Patent Application
20240384236
The disclosed invention is generally in the field of leukemia cells and 3D osteogenic niche (3DON) which recapitulate the leukemia bone marrow microenvironment. More specifically, it relates to the composition, materials, and methods of a novel 3DON that consists of stromal cells microencapsulated in extracellular matrix (ECM).
Acute myeloid leukemia (AML) is one of the most common acute leukemia (Brunetti, Gundry, and Goodell 2019). It is the most lethal hematopoietic cancer with a 5-year survival rate at around 27% (Duy et al. 2021; Marofi et al. 2021). AML has over 24 genetically defined subtypes (Vetrie, Helgason, and Copland 2020), making it a very heterogeneous disease and hence causing poor prognosis (HoushmandMohammad et al. 2017; Stanchina et al. 2020). Currently, the standard treatment for AML is a 7+3 regimen (7 days of cytarabine with 3 days of anthracycline) but it only results in 50% overall survival (Bertoli et al. 2017). This is followed by consolidation chemotherapy and hematopoietic stem cell transplantation for some high-risk patients (Yeung and Radich 2017). Relapse caused by chemoresistant leukemic cells occurs in more than 70% of patients (Villatoro et al. 2020). For aged individuals, who are the majority of AML patients, they may not tolerate the intensive chemotherapy or respond to the treatments due to their higher risk in complex karyotype and TP53 mutation (Stanchina et al. 2020). With the recent advancement in AML pharmacotherapies, tailor-made treatment for patients, especially the elderly, could be made possible by personalized medicine.
The bone marrow is a viscous tissue within the bone cavity responsible for hematopoiesis (Shafat et al. 2017). The two dynamic bone marrow niches-osteogenic niche (ON) that are made up of osteoblasts, osteocytes and osteoclasts; and perivascular niche, consist of endothelial cells and megakaryocytes, interact to regulate the functions of hematopoietic stem cells (HSCs) (Wang and Zhong 2018). Mesenchymal stromal cell (MSC) is an important cell type as they establish the hematopoietic environment by differentiating into many essential components of the BM niche, and they also affects the proliferation, differentiation, and homing of HSCs (Ladikou et al. 2020).
Leukemic stem cells (LSCs) are the precursors of AML cells which are genetically mutated from HSCs (Quek et al. 2016). In fact, leukemic cells interact with the BM microenvironment by activating bone morphogenetic protein 2 (BMP-2) and Smad1/5 signaling in MSCs to induce osteogenic differentiation, hence create a niche rich in pre-osteoblast to become a pro-tumoral environment (Ladikou et al. 2020) (Battula et al. 2017). However, one obstacle limiting AML for in vitro experimental study and drug screening application is that unlike AML cell lines, primary AML cells rapidly undergo spontaneous or stress-induced apoptosis in ex vivo cultures, which is highly patient dependent due to heterogeneity (Aasebø et al. 2021) (Brenner et al. 2019) (Cucchi et al. 2020). Opposingly, AML cells has prolonged survival in vivo (Abdul-Aziz et al. n.d.). This discrepancy between the in vitro and the in vivo behavior of AML cells, in particular, the spontaneous apoptosis and the poor survival of the AML cells in cultures (Ryningen et al. 2006) may be attributable to the lack of the pro-tumoral BM microenvironment consisting of soluble factors such as growth factors, soluble adhesion molecules and cell-cell/cell-extracellular matrix (ECM) interactions.
Different methods have been developed to enable ex vivo culture of primary AML cells (Cucchi et al. 2020), including the development of engineered conditioned medium with stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF), interleukin-3 (IL-3) (Lam et al. 2016) (Brenner et al. 2019); liquid tumor models such as 2D co-cultures with MSCs or endothelial cells to mimic the BM niche by secreting cytokines (Schuringa and Schepers 2009) (van Gosliga et al. 2007); and 3D co-cultures such as MSC-coated ceramic scaffolds (Antonelli et al. 2016), collagen I or fibronectin coated polylactic-co-glycolic acid (PLGA) and polyurethane (PU) scaffolds (Blanco et al. 2010), polyethylene glycol (PEG) hydrogels with HUVEC and MSCs (Bray et al. 2017). However, the preparation of the engineered conditioned medium is tedious, time-consuming, costly and inefficient because viral transfection of three different cell lines to over-express three different cytokines are necessary for producing the required medium (Hogge et al. 1996). Furthermore, the engineered conditioned medium and the 2D co-culture environment failed in mimicking the cell-cell or cell-ECM contacts, whereas the existing 3D cultures in a wide range of materials, although being more physiologically relevant, are yet to recapitulate the pro-tumoral osteogenic niche (Cucchi et al. 2020).
Any discussion of documents, acts, materials, devices, articles or the like which has been included in the present specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it existed before the priority date of each claim of this application.
Throughout this specification the word “comprise,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps.
Provided herein is a platform that comprised of leukemia cells co-cultured with biomimetic 3DON that support the survival of the leukemia cells. The 3DON is formed by encapsulating MSCs in naturally occurred ECM and differentiated towards osteogenic lineage. The 3DON is co-cultured with primary AML cells to support the survival, reduce the apoptosis and maintain the phenotype of AML cells for the applications of drug screening and chemotherapy adjuvant.
The composition of 3DON uses a formulation including cell component and ECM. In the preferred embodiment, the cell component that present in the bone marrow microenvironment, interact with the ECM and the co-culture cells, is healthy bone marrow (BM) derived MSCs or AML-derived MSCs, or other stromal cells that are present in the bone marrow microenvironment such as osteoblast, osteoclast, osteocyte, endothelial cells, adipocytes and immune cells. In the preferred embodiment, the natural ECM, being capable of providing support to the cells and interact with the cells, permitting cell migration and penetration, is collagen, or other material that supports cell growth and migration such as fibronectin, laminin and Matrigel. In the preferred embodiment, the cells being co-cultured with 3DON, is primary AML cells, or other leukemia cells including primary cells or cell lines.
The method of fabricating 3DON includes mixing of collagen, sodium hydroxide, culture medium and cells in a specific order, ratio, pH, concentration and volume; the temperature and incubation time is also controlled for gelation. The 3DON is followed by osteogenic differentiation which includes the use of osteogenic induction medium prepared with dexamethasone, β-glycerophosphate, ascorbic acid and bone morphogenic protein 2 (BMP2) in a specific concentration. In addition, the culture plate for forming 3DON is coated with pluronic F127 to reduce cell attachment. Leukemia cells were seeded into a well containing 3DON for a period of time before screening.
