Organoid culture of bone marrow with expansion of constituent normal and malignant hematopoietic cells

Abstract

Compositions and methods are provided for the culture of functional bone marrow, allowing analysis of the responsiveness to therapeutic agents, and for studying the interactions of cells in the bone marrow niche.

Description

ORGANOID CULTURE OF BONE MARROW WITH EXPANSION OF CONSTITUENTNORMAL AND MALIGNANT HEMATOPOIETIC CELLSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Pursuant to 35 U.S.C. § 1 19 (e), this application claims priority to the filing date of United States Provisional Patent Application Serial No. 63 / 543,257, filed October 9, 2023, the disclosure of which application is herein incorporated by reference.BACKGROUND

[0002] New blood cells are generated by bone marrow (BM) hematopoiesis, as orchestrated by extrinsically acting local niche signals and systemic growth factors. To ensure blood homeostasis, hematopoietic stem cells (HSC) self-renew and generate mature progeny via differentiation to multilineage progenitors that produce lineage-committed precursors. These precursors then differentiate to erythrocytes which transport oxygen, leukocytes which enable host defense and platelets which mediate coagulation. Approximately 200 x 109erythrocytes, 100 x 109leukocytes and 100 x 109platelets are produced in adults every day, but are shortlived. This continuous self-renewal and differentiation is activated or accelerated by pathology such as infection or blood loss, and regulated by lineage-specific cytokines.

[0003] HSCs and progenitors reside in the bone marrow microenvironment, whose specialized cell types form distinct microenvironmental niches that regulate HSC self-renewal versus differentiation, and provide relative hypoxia. Bone marrow niche cells, including mesenchymal stem cells (MSCs), endothelial cells (ECs), megakaryocytes (MKs) and osteoblasts (OBs), regulate HSCs by secreted factors, or direct cellular interactions. Bone marrow niche cells produce SCF, TGF|3, IL-6 and IL-7 that govern HSC proliferation and differentiation. Bone marrow capillary ECs express SCF and CXCL12 to regulate HSC maintenance, self-renewal, and proliferation. Mature megakaryocytes, proximal to HSCs, secrete factors regulating HSC quiescence or stress-induced proliferation. Also, MSCs, ECs, and osteoblast progenitors support HSC and progenitors in vitro, suggesting similar in vivo functions.

[0004] In leukemia, an accumulation of immature, abnormal BM progenitors proliferate independently of physiologic blood cell demand and exhibit impaired differentiation. Leukemogenesis is ‘evolutionary’ where genetic and epigenetic aberrations accumulate in a pre-leukemic cell, and subsequent hits confers significant heterogeneity. Leukemia is classified (WHO, FAB) by rapidity of progression (acute versus chronic) and affected hematopoietic lineage (myeloid or lymphoid). Acute leukemia is embodied by highly proliferative progenitors with impaired differentiation, versus slow onset and mature progenitors in chronic leukemia. Acute myeloid leukemia (AML) is the most common adultacute leukemia, with median onset at -70 years, >80000 yearly deaths globally, and poor 5- year survival of 30.5%, while acute lymphoid leukemia (ALL) is the most common childhood cancer with incidence of 1 / 20,000 children.

[0005] In addition to cell-intrinsic mutations, the microenvironment also exerts an essential role during leukemogenesis. Like healthy HSCs and progenitors, leukemic cells reside in the bone marrow microenvironment and specifically and interact with niche cells, receiving essential soluble factor signals and cell-cell interactions. By similar homing and anchoring signals, leukemia profoundly alters bone marrow stroma, impairing angiogenesis, inducing bone loss and creating a niche that interferes with HSC / progenitor function.

[0006] The bone marrow microenvironment exerts essential roles during leukemia initiation and progression. Further, the leukemic niche vitally impacts immune recognition and therapeutic response, and facilitates resistance to chemotherapy and targeted therapies. This has raised a pressing need for in vitro model systems that model leukemia holistically in its native bone marrow microenvironment. Chemically-defined 2D cultures to expand murine and human HSC are notably devoid of trilineage differentiation. Further, in vitro culture of primary leukemic blasts has been historically difficult, with typical duration of days to isolated reports of a few weeks.

