nuclei were counterstained with DAPI (Invitrogen). Images were captured with identical exposure and gain using a Nikon Eclipse Ni microscope.
In Vitro Chemosensitivity Studies
4,000 Met-1 cells were plated in quadruplicates in 96-well plates containing growth media. The next day, vehicle (PBS) or chemotherapy (doxorubicin: 0.33 nM-2.2 ∝M; paclitaxel: 14 μM-160 μM) was added to the plate and incubated for 72 hours. ATP levels were quantified as a surrogate measure for viability (CellTiter-Glo, Promega) using a luminometer (Perkin-Elmer).
Statistical Analyses
Data are represented as mean±SEM and analyzed by ANOVA, Student's t-test, and/or Mann-Whitney test as indicated using GraphPad Prism 7.0, unless otherwise stated. P<0.05 was considered statistically significant. Error bars represent standard deviation unless otherwise indicated.
Results
Selection of Her2+ and Estrogen Receptor-Negative Mammary Carcinoma Models
We aimed to design an approach that would enable us to generate appropriately matched control and CRISPR/Cas9 knockout cell lines, while taking into account the inherent functional heterogeneity present in nearly all breast tumors and tumor-derived cancer cell lines. We hypothesized that results from studies employing standard CRISPR/Cas9 approaches, which often require a subcloning and/or selection step, would be confounded by subclonal functional heterogeneity.
As a proof of concept, we chose to study Osteopontin (OPN), a protein that we have studied previously and that is relevant for breast cancer metastasis [18-25]. OPN plays an important role in metastasis and survival in many pre-clinical cancer studies, and is positively associated with metastasis as well as reduced progression-free and overall survival in breast cancer patients [24-26]. Additionally, OPN has been shown to play a role in chemoresistance in some cancer types types [18, 20, 22, 27-29], but it is unclear whether this is also true of breast cancer.
Hence, we determined that the breast cancer models that we would employ must meet the following criteria: secretion of detectable levels OPN both in vitro and in vivo, capacity to form primary and metastatic tumors in vivo, evidence of heterogeneity, and responsiveness to chemotherapy.
Transgenic mice that specifically overexpress oncogenic proteins in the mammary fat pad are commonly employed both for the study of spontaneous breast tumors and as a source for murine breast cancer cell lines that can be allografted orthotopically in immunocompetent animals. In this study, we utilized two such murine breast cancer cell lines: McNeuA, a HER2+ breast cancer cell line derived from a spontaneously arising mammary carcinoma in a MMTV-neu transgenic mouse [30], and Met-1, an estrogen receptor-negative (ER−) breast cancer cell line derived from a mammary carcinoma in a MMTV-PyMT transgenic mouse (FVB/N-Tg(MMTV-PyVmT) [31].
Characterization of McNeuA and Met-1 cell lines demonstrated their potential as models for this study, as they secreted detectable levels of OPN in culture as measured by ELISA (
In both models, the tumor bearing mice had significantly elevated plasma levels of OPN relative to cancer-free cohorts whereby average OPN levels were 8-fold and 15-fold higher in the McNeuA and Met1 tumor-bearing mice, respectively, at end stage (
Previous studies have demonstrated that both of these cell lines are capable of forming lung metastases [30, 31]. We were particularly interested in the Met-1 cell line, as women with metastatic ER-breast cancer most often experience pulmonary metastases [32]. We confirmed that the Met-1 cells formed pulmonary metastases, with 4 of 5 mice experiencing increased metastatic burden (˜15-300-fold increases) over the experimental time course (
We next tested responsiveness of Met-1 mammary carcinoma to combination doxorubicin (A), cyclophosphamide (C), and paclitaxel (T) chemotherapy, AC-T, a standard of care chemotherapy regimen for breast cancer patients with ER-negative disease. We first tested the sensitivity of Met-1 cells to doxorubicin and paclitaxel in vitro and performed an initial in vivo experiment to identify a therapeutically relevant, well-tolerated combinatorial dose.