The novel 3DON, when being co-cultured with primary AML cells, can increase the percentage of viable AML cells (60-70%) and reduce the percentage of AML apoptotic cells (5-10%). The phenotype of primary cells (CD33+ and CD34−) can also be preserved, which can be explained by the cell-cell and cell-ECM interaction between AML blasts and the 3DON, and secretion of cytokines by 3DON. Apart from this, drug test demonstrated that healthy 3DON can increase the chemosensitivity of primary AML cells towards daunorubicin and doxorubicin. AML cells were less sensitive to daunorubicin and doxorubicin upon co-culture with AML MSC-derived niches.
In summary, the use of novel 3DON for primary AML cell co-culture tackled the problem of spontaneous apoptosis of primary AML cells in ex vivo cultures and allowed the development of personalized drug screening that provide valid results. On the other hand, the 3DON increases the chemosensitivity of primary AML cells such that it can be applied as an adjuvant to chemotherapy by replenishing healthy BM-MSC derived 3DON into the bone marrow of the AML patients.
Additional advantages of the disclosed method and compositions will be set forth in part in the description which follows, and in part will be understood from the description, or can be learned by practice of the disclosed method and compositions. The advantages of the disclosed method and compositions will be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the invention as claimed.
The accompanying drawings illustrate several embodiments of the disclosed method and compositions and together with the description, serve to explain the principles of the disclosed method and compositions.
The disclosed method and compositions can be understood more readily by reference to the following detailed description of particular embodiments and the Example included therein and to the Figures and their previous and following description.
Disclosed are compositions, platforms, systems, and methods involving biomimetic 3D osteogenic niches (3DONs). In some forms, the 3DON is formed by encapsulating mesenchymal stromal cells (MSCs) in extracellular matrix (ECM) and differentiating the MSCs towards the osteogenic lineage. In some forms, the MSCs are bone marrow derived MSCs. In some forms, the MSCs are osteoblast cells, osteoclast cells, osteocytes, endothelial cells, adipocytes, immune cells, or combinations thereof. In some forms, the ECM is collagen, fibronectin, laminin, Matrigel, or combinations thereof.
In some forms, the 3DON are formed by mixing the ECM, the MSCs, sodium hydroxide, and culture medium and by inducing osteogenic differentiation. In some forms, ECM, MSCs, sodium hydroxide, and culture medium are mixed in a specific order, ratio, pH, concentration, volume, or combinations thereof. In some forms, the mixing is performed in a culture plate coated with pluronic F127. In some forms, the temperature and incubation time are chosen to allow for gelation. In some forms, the osteogenic differentiation is induced by use of osteogenic induction medium prepared with dexamethasone, β-glycerophosphate, ascorbic acid, and bone morphogenic protein 2 (BMP2) in a specific concentration.
Also disclosed are platforms comprising leukemia cells co-cultured with the disclosed 3DON. In some forms, the platform supports the survival of the leukemia cells. In some forms, the leukemia cells are primary acute myeloid leukemia (AML). In some forms, co-culturing the 3DON and leukemia cells supports the survival, reduces the apoptosis, and/or maintains the phenotype of the leukemia cells. In some forms, co-culturing the 3DON and leukemia cells increases the chemo-sensitivity of leukemia cells.
In some forms, the percentage of viable leukemia cells is increased and/or and the percentage of apoptotic leukemia cells is reduced in leukemia cells maintained in the platform as compared to leukemia cells not maintained in the platform. In some forms, the phenotype of primary cells (CD33+ and CD34−) is preserved to a greater extent in the cells maintained in the platform as compared to cells not maintained in the platform.
Also disclosed are methods of drug screening comprising exposing the leukemia cells of the disclosed platform to a test compound and comparing the effect of the test compound on the leukemia cells to a control platform that was not exposed to the drug.
Also disclosed are methods of treating a patient in need of chemotherapy with a chemotherapeutic and with the disclosed 3DON. In some forms, the 3DON is in a pharmaceutical formulation.
As used herein, “cells” refers to the smallest basic unit of an organism, which is typically microscopic and consists of cytoplasm enclosed in a membrane. “Cells” can be from cell lines or isolated from human or animals such as mouse, including but not limited to leukemia cells, MSCs, pre-osteoblasts, osteoblasts, osteoclasts, osteocytes, chondrocytes, endothelial cells, adipocytes and immune cells.
As used herein, “ECM” refers to the extracellular matrix materials, in pure, isolated, partially isolated, recombinant or synthetic form, including but not limited to collagen type I, II, IV, fibronectin, laminin, Matrigel, or a combination thereof.
The ECM used for fabricating 3DON must be able to provide support to the cells and interact with the cells to allow cell growth, permitting cell migration and penetration without introducing toxicity. The ECM used can be naturally occurring or synthetic such as collagen type I, II, IV, fibronectin, laminin, Matrigel, or a combination thereof. The ECM can be derived from either natural or synthetic sources, and it can be induced to solid form under specific conditions and support cellular survival and growth. The ECM can be produced from isolation or extraction from various animal sources, such as rat tail, porcine skin, bovine tendon, or human placenta.
The cells being microencapsulated in the 3DON can interact with the ECM surrounding them and the co-culture cells, they can be pre-osteoblasts, osteoblasts, osteoclasts, osteocytes, chondrocytes, endothelial cells, adipocytes and immune cells, preferably MSCs. The cells can survive and proliferate inside the 3DON. The cells can be derived from human bone marrow, peripheral blood, adipose tissue, umbilical cord tissue, amniotic fluid, or any other tissue or cells that can give rise to MSCs from healthy individuals or leukemia patients. The leukemia cells being co-cultured with 3DON can be of primary culture, or cell line culture, such as acute lymphocytic leukemia cells (ALL), chronic lymphocytic leukemia cells (CLL), chronic myeloid leukemia cells (CML), HL-60, KG-1, MOLM13 etc., preferably primary AML cells. The co-cultured cells can be obtained from peripheral blood or bone marrow from leukemia patients.
The 3DON is produced by encapsulating MSCs in an ECM, preferably rat-tail type I collagen. The MSC cell density can be 10000 cells/ml-300000 cells/ml preferably 50000 cells/ml. The collagen concentration can be 0.1 mg/ml-4 mg/ml preferably 2 mg/ml. The volume of 3DON can be 1 μl-200 μl, preferably 10 μl for drug test. The culture medium, IN sodium hydroxide, stock collagen and cell suspension are mixed according to this order and pipetted into the wells of a 384-well culture plate, or other culture plate such as 96-well or 1536-well plate without coating or pre-coated with pluronic F127 for an hour at room temperature. The 3DON is incubated at 37° C., 5% CO2 for 15 minutes to 120 minutes, preferably 60 minutes for gelation. The solidified 3DON is cultured for 3 days in Dulbecco's Modified Eagle Medium-low glucose (DMEM-LG) supplemented with 10% fetal bovine serum (FBS), 1% antibiotic-antimycotic (anti-anti). Osteogenic differentiation of the encapsulated MSCs is performed immediately or after 1 to 7 days culture, preferably 3 days culture. The 3DON is cultured in DMEM-LG supplemented with 10% FBS, 1% anti-anti, 100 nM dexamethasone (Sigma), 50 μM ascorbic acid 2-phosphate (BioChemika), 10 mM b-glycerophosphate (Calbiochem) and 10 ng/ml BMP-2 (PeproTech) as the osteogenic differentiation induction medium. The 3DON is cultured for 7 to 28 days, preferably 21 days in a 37° C. 5% CO2 incubator with regular medium replenishment every 2-3 days before co-culture.