[0007] Bone marrow model systems are of interest for understanding the interactions between cells, for production of blood cells, and for analysis of hematologic cancers such as leukemias and myelomas. A suitable model would be useful for screening of therapeutics, including agents to modify hematopoietic stem cells, and cancer therapeutics. The present disclosure addresses this need.SUMMARY

[0008] An in vitro adult stem cell bone marrow niche organoid model is provided, which organoids comprise functional hematopoietic stem cells, functional mesenchymal stem cells, stromal cells, bone cells, and intact trabecular bone architecture. The native bone architecture provides the structure for the cells of the niche to main viability and physiologic interactions. Intact bone fragments are grown within an air liquid interface transwell system. These intact bone fragments provide a environment composed of cells that locally act by producing growth factors that maintain mesenchymal and hematopoietic stem cells long term and that provide an adequate niche for continuous hematopoietic differentiation of normal blood progenitor cells into cells such as lymphocytes, erythrocytes, megakaryocytes, mature myeloid cells, e.g. neutrophils, etc. The cultures maintain functional bone marrow populations for extended periods of time, e.g. for up to 30 days, up to 45 days, up to 60 days, up to 90 days, or more.

[0009] In some embodiments the organoid is derived from normal human or mouse bone and bone marrow. In some embodiments the organoid is derived from an individual with ahematologic cancer, e.g. multiple myeloma, ALL, AML, etc. In other embodiments, the organoid is derived from a xenograft tissue, e.g. where a human hematologic malignancy is grown in an animal model. In further embodiments, cells from a human hematologic malignancy are combined in culture with an organoid from normal (non-transformed) individual.

[0010] In some embodiments, a media composition is provided that maintains functional hematopoietic, mesenchymal, endothelial, and stromal cells over extended periods of time. This allows the cells to be adequately nourished, so that paracrine interactions between the cells can maintain viability and differentiation properties. The medium may comprise, for example, an effective dose of an SCF agent, an EGF agent, an FGF agent, a TRO agent; an SCF agent, and a pyrimidoindole derivative, e.g. UM729. Optionally an EPO agent is included. Optionally one or more of an IL-7, IL-3 and a FLT3 ligand is included. Optionally PCL-PVAc- PEG is included. The factors are chosen to be suitable for the species of the tissue, e.g. human factors for human bone marrow, mouse factors for mouse bone marrow, etc.

[0011] In some embodiments the organoid model is used to test therapeutic agents for efficacy against a patient-derived hematologic malignancy. Therapeutic agents of interest include targeted agents, e.g. kinase inhibitors, venetoclax, ruxolitinib, etc., antibodies and cells specific for tumor antigens, e.g. anti-CD19, etc., and non-targeted agents, e.g. chemotherapeutic drugs, etc. Multiple agents can be tested to determine optimal efficacy against a cancer of interest, e.g. a patient-derived xenograft leukemia model, patient-derived leukemia biopsies, patient-derived multiple myeloma biopsies, and the like. Various methods are useful in determining the effectiveness of an agent, and for the specificity in killing cancer cells, for example using flow cytometry, live imaging, and the like. Parameters of interest include cell death, and phenotypic changes in the cancer cell and / or normal populations. Testing patient biopsy samples allows selection of a therapeutic agent for treatment of the patient.

[0012] In some embodiments, a culture comprising a bone and bone marrow sample, e.g. a patient-derived organoid, comprising functional hematopoietic stem cells, functional mesenchymal stem cells, stromal cells, bone cells, and intact trabecular bone architecture, is contacted with an effective dose of a cancer therapy, for example a targeted therapy specific for hematologic cancer cells. The response of may be monitored by determining viability of cancer cells, expression of biomarkers, including secreted factors; by phenotyping, e.g. by determining transcriptional responses, cell-surface phenotypes, and the like. Additional treatments may be used in comparison, or in combination therapy, in a personalized medicine approach. In some embodiments, the cells are labeled with a detectable marker, e.g. a fluorescent marker, an isotope tag, etc. to allow visualization by flow cytometry or live imaging following contacting the culture with a cancer therapy.