Treatment with both doxorubicin and paclitaxel significantly decreased viability of Met-1 cells in vitro. Cyclophosphamide, a pro-drug, requires activation into cytotoxic metabolites by liver enzymes in vivo and was therefore not tested in vitro. In vivo, a neoadjuvant combination dose of doxorubicin (5 mg/kg), paclitaxel (10 mg/kg), and cyclophosphamide (120 mg/kg) was well tolerated (no weight loss; data not shown) and had a cytostatic effect on Met-1 tumor growth (
To more closely emulate the clinical dosing regimen of AC-T chemotherapy, mice with Met1 mammary carcinoma were administered neoadjuvant AC-T every 2 weeks for 4 cycles. Interestingly, individual mice bearing Met-1 tumors exhibited differential responses to treatment, and in some cases, mice that initially experienced complete tumor regression eventually experienced local recurrence (
Collectively, our analyses indicated that the McNeuA and Met-1 cell lines met our criteria of OPN secretion in vitro and in vivo, efficient formation of primary orthotopic tumors, and evidence of phenotypic and functional heterogeneity in vivo. Moreover, the Met-1 cells met the criteria of metastatic capacity and, chemosensitivity. Hence, the McNeuA and Met-1 cell lines were ideal for our investigation into the effect of tumor heterogeneity on the generation of appropriately matched control and OPN-KO cell lines.
Heterogeneity Between Subclonal Populations Derived from McNeuA and Met-1
In order to better understand whether the inherent phenotypic heterogeneity of the McNeuA and Met-1 cells lines would potentially confound the results of an OPN-knockout study, we generated single cell-derived subclonal populations from both the McNeuA (50 clones) and Met-1 (42 clones) parental cell lines (
Levels of OPN secreted in vitro by the McNeuA and Met-1 subclones varied considerably. The McNeuA subclones secreted a range of OPN from 37.5-442.1 ng/ml per 106 cells (
Taken together, these results highlighted the phenotypic heterogeneity that exists within tumor-derived breast carcinoma populations in vitro. We therefore wondered if different clones would perform differently in vivo as well.
McNeuA and Met-1 Derived Clonal Populations Behave Differently In Vivo
To understand whether various subclones that displayed different phenotypes in vitro would also display functional heterogeneity with respect to tumorigenesis, we injected cohorts of FVB mice orthotopically with various McNeuA or Met-1 subclonal populations and monitored tumor growth parameters over a course of 64 or 49 days, respectively. We chose to use five subclones from each cell line that secreted the highest levels of OPN (MC-18, MC-22, MC-45, MC-47, MC-50 and MT-2, MT-3, MT-4, MT24, MT-29) (
Those clones that formed tumors displayed variability in latency and growth kinetics. For example, latency and growth kinetics were not statistically different between MC-22 and MC-50 when 106 cells were injected (
Similarly, the growth kinetics of the Met-1 subclonal populations was also variable. When 2.5×104 cells were injected, at the 28 day time point (when the MT-4 cohort had reached its endpoint), its growth kinetics were significantly different from the MT-2, MT-4, MT-24 and MT-29 subclones (p<0.0002,
The subclones derived from the human melanoma cell line also varied in incidence of subcutaneous tumor formation in NOD-SCID mice, with some clones (i.e. 11, 28, 29, 30) unable to form tumors in vivo. Moreover, tumor mass at the experimental end point varied considerably among these subclones.
Critically, a number of individual subclonal populations from each tumor model exhibited different tumor formation capabilities than the respective bulk parental population from which they were derived. For example, while the parental Met-1 tumor cell line formed orthotopic tumors with 100% incidence, the MT-3 subclonal cell line formed tumors with only 60% incidence when the same number of cells was injected (
These observations revealed the considerable subclonal heterogeneity that exists within human and murine mammary carcinoma cell lines and that the behavior of individual subclones differs from their respective parental populations.
Evidence that Identification of Proper Controls is Necessary for Correct Interpretation of Experimental Findings
Traditional CRISPR/Cas9 editing protocols begin with infection or transfection of the bulk parental population [3-7]. For this reason, the unedited or mock-infected parental cell line is typically used as a control. Due to the inefficiency of infection and/or editing in certain cell lines (especially tumor cell lines that are hyperploid), there is often a subclonal selection step that follows the initial infection and then a validated, edited subclone is used for subsequent experimentation. Our initial characterizations of the McNeuA and Met-1 parental and subclonal populations demonstrate why one must use caution when considering this commonly used approach.