The leukemia cells are then seeded to the wells containing 3DON with percentage of leukemia cells being 10%-90%, preferably 50% of the total cell number in the well. The medium used for culture is 1:1 mixture of DMEM-LG medium (supplemented with 10% FBS and 1% anti-anti) and Roswell Park Memorial Institute (RPMI) 1640 medium (supplemented with 20% FBS and 1% anti-anti).
Leukemia cells are co-cultured with the 3DON for 1 to 7 days, preferably 3 days. The drugs applied can be FDA approved for leukemia, such as chemotherapeutic drugs, or chemicals under development for treating leukemia patients. Drugs of 5-30 different concentrations are administered into the culture wells. The leukemia cells are exposed to drugs on any day of the co-culture, preferably the second day, for 0.5 hours-96 hours, preferably 24 hours in a 37° C. 5% CO2 incubator and cells are aspirated into another culture plate for viability assay such as PrestoBlue, Resazurin assay, alamarBlue, (3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) (MTT) assay etc. Graphs plotting the response of AML cells versus the concentration of drug can be used for calculating IC50 values for each type of drug.
The 3DON is designed to mimic the bone marrow microenvironment, specifically the osteogenic niche, to support the survival and phenotype maintenance of primary AML cells. To test for the effects of 3DON in AML co-culture, nine culture groups are designed for comparison. Primary AML cells are cultured in three control groups, I: negative control 1 (AML medium) (NC1); II: negative control 2 (MSC medium) (NC2); and III: positive control (engineered growth factor cocktail medium for hematopoietic cells) (PC), and co-cultured in six experimental groups: IV: healthy 2D mesenchymal niche (MSC feeder layer) (2DMN); V: healthy 3D mesenchymal niche (3DMN); VI: healthy 3D osteogenic niche (3DON); VII: AML 2DMN; VIII: AML 3DMN; IX: AML 3DON. The NC groups are used to confirm that the differences between the co-culture groups and the NC groups are indeed due to the effect of the co-culture but not the effect of the medium. The engineered growth factor cocktail medium has been reported to significantly reduce apoptosis of AML cells. (Hogge et al. 1996; Lam et al. 2016) as it contains conditioned medium from engineered murine stromal cell lines that produce stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF), interleukin-3 (IL-3) and fms-like tyrosine kinase 3 ligand (FLT3 ligand). (Hogge et al. 1996; Lam et al. 2016) AML medium and MSC medium are the negative controls because of the absence of stromal cells and supplementation of cytokines. Human MSCs are used as the niche cells to support AML cultures in the six co-culture experimental groups. 2DMN is prepared by seeding MSCs as a 2D feeder layer. 3DMN is prepared by encapsulating MSCs in collagen microspheres. 3DON is prepared by differentiating the 3DMN into osteogenic lineage. The primary AML cells are cultured under different conditions and collected for subsequent evaluation of their viability, apoptosis, phenotype maintenance, and drug response. Medium from 3DON and PC before co-culture are also collected for cytokine secretion assay.
Results demonstrated that the reconstituted AML 3D BM niches derived from AML patients provided the optimal niche to support the primary AML in cultures. Specifically, the viability of AML cells co-cultured with the AML 3DMN and AML 3DON groups were the highest, while the percentages of apoptotic cells in them were the lowest. Cytokine array analysis revealed that the enhanced secretion of anti-apoptotic cytokines such as IL-8, GROα and OPG in the AML 3DMN and AML 3DON groups. In addition, the AML phenotype of CD33+ and CD34-were nicely preserved in these groups. The intrinsically high chemoresistance of AML was well retained by the AML niches (2DMN, 3DMN, and 3DON groups) as compared to the healthy niches and other control groups, using daunorubicin and doxorubicin as examples, suggesting the physiological resemblance of the AML niches and the necessity to reconstitute AML BM niches for valid drug screening. Moreover, the healthy BM niches (2DMN, 3DMN, and 3DON), while equally supporting the AML cell survival and phenotype maintenance in vitro, showed increased drug sensitivity to the two sample drugs used, indicating that reconstituting a healthy BM niche in leukemic patients may overcome the intrinsic chemoresistance problem in leukemia patients. This work contributes to the application of the AML 3DON in personalized drug screening and suggests the potential of using the healthy 3DON as a chemotherapeutic adjuvant for better AML chemotherapies with enhanced drug sensitivity.
AML primary cells were cultured as previously reported [20]. AML primary cells isolated from 6 patients (1 male and 5 females, ages 51-71) with FAB stage M1 or M5 were used, Department of Medicine, the University of Hong Kong. All procedures were approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (HKU) (UW 05-183 T/846). Informed written consent from all participants was obtained prior to conducting the research. Primary cells were maintained in primary culture medium which was composed of 50% Iscove's Modified Dulbecco's Medium (IMDM) full medium and 50% conditioned medium collected from engineered murine fibroblast cell lines [20]. IMDM full medium was prepared from IMDM supplemented with 10% fetal bovine serum (FBS) (Gibco) and 1% antibiotic-antimycotic (Gibco). The primary cells were thawed in primary culture medium and immediately seeded in different culture conditions.
To discover the differences in behaviours of AML cells in healthy niche environment and AML niche environment, human bone marrow-derived mesenchymal stem cell or stromal cells (MSCs) from 3 healthy donors (M, 38 years old; M, 25 years old; F, 24 years old) and 1 AML patients (F, 54 years old) were obtained. All procedures were approved by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (HKU) (UW 05-183 T/846) The cells were grown in Dulbecco's modified Eagle medium-low glucose (DMEM-LG) (Gibco), supplemented with 10% FBS, 1% antibiotic-antimycotic and 1% GlutaMax (Gibco). MSCs were maintained in humidified conditions with 5% CO2 at 37° C. and medium was refreshed every 3-4 days until 80-90% confluence. Cells were trypsinized using 0.05% trypsin-EDTA (Gibco) and cells at passage 6 were used for microencapsulation.