[0013] A feature of the bone marrow niche organoid model is the ability to maintain and produce functional stem cells, e.g. HSC and MSC. For example, long term-HSC are produced in the culture model. Differentiated hematopoietic cells are produced in the culture, including progenitor cells such as CFU-G, granulocyte colony-forming unit; CFU-M, monocyte colonyforming unit; CFU-GM, granulocyte-monocyte colony-forming unit; CFU-GEMM, granulocyte- erythrocyte-monocyte-megakaryocyte colony-forming unit; BFU-E, burst forming unit erythrocytes. These progenitor cells can also give rise to differentiated effector cells, e.g. by producing de novo neutrophils.BRIEF DESCRIPTION OF THE DRAWINGS

[0014] The invention is best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings are not to-scale. On the contrary, the dimensions of the various features are arbitrarily expanded or reduced for clarity. Included in the drawings are the following figures.

[0015] FIGS. 1 A-1 G. Normal murine air-liquid interface (ALI) bone marrow organoids (mBMO). (a) Schematic, (b) Overview (upper) and zoomed (lower) of identical brightfield view of mBMO culture, d1 versus d19. (c) Brightfield imaging of mBMO, d1 -25. from 5 C57 / BL6 mice, (d) Flow cytometry (left) and histology (right), mBMO d47 with hematopoietic progenitors (HPC), HSC and progenitors (HSPC), long-term HSC (LT-HSC), erythroblasts, megakaryocytes, and myeloid cells with mature murine neutrophils with characteristic ring chromatin, (e) Total mBMO cells, (f) Colony forming unit (CFU) assays from 50,000 mBMO- derived cells, (g) Total # of BMO CFUs, granulocyte (CFU-G), monocyte (CFU-M), granulocyte-macrophage (CFU-GM), granulocyte-erythrocyte-monocyte-megakaryocyte (CFU-GEMM), erythrocyte (BFU-E). Two-way ANOVA,‘ p<0.05, **** p<0.0001 .

[0016] FIGS. 2A-2D. mBMO preserve diverse stromal populations, (a) Quantification of flow cytometry of mBMO stroma with endothelium (CD31 +), MSC (LEPR+ and / or CD105+), fibroblasts (PDGFRA+), osteoblasts (CD51 +). (b) BMO (d35) immunostained for vimentin (stroma), CD45 (hematopoietic) and CD31 (endothelium), (c) MSCs were harvested from BMO at d42, and differentiated to adipocytes, osteocytes and chondrocytes, (d) BMO secrete numerous cytokines in culture supernatants, d20-130, Luminex, Iog2 fold change.

[0017] FIGS 3A-3C. BMO-derived cells rescue lethally irradiated C57 / BL6 recipient mice. Cells from 3 week-old BMO (Ly5.2) were transplanted into lethally irradiated Ly5.1 recipient mice, (a) Survival of 4 BMO-transplanted mice at day 112 vs demise at day 12 without transplant, (b) Hemoglobin (Hb), platelets and white blood cells (WBC) in peripheral blood (PB) after transplant vs control animals (lethally irradiated and transplanted with naive donor unirradiated bone marrow cells and PB support) from (a), d84 after transplantation, (c)Analysis of (a) for % lineage contribution in PB of Ly5.2(+) B, T and myeloid cells from mBMO- transplanted cells.

[0018] FIGS. 4A-4D. Normal human ALI BMO. A. Strategy 1 : Direct culture of normal human bone marrow biopsies in ALI with schematic and implementation, where (B) normal human ALI BMO from a core biopsy of a healthy individual shows outgrowth of hematopoietic cells and phenotypic HSCs and HPCs (culture day 34). Flow cytometry analysis shows the presence of stromal cells. The hematopoietic component includes HSC, and progenitor cells (HPC). C. Schematic illustration of strategy 2, where human HSPCs are cultures in mouse bone marrow. D. Results of strategy 1 human bone cultures at days 1 and 27, showing D27 retention of erythroid, myeloid, lymphoid, progenitor and stem cell components.

[0019] FIGS. 5A-5C. Human leukemia PDX bone marrow organoids, (a) Organoids from an adult AML PDX model show outgrowth of leukemic blasts in bright field (day 32) and FACS (day 42). Exome-seq and mutant allele frequency (MAF) showed maintenance of the major driver mutations (right, day 29). (b) Organoids (day 57) from a pediatric AML PDX show leukemic blast outgrowth in brightfield, histology and FACS, conserved copy number variation profile (whole genome sequencing, arrows = deletions in chromosomes 19 and 20) and day 50 organoid re-engraftment of a naive NSG mouse with human CD45 (hCD45) dominance in mouse peripheral blood.