In some scenarios, subclonal heterogeneity could confound interpretation of knockout efficiency. For example, 23% of the Met-1 subclones have low or no detectable secreted OPN (
In another scenario, if the clonal population that was selected after CRISPR/Cas9 OPN-knockout happened to be clone MT-3 and its orthotopic tumor penetrance was compared to that of the parental Met-1 population, then one could erroneously interpret the necessity of OPN for primary tumor formation, when in fact this clone, prior to OPN knockout, already inherently forms tumors with lower incidence (˜66%) than the parental population (100%) (
Likewise, comparing two subclonal populations, even those that secrete similar levels of OPN and form tumors with the same incidence, could also lead to spurious results. For example, if one randomly selected MT-29 as an OPN KO clone and MT-4 as a control, then incorrect conclusions could be drawn about the role of OPN in tumor growth. This is because prior to OPN KO, both clones express similar levels of OPN (˜225 ng/ml;
Our results provided evidence that neither the parental population nor other subclones would represent an appropriately matched wild-type control for a CRISPR/Cas9 knockout cell line that was selected after the gene editing step. The only appropriate control would be to compare the behavior of edited and unedited cells derived from the same clonal population. We therefore concluded that a modified strategy should be developed to account for heterogeneity and enable the generation of appropriately matched cell lines.
Generating spp1 Knockout Clonal Populations Via CRISPR/Cas9
One would not have known a priori about differences in subclonal biological phenotypes and experimental outcomes by taking traditional approaches to gene editing. Therefore, we developed a modified CRISPR-Cas9 editing protocol for generating matched control and knockout cells. Appropriate subclonal populations that we had generated and characterized were chosen for CRISPR/Cas9 gene targeting based on desired biological properties of high intrinsic levels of OPN secretion and orthotopic tumor incidence of 100%. We identified three clonal populations that fit these criteria: MC-22, MC-50, and MT-2 (
We used our modified CRISPR/Cas9 editing strategy to delete the spp1 gene (which encodes Osteopontin) in each subclonal population in order to generate OPN KO cell lines. To do so, the individual subclonal populations were transiently co-transfected with a human codon-optimized spCas9-2A-GFP fusion protein expression plasmid (Addgene plasmid #44719) and a plasmid harboring a sgRNA targeting exon 2 of spp1 (
Due to the transient nature of our transfection protocol, only cells in which the Cas9-GFP fusion protein had been randomly integrated would maintain GFP expression past this point. In order to avoid random integration of the Cas9 expression plasmid into the genome, a second round of single-cell sorting by FACS was employed to isolate cells that had not undergone a Cas9 integration event by sorting and selecting for GFP-negative cells (
We next employed Sanger sequencing to identify the edited subpopulations from among the recovered subclones. Of the recovered subclones from the MC-22, MC-50, and MT-2 lines, a subset of the single cell clones contained either a hemizygous or homozygous mutation in the spp1 gene, representing editing efficiencies of 42%, 55%, and 53%, respectively (
We validated loss of OPN protein expression in each of the OPN KO clones compared to its appropriately matched control using western blotting, ELISA of conditioned media, or immunocytochemistry. We observed no detectable OPN protein (
Osteopontin is Dispensable for Primary Tumor Growth
Most studies, including our own, report that OPN is dispensable for primary tumor growth, but is critical for metastasis due to its effects on tumor cells, the host systemic environment, and the tumor microenvironment [19, 21, 23]. Therefore, successful generation of appropriately matched KO and WT cell lines should also reflect these properties (e.g., loss of OPN should have no effect on primary tumor growth, but should alter metastatic ability). This makes OPN an ideal protein to test our concept because its dispensability for primary tumor growth means that WT and OPN KO clones should exhibit similar primary tumor growth kinetics and incidence. Therefore, we tested the tumor formation capabilities of the matched clones.