To mimic the 3D bone marrow niche consisting of mesenchymal cells, 3DMN were prepared as reported previously [29]. Briefly, rat-tail collagen type I (BD Biosciences) was neutralized with IN sodium hydroxide (Sigma-Aldrich) and MSCs were suspended in the neutralized collagen solution in an ice bath. The cell mixture was diluted with culture medium to reach a cell density of 5×105 cells/ml and a collagen concentration of 2 mg/ml. Cell mixture containing 5×104 cells was dispensed into 96 well plates pre-coated with Pluronic F-127 (Sigma) to make the plate non-adhesive such that the MSCs would not migrate from the 3DMN to attach at the bottom of the plate (for flow cytometry and viability test), and 5×103 cells in 384 well plates also pre-coated with F-127 (for drug test). They were then incubated at 37° C. 5% CO2 environment for 45 minutes to induce gelation. The collagen microencapsulated MSC produced is the 3DMN. Healthy 3DMN was formed by healthy MSCs while AML 3DMN was composed of AML-patient derived MSC. The 3DMN was ready for subsequent experiments after 3 days of culture.
To mimic the 3D bone marrow niche that consists of osteolineage cells, 3DON was prepared as follows. The 3DON was formed from the osteogenic differentiation of the 3DMN 3 days after cell encapsulation as previously described [31]. The 3DMN was cultured in DMEM-LG supplemented with 10% FBS, 1% antibiotic-antimycotic, 100 nm dexamethasone (Sigma), 50 μm ascorbic acid 2-phosphate (BioChemika), 10 mm b-glycerophosphate (Calbiochem) and 10 ng/ml BMP-2 (PeproTech) as the osteogenic differentiation induction medium. The 3DMN was cultured for 21 days with osteogenic medium replenishment every 2-3 days before co-culture. These 3DMN that had undergone osteogenic differentiation were called 3DON. Healthy 3DON was formed by healthy MSCs while AML 3DON was composed of AML-patient derived MSC. The 3DON was ready for subsequent experiments once completed 21 days of osteogenic differentiation.
To compare the difference between 2D and 3D culture, 2DMN was prepared by seeding 5×103 cells into each well of a 384-well plate, and 5×104 cells into each well of a 96-well plate. The seeded cells were cultured in DMEM-LG for 3 days before co-culture with AML cells.
To observe how different niches affect the viability, apoptosis, and phenotypes of AML cells, co-culture experiments were performed. For the co-culture experiment, there were six co-culture groups, healthy and AML 3DON, 3DMN and 2DMN, the control groups included AML cultured in AML cell line medium (medium (Roswell Park Memorial Institute 1640 medium (RPMI1640) supplemented with 10% FBS and 1% antibiotic-antimycotic) (NC1), MSC medium (NC2), and engineered growth factor cocktail medium for hematopoietic cells (PC). For viability test, apoptosis test, and CD33, CD34 phenotypes test, AML cells were seeded into 96-well plate, each well contained one 3DON/3DMN and each 3DON/3DMN contained 5×104 cells. Specifically, the AML cells in co-culture groups were maintained in 1:1 AML cell line medium:MSC medium as the cell number of AML:MSC were 1:1. The 3DON was differentiated for 21 days before co-culture, 3DMN was formed 3 days before co-culture for the 3DMN to contract and 2DMN was seeded 3 days before co-culture as a fair comparison to the 3DMN. All the groups were seeded with AML cells on the same day. In a 96-well plate, each well was seeded with 5×104 primary AML cells. Trypan blue viability assay on AML cells was performed after three days of co-culture with different niches for the AML cells to interact with the environment. The experiments were performed in triplicates, and a total of three independent experiments were conducted.
Maintaining the phenotype of AML cells in drug screening platform is necessary to ensure valid screening results. Phenotypes of AML cells were analyzed by flow cytometry. For flow cytometry, each well was seeded with 5×104 primary AML cells. Cells were aspirated after 3 days of co-culture and washed. Cells were stained with allophycocyanin-Cy7 (APC/Cy7) or fluorescein isothiocyanate (FITC) isotype control and monoclonal antibodies (CD33-APC/Cy7 (Abcam), an AML myeloid lineage marker and CD34-FITC (Abcam), a hematopoietic stem cell marker) for 30 minutes on ice. The cells were then washed and resuspended in 5% FBS (200 μL) and passed through 70 μm cell strainer and kept on ice before analysis. The samples were analyzed by LSRFortessa (Bdbiosciences) and ten thousand events per sample were recorded using BD FACSDiva software. Gatings were created to exclude dead cells, debris, and doublets such that only live singlets were analyzed.
It is reported that primary AML cells would undergo spontaneous apoptosis rapidly when being cultured ex vivo, therefore, apoptosis detection was performed to analyze whether the in vivo niche could reduce apoptosis [16-18]. Apoptosis was detected using TUNEL assay kit (Abcam). Cells were fixed and stained according to the manufacturer's protocol. In brief, each sample was prepared by pooling primary AML cells from 2 wells (approximately 100000 cells) and were fixed with 1% paraformaldehyde for 15 mins after being cultured for 3 days in different culture conditions. The cells were then washed and transferred to 70% ethanol and incubate for a minimum of 30 minutes at −20° C. Cells were washed again and resuspended in the DNA labelling solution consisting of reaction buffer, TdT enzyme, FITC-dUTP, and ddH2O and incubated for 1 hour at 37° C. Next, cells were counterstained with Propidium Iodide/rNase A. Cells were analyzed by flow cytometer LSRFortessa (Bdbiosciences). The FITC-labelled TUNEL-positive cells were recognized as apoptotic cells. Gatings were created to exclude debris and doublets such that only single cells were analyzed. Ten thousand events per sample were recorded for analysis using BD FACSDiva software.
ALP staining and von Kossa staining was performed to verify successful osteogenic differentiation of the MSCs in 3DON, using 3DMN as the control without differentiation. The 3DON or 3DMN groups were fixed with 4% paraformaldehyde (PFA) to preserve the sample and cut into 10 μm sections. The samples were stained using alkaline Phosphatase Kit (Sigma) following the manufacturer's protocol with nuclear fast red as counter stain. Von Kossa staining was performed to reveal the calcium deposits. Slides were immersed in 1% silver nitrate (Sigma) solution under ultraviolet for 1 hour and then immersed in 2% sodium thiosulphate (Sigma) solution for 3 minutes to remove unreacted silver according to the previous protocol [101].
Immunofluorescence staining of the osteogenic markers was performed to verify successful osteogenic differentiation of the MSCs in 3DON. The fixed frozen sections of samples were permeabilized by adding 0.5% Tween-20 for 10 minutes and washed with PBS 3 times, 5 minutes each. Blocking of nonspecific sites was performed by adding 5% of bovine serum albumin (Sigma) for 30 minutes. Primary antibodies osteopontin (ab8448, abcam) and osteocalcin (ab13420, abcam) were diluted in 1:200 concentration and incubated at 4° C. overnight. After washing, secondary antibody was diluted to 1:200 and incubated at room temperature for 1-hour, washed, and mounted with fluorogel II mounting medium with DAPI (Electron microscopy Sciences).