[0020] FIG. 6A-H. BMO from patient-derived acute leukemia core biopsies, (a-g) Leukemia BMO from bone marrow core biopsies from a pediatric AML (a-d) and two different B-ALL patients (e-g) and (h). Bright field (a, e), flow cytometry (b, f), cytosmears (c, g), immunostaining (d) and copy number profile (h). BMO maintain leukemic blast phenotype and RAS4'467mutation (b-c, f-h). Also present are normal hematopoiesis (b) and leukemic niche stroma and vascular structures (d).

[0021] FIGS. 7A-B. In vitro AML BMO treatment with ruxolitinib and venetoclax. Trabecular bone fragments of n = 3 AML PDXs, NPM1 mutant (a) or EVI1 rearranged (b) were cultured as 24 well ALI BMO + / - venetoclax (Ven), ruxolitinib (Rux) or both for 14 days. Flow cytometry was gated on original leukemia immunophenotype and viable cells (red boxes). Bar graphs show % leukemic blasts per all events vs. DMSO. Note AML eradication in green boxed conditions, Two-way ANOVA, * p<0.05, *** p<0.001.

[0022] FIGS 8A-8B. Media optimization in mBMO. (a) 6-week-old mBMO (Ly5.2+) cultured in base media (BMOM) + / - EPO (1 U / ml) and IL7 (10 ng / ml) were transplanted in a Ly5.1 mouse. Survival analysis of the first 84 days, (b) Peripheral blood hemoglobin (Hb) at week 12 after transplantation corresponding to erythropoiesis, showing individual biological replicate cultures, (c) Single cells (50,000) from mBMO cultured with BMOM, high TPO (100 ng / ml), or with EPO / IL7, were plated in methylcellulose for CFU assay and counted after 10-14 days.Left: representative brightfield of end-point colonies. Right: quantification (n=4 biological replicates) of CFU subtypes. Two-way ANOVA, *p<0.05, “ p<0.01 .

[0023] FIGS. 9A-9D, A. trabecular bone fragments from C57 / BL6 mice (femur heads, tibia plateau and sternum combined) were cultured in porcine-derived collagen type 1 A in an airliquid-interface under hypoxic conditions for 47 days. Single cells were harvested at day 47 of culture from the bone marrow organoids and stained with FVS780, CD45, CD1 17 (cKit), Seal , CD48, CD150, CD71 (erytroid progenitors), Ter119 (erytroid cells), CD19 (B cells), B220 (B cells), CD3 (T cells), Gr-1 (myeloid cells) within multiple panels to stain both hematopoietic stem- and progenitor cells and differentiated hematopoietic cells. After 47 days of culture, flow cytometric analysis show the maintenance of hematopoietic stem- and progenitor cells in these cultures. Also multilineage differentiation was confirmed by surface marker expression. B, Quantification of the flow cytometry panels described in a. Figure shows the percentage of subpopulations within all live events. C. Before staining, a cell smear was created of part of the single cell suspension and stained with May-Grunwald Giemsa to show phenotypic multilineage differentiation within the bone marrow organoids. D. Cartoon showing the differences between human and murine neutrophil differentiation to emphasize that after 47 days of culture bone marrow organoids still produce mature neutrophils (which are very shortlived cells) without any addition of myeloid growth factors.

[0024] FIGS. 10A-10D. A. Trabecular bone fragments from C57 / BL6 mice (femur heads, tibia plateau and sternum combined) were cultured in porcine-derived collagen type 1 A in an airliquid-interface under hypoxic conditions for 47 days. Single cells were harvested at day 47 of culture from the bone marrow organoids and stained with FVS780, lineage cocktail, CD45, CD1 17 (cKit), Seal , CD48, CD150 to be able to quantify hematopoietic stem- and progenitor cells. After 47 days of culture, flow cytometric analysis show the maintenance of hematopoietic stem- and progenitor cells in these cultures. B. Single cells suspensions derived from bone marrow organoid cultures were plated in methylcellulose to start a colony-forming unit assay (according to manufacturers protocol). In this assay, every progenitor cell or stem cell makes one colony. 50.000 single cells were plated in the CFU assay and colony forming units were counted after 10-14 days of growth. C. Quantification of CFUs was performed by both manual count and by Image J software and plotted per CFU type. CFU-G, granulocyte colony-forming unit; CFU-M, monocyte colony-forming unit; CFU-GM, granulocyte-monocyte colony-forming unit; CFU-GEMM, granulocyte-erythrocyte-monocyte-megakaryocyte colony-forming unit; BFU-E, burst forming unit erythrocyte. D. Quantification of total CFU per timepoint of culture shows a significant enrichment of stem- and progenitor cells during bone marrow organoid culture. Statistical differences between groups were tested with a two-way ANOVA, * p<0.05.