WT and OPN KO MC-50 cells (2×105), MC-22 cells (1×105) or MT-2 cells (2.5×104) were orthotopically injected into FVB mice and were allowed to grow until tumors reached ˜1 cm3. Loss of tumor-derived OPN did not significantly affect growth kinetics or the final mass of any of the tumors derived from matched subclonal cell lines (
As a control, we tested the concentration of circulating plasma mOPN in the tumor-bearing mice and cancer-free controls. As expected, mOPN plasma levels were elevated in the mice bearing WT tumors relative to the cancer-free cohort, and plasma OPN levels were significantly reduced in the mice bearing KO tumors relative to WT (
It is important to note that if we had used the parental McNeuA cell line as a WT control rather than the appropriately matched WT MC-22 cell line, we would have failed to see a significant difference in the circulating OPN levels between cohorts (
Finally, we visualized OPN expression in the tumors that formed in each cohort using immunohistochemical staining. We observed positive staining for OPN in the WT MC-22, MC-50, and MT-2 tumors, but did not detect any OPN+ cells in the corresponding OPN KO tumors (
Together, these results demonstrated that our modified CRISPR/Cas9 gene editing protocol can be successfully used for studies examining the role of a gene in primary tumor outgrowth.
Loss of Osteopontin Reduces Multifocal Metastatic Outgrowth
Osteopontin is considered a biomarker for tumor progression and is detected at higher levels in more aggressive tumors than their low-grade counterparts, is elevated in the serum of patients with metastatic disease, and is included in gene lists predicting poor prognosis for many cancer types [25, 33-39]. Although OPN is most often dispensable for primary tumor growth, OPN is necessary for metastasis [21, 40-42].
Met-1 cells are highly metastatic [31] (
Metastatic burden was decreased in the MT-2 OPN KO cohort relative to that of the MT-2 WT cohort, as indicated by reduced bioluminescent signal over the course of the experiment (p=0.0085 at day 7, p>0.05 for all other time points; FIG. 6B,6C). As further confirmation, we analyzed H&E lung sections at the experimental end point and quantified the numbers of single and multifocal metastases. There were significantly fewer total and multifocal pulmonary metastases in mice that had been injected with the OPN KO cells compared to mice that had been injected with OPN WT cells (
Collectively, our results established that by using appropriately matched cells, we could confidently conclude that OPN is necessary for metastatic colonization and that our CRISPR/Cas9 protocol is useful for pre-clinical metastasis studies.
Loss of Osteopontin Enhances Chemosensitivity
Resistance to standard chemotherapies remains a significant clinical problem, particularly for triple-negative breast cancer [43]. In order to interrogate whether OPN contributes to chemoresistance in breast cancer models, we tested the MT-2 WT and KO cell lines for sensitivity to AC-T chemotherapy in vivo.
We injected 2.5×104 MT-2 WT or matched OPN KO tumor cells into the mammary fat pads of FVB mice. When established tumors reached ˜60-80 mm3 in volume (14 days), animals were randomized based on tumor volume and enrolled into either vehicle control (PBS) or AC-T chemotherapy treatment cohorts (
MT-2 WT and MT-2 KO tumors exhibited sensitivity to AC-T treatment relative to their respective vehicle-treated cohorts (
Together, these data established that elimination of OPN expression enhances chemosensitivity of the MT-2 breast cancer population.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application is a continuation of PCT/US2019/036554, filed on Jun. 11, 2019, which claims the benefit of U.S. Provisional Application Ser. No. 62/683,225, filed on Jun. 11, 2018. The entire contents of the foregoing are incorporated herein by reference.
This invention was made with Government support under Grant No. CA166284 awarded by the National Institutes of Health and Grant No. W81XWH-14-1-0191 awarded by the Department of Defense. The Government has certain rights in the invention.
| Number | Date | Country | |
|---|---|---|---|
| 62683225 | Jun 2018 | US |
| Number | Date | Country | |
|---|---|---|---|
| Parent | PCT/US2019/036554 | Jun 2019 | US |
| Child | 17119245 | US |