Live and dead staining was performed on the 3DMN and 3DON to assess the viability of the cells being encapsulated. The live/dead viability kit (L3224, Invitrogen) was used. The samples were washed with PBS thrice and incubated in calcein-AM and ethidium homodimer-1 according to the manufacturer's protocol.
SEM imaging was performed to observe the structure inside the 3DON. The fabricated 3DONs were rinsed with PBS, then fixed with 4% PFA at 4° C. overnight. They were washed with PBS and followed by serial dehydration (10%, 30%, 50%, 70%, 90%, 95%, and 100%, 15 minutes each). Samples were then undergone critical point drying at the Electron Microscopy Unit (the University of Hong Kong). The internal structure of the coprecipitates was revealed by fracture and sputter-coated by gold. Scanning electron microscopy (SEM) images were captured by Hitachi S-4800. The EDX analysis was performed to map the calcium (Ca) and phosphorus (P) weight content in the samples.
For sample drug test, AML cells were cultured in nine different conditions (same as co-culture analysis) in 384 wells for 3 days. For the 3DON/3DMN groups, each well contained one 3DON/3DMN and each 3DON/3DMN contained 5000 cells. For other groups, AML cells of density 5000 cells/well were seeded into each well. On the second day of co-culture, daunorubicin (Sigma) of ten different working concentrations (0-426 UM) and doxorubicin (Sigma) of fifteen different working concentrations (0-2.559 mM) was administered into the culture wells. After 24 hours, AML cells were aspirated into a blank 384 well plate for viability assay using PrestoBlue (Invitrogen) and measured by a fluorescence plate reader (SpectraMax iD3, Molecular Devices) after 3 hours of incubation. The experiments were performed in triplicate, and a total of three independent experiments were conducted.
The cytokine array was used to detect the level of cytokines secreted in different niches. Conditioned medium from 3DON was collected for 3 days post ostoegenic differentiation while that from 3DMN and 2DMN were collected for 3 days post-culture. The collected medium was passed through a 0.2 μm filter and stored at −80° C. Uncultured DMEM-LG full medium was used as a control. The soluble cytokines in the medium were detected by human cytokine antibody array (Abcam). The analysis was performed according to the manufacturer's instructions. Briefly, membranes were blocked in 1× blocking buffer at room temperature for 30 minutes and washed. Undiluted samples were added and incubated overnight at 4° C. and washed. Then, 1× biotin-conjugated anti-cytokines were pipetted onto each membrane and incubated for 2 hours at room temperature and washed. 1×HRP-conjugated streptavidin was then added and incubated for another 2 hours at room temperature and washed. Detection buffer mixture was pipetted onto the membrane and incubated for 2 minutes at room temperature followed by chemiluminescence detection using Azure c300 gel imaging system (Azure Biosystems). Densitometric data were obtained using ImageJ software.
Statistical calculations were performed with GraphPad Prism 9 (GraphPad Software). The statistical analyses were performed by one-way ANOVA followed by Bonferroni's multiple comparisons test, except for viability test, CD33 CD34 phenotype maintenance, cytokine level comparisons, and IC50 comparisons were analysed by non-parametric one-way ANOVA Kruskal-Wallis test followed by Dunn's multiple comparisons test to reveal the statistically significant differences among different groups with a significance level of 0.05. The IC50 values were determined from the dose-response model built-in to Prism.
The primary AML cells were co-cultured with the 3DON for 3 days in different conditions. Primary AML cells were cultured in three control groups, I: negative control 1 (AML medium) (NC1); II: negative control 2 (MSC medium) (NC2); and III: positive control (engineered growth factor cocktail medium for hematopoietic cells) (PC), and co-cultured in three experimental groups: IV: healthy 2D mesenchymal niche (MSC feeder layer) (2DMN); V: healthy 3D mesenchymal niche (3DMN); VI: healthy 3D osteogenic niche (3DON); VII: AML 2DMN; VIII: AML 3DMN; IX: AML 3DON.
The 3DON was produced by encapsulating MSCs in rat-tail type I collagen. The MSC cell density was 50000 cells/ml, collagen concentration 2 mg/ml, volume of 3DON was 100 μL. The culture medium, IN sodium hydroxide, stock collagen and cell suspension were mixed and pipetted into the wells of a 96-well plate, pre-coated with F127 for an hour at room temperature. Osteogenic differentiation of the encapsulated MSCs were performed 3 days after cell encapsulation. The microspheres were cultured in DMEM-LG supplemented with 10% FBS, 100 nM dexamethasone (Sigma), 50 μM ascorbic acid 2-phosphate (BioChemika), 10 mM b-glycerophosphate (Calbiochem) and 10 ng/ml BMP-2 (PeproTech) as the osteogenic differentiation induction medium. The microspheres were cultured for 21 days with regular medium replenishment every 2-3 days before co-culture.
The 3DMN were produced the same way as 3DON except without the 21 days osteogenic differentiation. The 2DMN group was prepared by seeding 50000 cells in each well of the 96-well plate. The medium used for NC1 was RPMI 1640 medium supplemented with 20% FBS and 1% anti-anti. The medium used for NC2 was DMEM-LG medium supplemented with 10% FBS and, 1% GlutaMAX and 1% anti-anti. For the co-culture groups, the medium was a 1:1 mixed medium of NC1 and NC2 medium.
ALP staining and von Kossa staining was performed to verify successful osteogenic differentiation of the MSCs in 3DON, using 3DMN as the control without differentiation. The 3DON or 3DMN groups were fixed with 4% paraformaldehyde (PFA) to preserve the sample and cut into 10 μm sections. The samples were stained using alkaline Phosphatase Kit (Sigma) following the manufacturer's protocol with nuclear fast red as counter stain. Von Kossa staining was performed to reveal the calcium deposits. Slides were immersed in 1% silver nitrate (Sigma) solution under ultraviolet for 1 hour and then immersed in 2% sodium thiosulphate (Sigma) solution for 3 minutes to remove unreacted silver according to the previous protocol [101].
SEM imaging was performed to observe the structure inside the 3DON. The fabricated 3DONs were rinsed with PBS, then fixed with 4% PFA at 4° C. overnight. They were washed with PBS and followed by serial dehydration (10%, 30%, 50%, 70%, 90%, 95%, and 100%, 15 minutes each). Samples were then undergone critical point drying at the Electron Microscopy Unit (the University of Hong Kong). The internal structure of the coprecipitates was revealed by fracture and sputter-coated by gold. Scanning electron microscopy (SEM) images were captured by Hitachi S-4800. The EDX analysis was performed to map the calcium (Ca) and phosphorus (P) weight content in the samples.