[0025] FIGS. 1 1 A-1 1 B. A. Bone marrow organoids were stained with live surface marker- directed antibodies (designed for flow cytometry) overnight within their collagen matrix andsubsequently washed with culture medium for >3 times by letting medium pass through the collagen matrix. Bone marrow organoids were stained with CD31 (endothelial cells), CD45 (hematopoietic cells), CD105 (subpopulation of MSCs), CD11 b (myeloid cells), and / or CD90 (subpopulation of MSCs and fibroblasts, but also hematopoietic progenitors). Organoids were subsequently imaged using a Zeiss LSM900 confocal microscope after which Z-stacked images were analyzed using Imaris software. Images are representative images of multiple organoids from multiple mice on day 8, day 21 and day 28 of organoid culture. 6B. Bone marrow organoids were stained as described above after which Z-stacked imaging was performed overnight in an environmental chamber with 5% CO2 at 37 degrees Celsius. Representative live imaging shows living vascular structures and interaction between stromal cells and hematopoietic cells within bone marrow organoids.

[0026] FIGS. 12A-12G. A. Patient-derived leukemic blasts from a pediatric patient with an AML with a Evil rearrangement (t2;3) were transplanted and engrafted in immunocompromised mice (NSG). After engraftment, mice were held until disease symptoms appeared in order to mimick the equivalent of a full blown leukemia. Trabecular bone fragments (femur heads, tibia plateau and sternum) from these mice were cultured in porcine- derived collagen 1 A in an air-liquid-interface under hypoxic conditions. Brightfield imaging of the entire insert (12mm) was performed with a Keyance brightfield microscope at day 57 after initiation of cultures. B. Single cells harvested from the leukemia bone marrow organoids were used to make a cell smear on a glass slide. Cells were fixed with ice-cold 100% methanol and subsequently stained with May-Grunwald and Giemsa to show phenotypic blasts within the leukemia bone marrow organoids. C. Flow cytometric analysis of the single cells harvested from the leukemia bone marrow organoids were stained with moiuse CD45, human CD45, and human CD34 antibodies. We confirmed that the leukemia-associated immune phenotype (LAIP) of the patient was similar to the LAIP of the organoid-derived cells. D. Single cells derived from 50 day-old leukemia bone marrow organoids were used to inject a sublethally irradiated NSG mouse to show leukemic potential after culture. We show the engraftment and outgrowth of leukemia within this mouse. E. We studied the LAIP described in (c) over time, and show long-term persistence of phenotypical leukemic blasts in multiple animals (n=4) in leukemic niche organoids, even after engraftment in a new animal. F. Trabecular bone fragments of n = 3 PDXs were cultured in a 24 wells format using 12 mm inserts. This allowed the study of targeted therapy. We treated organoids for 14 days with the drugs venetoclax (a BCL2 inhibitor used in multiple phase 1 / 2 trials for primary, refractory or relapsed AML) and Ruxolitinib (a JAK1 / 2 inhibitor). Human CD45+ population was unaffected by two concentrations of Ruxolitinib, which was expected since this leukemia does not have an activating mutation in the JAK / STAT pathway. In contrast, Venetoclax irradicated human leukemic blasts in clinically relevant concentrations (serum levels of Venetoclax in patientscan reach 1000 nM). G. Relative amount of leukemic blasts per all events compared to the DMSO control condition. Statistical differences between groups were tested with a two-way ANOVA, *” p<0.001.