Live and dead staining was performed on the 3DMN and 3DON to assess the viability of the cells being encapsulated. The live/dead viability kit (L3224, Invitrogen) was used. The samples were washed with PBS thrice and incubated in calcein-AM and ethidium homodimer-1 according to the manufacturer's protocol.
From
Von Kossa staining reveals calcium deposition as a late-stage osteogenic differentiation marker. After 21 days in osteogenic differentiation (OD) medium, the healthy 3DON were heavily stained black (
Scanning electron microscopy (SEM) revealed the microstructure of 3DON and 3DMN. Low magnification (4 kX) images revealed that the cells (circled) being encapsulated were settling on a collagen fiber meshwork (
The presence of osteogenic markers ostcopontin were confirmed by immunofluorescence staining presented in
The bright field images showing the gross appearance of the healthy and AML 3DON in
Live dead staining in
To analyze the viability of AML cells upon co-culture with different groups, trypan blue exclusion test was conducted after three days of co-culture. The preparation of all the culture groups were described in example 1. Trypan blue counting was performed by aliquoting 10 μL sample of cell suspension from each well for cell counting.
Apoptosis was detected using TUNEL assay kit (Abcam). Cells were fixed and stained according to manufacturer's protocol. In brief, each sample was prepared by pooling primary AML cells from 2 wells (total approximately 100000 cells), and were fixed with 1% paraformaldehyde for 15 mins after being cultured for 3 days in different culture conditions. The cells were then washed and transferred to 70% ethanol. Cells were washed and resuspended in the DNA labelling solution that consist of reaction buffer, TdT enzyme, FITC-dUTP, and ddH2O and incubated for 1 hour at 37° C. Next, cells were counterstained with Propidium Iodide/RNase A. Cells were analyzed by flow cytometer LSRFortessa (Bdbiosciences). The FITC-labelled TUNEL-positive cells were recognized as apoptotic cells. Ten thousand events per sample were recorded using BD FACSDiva software.
For viability, as
To quantify the population of apoptotic cells, fragmented DNA was stained by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay, and
A cytokine array screening of 80 cytokines and growth factors was performed to identify the type and quantity of secreted proteins by the experimental groups, against the NC2 group (uncultured DMEM-LG medium) and the PC group (uncultured engineered growth factor cocktail medium).
The preparation of 3DON were described in example 1. Conditioned medium from 3DON was collected for 3 days post ostoegenic differentiation while that from 3DMN and 2DMN were collected for 3 days post-culture. The collected medium was passed through a 0.2 μm filter and stored at −80° C. Uncultured DMEM-LG full medium was used as a control. The soluble cytokines in the medium were detected by human cytokine antibody array (Abcam). The analysis was performed according to the manufacturer's instructions. Briefly, membranes were blocked in 1× blocking buffer at room temperature for 30 minutes and washed. Undiluted samples were added and incubated overnight at 4° C. and washed. Then, 1× biotin-conjugated anti-cytokines were pipetted onto each membrane and incubated for 2 hours at room temperature and washed. 1×HRP-conjugated streptavidin was then added and incubated for another 2 hours at room temperature and washed. Detection buffer mixture was pipetted onto the membrane and incubated for 2 minutes at room temperature followed by chemiluminescence detection using Azure c300 gel imaging system (Azure Biosystems). Densitometric data were obtained using ImageJ software.
The 10 proteins with the highest fold changes in healthy 3DON were interleukin-8 (IL-8) (37.94), osteoprotegerin (OPG) (35.48), tissue inhibitor of metalloproteinases-2 (TIMP-2) (22.77), monocyte chemoattractant protein-1 (MCP-1) (18.87), interleukin-10 (IL-10) (17.43), growth-related oncogene-α (GRO-α) (16.58), interleukin-6 (IL-6) (15.00), growth-related oncogene (GRO) (14.18), interleukin-7 (IL-7) (12.84), and angiogenin (10.61). In AML 3DON, the 10 highest fold changes were MCP-1 (22.52), IL-8 (21.17), TIMP-2 (18.69), IL-6 (18.35), vascular endothelial growth factor (VEGF) (11.34), epithelial neutrophil-activating peptide 78 (ENA-78) (8.18), GRO (6.25), angiogenin (5.97), macrophage inflammatory protein-3α (MIP-3α) (5.69) and GRO-α (5.65). In AML 3DMN, the 10 proteins with the highest fold changes were IL-6 (46.79), MCP-1 (24.3), TIMP-2 (20.63), IL-8 (19.51), VEGF (11.48), hepatocyte growth factor (HGF) (9.93), Angiogenin (9.01), GRO (8.15), TIMP-1 (6.94), GRO-α (5.96) (Table 1).
In healthy 3DON, there were 64 out of the total 80 in the cytokine array that had a higher fold change compared to PC and most of them had functions related to cell growth, differentiation or migration (
In AML 3DON, which best supports AML growth, there were 23 cytokines that had a higher fold change compared to PC and were mostly related to cell growth, differentiation or migration and also hematopoiesis or angiogenesis-related. (
In AML 3DMN, there were 34 cytokines that had a higher fold change compared to PC and were mostly related to cell growth, differentiation or migration and also hematopoiesis or angiogenesis-related (
The PC group was prepared by pooling the medium from three engineered murine fibroblast cell lines, M210B4+JGCSF+JIL3 HyTK, sl/sl SCF+IL3 HyTK, and sl/sl FLT3L irradiated at 80 Gy, mixed in 1:1:1 ratio for culture. The medium collected after 3 days was the engineered growth factor cocktail medium. The highest fold changes in PC group as compared to the NC2 group (MSC medium) were IL-6 (46.94), MCP-3 (42.34), MCP-2 (32.15), IL-8 (27.93), MIP-3α (25.76), IL-10 (23.06), MCP-1 (17.35), GRO (16.61), ENA-78 (14.37), and OPG (13.70). These cytokines all belong to inflammatory responses except for OPG which is for bone metabolism.
Most of the cytokines that had a lower fold change in AML 3DON and healthy 3DON compared to PC were related to the inflammatory response (
In addition, the engineered cell lines were designed to produce SCF, G-CSF, IL-3, and FLT-3 ligand, and among the detected levels of these four proteins in the medium (1.83-fold, 4.98-fold, 2.16-fold and 1.54-fold respectively), SCF was lower than that released by AML 3DON (2.27-fold), and all were lower than that released by healthy 3DON (2.20-fold, 5.84-fold, 4.00-fold, and 6.90-fold respectively), which may be the reason why the healthy and AML 3DON are better than the PC in supporting the survival of AML cells in vitro.