[0027] FIGS. 13A-13G. A. A trabecular bone biopsy (core) was obtained from a pediatric patient with an inversion-16 (inv16) positive AML at diagnosis. The core biopsy was cut into small trabecular bone fragments and cultured in the above described ALI system in porcine- derived collagen 1 A in under hypoxic conditions. Brightf ield imaging of the entire insert (12mm) was performed with a Keyance brightfield microscope at day 10 and day 31 after initiation of cultures. B-C. After 18 days one of the inserts was sacrificed to perform flow cytometry. Single cells harvested from the leukemia bone marrow organoids were stained with FVS780 (live / dead), CD45, CD3, CD19, CD38, and CD34 antibodies. We show presence of both stromal and hematopoietic cells and show that the hematopoietic population mainly consists of CD3 negative, CD19 negative, CD38 positive, partially CD34 positive leukemic blasts. Next to this leukemic population, we also see the presence of healthy B cells and T cells. D. After 31 days of culture, bone marrow organoids were fixed with 4% paraformaldehyde and permeabilized within their collagen matrix and subsequently stained with primary antibodies against CD45 (hematopoietic cells), Vimentin (stromal cells) and CD31 (endothelial cells). Nuclei were stained with DAPI. After 35 days of organoid culture, we confirm the presence of leukemic cells, vascular structures and stromal cells and their intact 3-dimensional architecture. E. A trabecular bone biopsy (core) was obtained from a pediatric patient with a B cell acute lymphoblastic leukemia (B-ALL) at diagnosis. The core biopsy was cut into small trabecular bone fragments and cultured in the above described ALI system in porcine-derived collagen 1 A in under hypoxic conditions. Brightfield imaging of the entire insert (12mm) was performed with a Keyance brightfield microscope at day 11 and day 24 after initiation of cultures. F. After 24 days single cells harvested from the primary leukemia bone marrow organoids and stained with FVS780 (live / dead), CD45, CD19, and CD34 antibodies. This patient’s blasts were CD45 negative, CD19 positive, CD34 positive at diagnosis. We show the preservation of this LAIP in our organoid cultures. G. The same cell suspension (as f.) was used to make a cell smear on a glass slide. Cells were fixed with ice-cold 100% methanol and subsequently stained with May-Grunwald and Giemsa to show phenotypic lymphoid blasts within the leukemia bone marrow organoids.

[0028] FIGS. 14A-14H. A. A trabecular bone biopsy (core) was obtained from adult patient with multiple myeloma (MM) at diagnosis. The core biopsy was cut into small trabecular bone fragments and cultured in the above described ALI system in porcine-derived collagen 1 A in under hypoxic conditions. Brightfield imaging of the entire insert (12mm) was performed with a Keyance brightfield microscope at day 6 and day 20 after initiation of cultures and zoomed in for increased detail. B. After 26 days single cells harvested from the primary MM bonemarrow organoids and stained with FVS780 (live / dead), CD45, CD19, CD3, CD38, and CD138 antibodies. This patient’s blasts were CD45, CD19, CD3 negative, and CD18 CD138 positive at diagnosis. We show the preservation of this LAIP in our organoid cultures. C. A trabecular bone biopsy (core) was obtained from adult patient with Waldenstrom Macroglobulinemia (WM) at diagnosis. The core biopsy was cut into small trabecular bone fragments and cultured in the above described ALI system in BME2 under hypoxic conditions. Brightfield imaging of the entire insert (12mm) was performed with a Keyance brightfield microscope at day 1 , day 8, and day 36 after initiation of cultures and zoomed in for increased detail. D. After 36 days single cells harvested from the primary WM bone marrow organoids and stained with FVS780 (live / dead), CD45, CD19, CD10, CD38, and IgM antibodies. This patient’s blasts were CD45, CD19, CD38, IgM positive at diagnosis. We show the preservation of this LAIP in our organoid cultures. E. Patient-derived MM bone marrow organoids were stained with live surface marker- directed antibodies (designed for flow cytometry) overnight within their collagen matrix and subsequently washed with culture medium for >3 times by letting medium pass through the collagen matrix. Bone marrow organoids were stained with CD38 (MM marker), CD45 (hematopoietic cells), and CD105 (subpopulation of MSCs). Organoids were subsequently imaged using a Zeiss LSM900 confocal microscope after which Z-stacked images were analyzed using Imaris software, e. shows a 360 degrees turn of the Z stack to show the 3D structure of the organoid. F. Live imaging of the MM blasts within their microenvironment overnight. G. Patient-derived WM bone marrow organoids were stained with TMRM (a dye accumulating within mitochondria without affecting cell survival and only fluorescing when a cell is alive) and NucDeadRed-647 (a dye staining DNA and hence dead cells). These dyes were added to the medium 3 hours before start op imaging with a LSM900 confocal microscope. H. Live imaging of living WM blasts (Z-stacked) and other living cells within the WM microenvironment overnight.

[0029] FIGS. 15A-15B. A. Normal human trabecular bone fragments were obtained during a diagnostic procedure from a child suffering from lymphoma without any bone marrow involvement. B. Trabecular bone fragments were cultured in porcine-derived collagen type 1 A in an air-liquid-interface under hypoxic conditions for 34 days. Single cells were harvested at day 34 of culture from the bone marrow organoids and stained with FVS780, CD45, lineage cocktail, CD38 and CD34. After 34 days of culture, flow cytometric analysis show the maintenance of hematopoietic stem (CD34+ CD38 -) and progenitor cells (CD34+, CD38+) in these cultures.DETAILED DESCRIPTION

[0030] Before the present methods and compositions are described, it is to be understood that this invention is not limited to particular method or composition described, as such may,of course, 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 be limiting, since the scope of the present invention will be limited only by the appended claims.

[0031] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0032] Unless defined otherwise, 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. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It is understood that the present disclosure supercedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0033] It must be noted that as used herein and in the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" includes a plurality of such cells and reference to "the peptide" includes reference to one or more peptides and equivalents thereof, e.g. polypeptides, known to those skilled in the art, and so forth.

[0034] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0035] The term "cell culture" or "culture" means the maintenance of cells in an artificial, in vitro environment. It is to be understood, however, that the term "cell culture" is a generic term and may be used to encompass the cultivation not only of individual cells, but also of tissues or organs.

[0036] The term “culture system" is used herein to refer to the culture conditions in which explants are grown that promote prolonged tissue expansion with proliferation, multilineage differentiation and recapitulation of cellular and tissue ultrastructure.

[0037] “Gel substrate”, as used herein has the conventional meaning of a semi-solid extracellular matrix. Gel described here in includes without limitations, collagen gel, matrigel, extracellular matrix proteins, fibronectin, collagen in various combinations with one or more of laminin, entactin (nidogen), fibronectin, and heparin sulfate; human placental extracellular matrix.

[0038] An “air-liquid interface" is the interface to which the explant cells are exposed to in the cultures described herein. The primary tissue may be mixed with a gel solution, e.g. a collagen gel, which is then poured over a layer of gel formed in a container with a lower semi-permeable support, e.g. a membrane. This container is placed in an outer container that contains the medium such that the gel containing the tissue in not submerged in the medium. The primary tissue is exposed to air from the top and to liquid medium from the bottom, see for example US Patent no. 9,464,275 herein specifically incorporated by reference.

[0039] By "containet1’ is meant a glass, plastic, or metal vessel that can provide an aseptic environment for culturing cells.

[0040] The term “sample” with reference to a patient encompasses solid tissue samples such as a biopsy specimen or cells derived therefrom and the progeny thereof. The term also encompasses samples that have been manipulated in any way after their procurement, such as by treatment with reagents; washed; or enrichment for certain cell populations, such as diseased cells. The definition also includes samples that have been enriched for particular types of molecules, e.g., nucleic acids, polypeptides, etc. The term “biological sample” encompasses a clinical sample, and also includes tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, and the like.

[0041] The term “explanf is used herein to mean a piece of tumor tissue, and the immune and stromal cells present in that tissue; and the cells thereof originating from the tumor tissue that is cultured in vitro, for example according to the methods of the invention. The tissue from which the explant is derived is obtained from an individual.

[0042] The term “organoid' is used herein to mean a 3-dimensional growth of tumor tissue in culture that retains characteristics of the tumor in vivo, e.g. recapitulation of cellular and tissue ultrastructure, immune cell interactions, etc.

[0043] As used herein, the term “immune cell” includes cells that are of hematopoietic origin and that play a role in the immune response. Immune cells include lymphocytes, such as B cells and T cells; natural killer cells; dendritic cells; myeloid cells, such as monocytes, myeloid cells, eosinophils, mast cells, basophils, and granulocytes.