Besides, the 3DON and 3DMN niche groups, but not the PC group, also involved contact-mode interactions with the AML cells during co-cultures. Research has found that the protective effect on leukemia cells was reduced in a culture system lack of cell-cell contact [39]. AML-collagen interactions such as through integrin α2β1 binding [40], AML-MSC or AML-osteoblast interactions by integrin α4β1 binding would reduce the apoptosis of AML cells, which may be another explanation of the increased viability in the 3DON and 3DMN groups compared to the PC group.
The fold changes of cytokines detected in the conditioned medium of AML 2DMN, AML 3DMN, and AML 3DON were shown in
To further understand how the cytokines present in the PC, healthy 3DON, AML 3DMN, and AML 3DON groups aided the survival of the AML cells, protein annotation through evolutionary relationship gene ontology-slim (PANTHER GO-Slim) analysis was performed. For protein class, the enriched cytokines were mostly belonged to intercellular signal molecules (75%) and protein-binding activity modulators (12%) (
Gene ontology term enrichment analysis (
Only 80 cytokines were assessed in this experiment, there could be more proteins released by the 3DON that were pro-tumoral but not covered by the cytokine chip here. For instance, a study demonstrated that AML cells in fact induce osteogenic differentiation in bone marrow-derived MSCs by secreting BMP-2 to activate Smad1/5 phosphorylation and connective tissue growth factor (CTGF) expression which in turn enhances AML engraftment [14]. It has been demonstrated that BMP-2 was found to be present in the healthy 3DON, therefore BMP-2 may be another factor that associates with in the survival of AML but not included in the cytokine chip [31], Systemic proteomics study is therefore needed in the future to analyze the secretome from the 3DON thoroughly.
The preparation of all the culture groups were described in example 1. To check for any changes in phenotype, cells were aspirated after 3 days of co-culture and washed. Cells were stained with allophycocyanin-Cy7 (APC/Cy7) or fluorescein isothiocyanate (FITC) isotype control and monoclonal antibodies (CD33-APC/Cy7 (Abcam) and CD34-FITC (Abcam)) for 30 minutes on ice. The cells were then washed and resuspended in 200 μl 5% FBS and passed through 70 μm cell strainer and kept on ice before analysis. The samples were analyzed by LSRFortessa (Bdbiosciences) and ten thousand events per sample were recorded using BD FACSDiva software.
To check whether the phenotype of AML cells were maintained in the BM organoid, immunophenotyping by flow cytometry was performed by staining two leukemia associated immunophenotypes (LAIPs), CD33 and CD34. (Kern et al. 2010) The cut-off was defined as follows: “positive” (≥40%), “dimly positive” (20-40%) and “negative” (<20%). (Zeijlemaker, Gratama, and Schuurhuis 2014).
CD33 is a myeloid marker while CD34 is a hematopoietic stem cell marker. According to the French-American-British classification (FAB), myeloid cell differentiation were categorized into seven stages, M0-M7. (Plesa et al. 2008) Stages having both CD33+ and CD34+ cells could be myeloid progenitor cells (M0), monoblast (M4), or immature megakaryoblast (M7). Stages with CD33+ and CD34− cells are myeloid monocytic progenitor cells (M1), myeloblast (M2), promyelocyte (M3), or promonocyte (M5). No stage has CD33− and CD34+ cells. (Plesa et al. 2008) The patient samples used for this experiment were clinically classified as FAB stages M1 and M5. Cell markers involved for both of the stages therefore should be CD33 positive and CD34 negative (CD33+CD34−). (Casasnovas et al. 2003; Khalidi et al. 1998; Plesa et al. 2008).
Representative flow cytometry plots were presented in
For drug screening, AML cells were cultured in six different conditions. The preparation of all the culture groups were described in example 1 except that the 384 well-plate was used instead of 96-well plate, and also AML cells of density 5000 cells/well and 3DON of 10 μl were seeded into each well. On the second day of co-culture, daunorubicin (Sigma) of ten different working concentrations (0-320 μM) was administered into the culture wells. After 24 hours, AML cells were aspirated into a blank 384 well plate for viability assay using PrestoBlue (Invitrogen) and measured by a fluorescence plate reader (SpectraMax iD3, Molecular Devices) after 3 hours of incubation. The IC50 was calculated as a measure of drug potency in different groups. Daunorubicin, which is one of the drugs being used as a standard treatment clinically, was used as the sample drug (Bertoli et al. 2017).
Acute myeloid leukemia (AML) is one of the most prevalent acute leukaemia [1]. It is the most lethal hematopoietic cancer with a 5-year survival rate of around 27% [2,3]. AML has over 24 genetically defined subtypes [4], making it a very heterogeneous disease and hence causing poor prognosis [5,6]. Currently, the standard treatment for AML is a 7+3 regimen (7 days of cytarabine with 3 days of anthracycline such as daunorubicin or doxorubicin) but it only results in 50% overall survival [7]. This is followed by consolidation chemotherapy and hematopoietic stem cell transplantation for some high-risk patients [8]. Relapse caused by chemoresistant leukemic cells occurs in more than 70% of patients [9]. Aged individuals, who are the majority of AML patients, may not tolerate the intensive chemotherapy or respond to the treatments due to their higher risk of complex karyotype and TP53 mutation [5]. With the recent advancement in AML pharmacotherapies, tailor-made treatment for patients, especially the elderly, could be made possible by personalized medicine, with potential benefits such as less side effects and improvement in treatment outcomes.
Bone marrow (BM) is a viscous tissue within the bone cavity responsible for haematopoiesis [10]. The two dynamic bone marrow niches-osteogenic niche (ON) that are made up of osteoblasts, osteocytes, and osteoclasts; and the perivascular niche, which consists of endothelial cells and megakaryocytes, interact to regulate the functions of haematopoietic stem cells (HSCs) [11]. Mesenchymal stromal cell (MSC) is an important cell type as they establish the hematopoietic environment by differentiating into many essential components of the BM niche, and they also affect the proliferation, differentiation, and homing of HSCs [12]. Leukemia cells are genetically mutated from HSCs [13]. In fact, leukemic cells interact with the BM microenvironment by different mechanisms, for example they would activate bone morphogenetic protein 2 (BMP-2) and Smad1/5 signaling in MSCs to induce osteogenic differentiation to build their own favorable niche for supporting leukemogenesis [12,14].
AML cells have prolonged survival in vivo [15]. However, primary AML cells rapidly undergo spontaneous or stress-induced apoptosis in ex vivo cultures, possibly owing to the change in the microenvironment, albeit this phenomenon is highly patient-dependent due to heterogeneity [16-18]. As a result, AML cells in conventional cultures can hardly be used to screen for drugs and therapies, because cell death may not be attributable to the drug being screened and evaluated. This discrepancy between the in vitro and the in vivo behavior of AML cells, in particular, the spontaneous apoptosis and the poor survival of the AML cells in cultures may be attributable to the lack of the pro-tumoral BM microenvironment consisting of soluble factors such as growth factors, soluble adhesion molecules and cell-cell/cell-extracellular matrix (ECM) interactions in native cancer tissues.
Different methods have been developed to improve ex vivo culture of primary AML cells [18], including the development of engineered conditioned medium with stem cell factor (SCF), granulocyte colony-stimulating factor (G-CSF), interleukin-3 (IL-3) [17,20]; liquid tumor models such as 2D co-cultures with MSCs or endothelial cells to mimic the BM niche by secreting cytokines [21,22]; and 3D co-cultures such as MSC-coated ceramic scaffolds [23], collagen I or fibronectin coated polylactic-co-glycolic acid (PLGA) and polyurethane (PU) scaffolds [24], polyethylene glycol (PEG) hydrogels with HUVEC and MSCs [25]. However, the preparation of the engineered conditioned medium is tedious, time-consuming, costly, and inefficient because viral transfection of three different cell lines to over-express three different cytokines is necessary for producing the required medium [26]. Furthermore, the engineered conditioned medium and the 2D co-culture environment failed in mimicking the cell-cell or cell-ECM contacts, whereas the existing 3D cultures in a wide range of materials, although being more physiologically relevant, they are yet to recapitulate the pro-tumoral osteogenic niche [18]. In addition, studies have found AML-derived MSCs are different from healthy donors' MSCs in terms of differentiation ability and hematopoiesis support ability [27] [28], however, a leukemia-derived osteogenic niche 3D model is lacking for AML studies.
To address the inadequacy of the existing AML drug screening platforms, reconstituting biomimetic 3D osteogenic niche (3DON) for AML culture is warranted. Progress is underway in developing a 3D microencapsulation platform using naturally occurring extracellular matrix to fabricate physiologically relevant and ECM-based 3D microtissues including those derived from stem cells such as chondrogenic and osteogenic organoids, mature cells such as nucleus pulposus cells and cancer cells such as neuroblastoma cells [33]. Here, it was hypothesized that reconstituting a leukemic 3DON through BM MSC-derived osteogenic organoids, will support the survival and phenotype maintenance of primary AML cells upon co-culture, therefore providing a physiologically relevant in vitro model for personalized chemotherapy drug screening. Moreover, MSCs derived from both healthy individuals and AML patients will be used for forming the bone marrow niches to investigate their differences in supporting AML survival for drug screening applications.
Results demonstrated that the reconstituted AML 3D BM niches derived from AML patients provided the optimal niche to support the primary AML in cultures. Specifically, the viability of AML cells co-cultured with the AML 3DMN and AML 3DON groups were the highest, while the percentages of apoptotic cells in them were the lowest. Cytokine array analysis revealed that the enhanced secretion of anti-apoptotic cytokines such as IL-8, GROα and OPG in the AML 3DMN and AML 3DON groups. In addition, the AML phenotype of CD33+ and CD34-were nicely preserved in these groups. The intrinsically high chemoresistance of AML was well retained by the AML niches (2DMN, 3DMN, and 3DON groups) as compared to the healthy niches and other control groups, using daunorubicin and doxorubicin as examples, suggesting the physiological resemblance of the AML niches and the necessity to reconstitute AML BM niches for valid drug screening. Moreover, the healthy BM niches (2DMN, 3DMN, and 3DON), while equally supporting the AML cell survival and phenotype maintenance in vitro, showed increased drug sensitivity to the two sample drugs used, indicating that reconstituting a healthy BM niche in leukemic patients may overcome the intrinsic chemoresistance problem in leukemia patients. This work contributes to the application of the AML 3DON in personalized drug screening and suggests the potential of using the healthy 3DON as a chemotherapeutic adjuvant for better AML chemotherapies with enhanced drug sensitivity.
It is understood that the disclosed method and compositions are not limited to the particular methodology, protocols, and reagents described as these can vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims.
“Optional” or “optionally” means that the subsequently described event, circumstance, or material may or may not occur or be present, and that the description includes instances where the event, circumstance, or material occurs or is present and instances where it does not occur or is not present.
Unless the context clearly indicates otherwise, use of the word “can” indicates an option or capability of the object or condition referred to. Generally, use of “can” in this way is meant to positively state the option or capability while also leaving open that the option or capability could be absent in other forms or embodiments of the object or condition referred to. Unless the context clearly indicates otherwise, use of the word “may” indicates an option or capability of the object or condition referred to. Generally, use of “may” in this way is meant to positively state the option or capability while also leaving open that the option or capability could be absent in other forms or embodiments of the object or condition referred to. Unless the context clearly indicates otherwise, use of “may” herein does not refer to an unknown or doubtful feature of an object or condition.
Ranges can be expressed herein as from “about” one particular value, and/or to “about” another particular value. When such a range is expressed, also specifically contemplated and considered disclosed is the range from the one particular value and/or to the other particular value unless the context specifically indicates otherwise. Similarly, when values are expressed as approximations, by use of the antecedent “about,” it will be understood that the particular value forms another, specifically contemplated embodiment that should be considered disclosed unless the context specifically indicates otherwise. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint unless the context specifically indicates otherwise. It should be understood that all of the individual values and sub-ranges of values contained within an explicitly disclosed range are also specifically contemplated and should be considered disclosed unless the context specifically indicates otherwise. Finally, it should be understood that all ranges refer both to the recited range as a range and as a collection of individual numbers from and including the first endpoint to and including the second endpoint. In the latter case, it should be understood that any of the individual numbers can be selected as one form of the quantity, value, or feature to which the range refers. In this way, a range describes a set of numbers or values from and including the first endpoint to and including the second endpoint from which a single member of the set (i.e. a single number) can be selected as the quantity, value, or feature to which the range refers. The foregoing applies regardless of whether in particular cases some or all of these embodiments are explicitly disclosed.
Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of skill in the art to which the disclosed method and compositions belong. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present method and compositions, the particularly useful methods, devices, and materials are as described. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. No admission is made that any reference constitutes prior art. The discussion of references states what their authors assert, and applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of publications are referred to herein, such reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art.
Those skilled in the art will recognize or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the method and compositions described herein. Such equivalents are intended to be encompassed by the following claims.
This application claims the benefit of and priority to U.S. Provisional Application No. 63/467,291 filed May 17, 2023, the entire content of which is incorporated herein by reference for all purpose in its entirety.
| Number | Date | Country | |
|---|---|---|---|
| 63467291 | May 2023 | US |