[0044] Methods are provided for the culture of small amounts of clinical specimens. Samples of interest include human tissue, e.g. solid tumor microbiopsy samples such as needle or fine needle aspirate. Samples may be taken at a single timepoint, or may be taken at multiple timepoints. Samples may be as small as 107cells, 106cells, 105cells, or less.

[0045] The phrase “mammalian cells” means cells originating from mammalian tissue. Typically, in the methods of the invention pieces of tissue are obtained surgically, e.g. biopsy, needle biopsy, etc. and minced to a size less than about 1 mm3, and may be less than about 0.5 mm3, or less than about 0.1 mm3. “Mammalian” used herein includes human, equine, bovine, porcine, canine, feline, rodent, e.g. mice, rats, hamster, primate, etc. “Mammalian tissue cells” and “primary cells” have been used interchangeably.

[0046] “Ultrastructure” refers to the three-dimensional structure of a cell or tissue observed in vivo. For example, the ultrastructure of a cell may be its polarity or its morphology in vivo, while the ultrastructure of a tissue would be the arrangement of different cell types relative to one another within a tissue.

[0047] The term “candidate cells” refers to any type of cell that can be placed in co-culture with the tissue explants described herein. Candidate cells include without limitations, genetically engineered T cells including without limitation CAR-T cells, dendritic cells, phagocytic cells T cells, B cells, etc.

[0048] The term “candidate agent” means any oligonucleotide, polynucleotide, siRNA, shRNA, gene, gene product, peptide, antibody, small molecule or pharmacological compound that is introduced to an explant culture and the cells thereof as described herein to assay for its effect on the explants.

[0049] The term "contacting" refers to the placing of candidate cells or candidate agents into the explant culture as described herein. Contacting also encompasses co-culture of candidate cells with tissue explants for at least 1 hour, or more than 2 hrs or more than 4 hrs in culture medium prior to placing the tissue explants in a semi-permeable substrate. Alternatively, contacting refers to injection of candidate cells into the explant, e.g. into the lumen of an explant.

[0050] “Screening” refers to the process of either co-culturing candidate cells with or adding candidate agents to the explant culture described herein and assessing the effect of the candidate cells or candidate agents on the explant, including without limitation immune cells present in the explant. The effect may be assessed by assessing any convenient parameter, e.g. phenotypic changes, protein expression, mRNA expression, etc.

[0051] The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a mammal being assessed for treatment and / or being treated. In some embodiments, the mammal is a human. The terms “subject,” “individual,” and “patient” encompass, without limitation, individuals having a disease. Subjects may be human, but also include othermammals, particularly those mammals useful as laboratory models for human disease, e.g., mice, rats, etc.

[0052] The term “diagnosis” is used herein to refer to the identification of a molecular or pathological state, disease or condition in a subject, individual, or patient.

[0053] The term “prognosis” is used herein to refer to the prediction of the likelihood of death or disease progression, including recurrence, spread, and drug resistance, in a subject, individual, or patient. The term “prediction” is used herein to refer to the act of foretelling or estimating, based on observation, experience, or scientific reasoning, the likelihood of a subject, individual, or patient experiencing a particular event or clinical outcome. In one example, a physician may attempt to predict the likelihood that a patient will survive.

[0054] As used herein, the terms “treatment,” “treating,” and the like, refer to administering an agent, or carrying out a procedure, for the purposes of obtaining an effect on or in a subject, individual, or patient. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of effecting a partial or complete cure for a disease and / or symptoms of the disease. “Treatment,” as used herein, may include treatment of infection in a mammal, particularly in a human, and includes one or more of: (a) preventing disease; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease or its symptoms, i.e., causing regression of the disease or its symptoms. Treating may also refer to any indicia of success in the treatment or amelioration or prevention of a disease, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of an examination by a physician. The term "therapeutic effect" refers to the reduction, elimination, or prevention of the disease, symptoms of the disease, or side effects of the disease in the subject.

[0055] As used herein, a "therapeutically effective amount" refers to that amount of the therapeutic agent sufficient to treat or manage a disease or disorder. A therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the onset of disease, e.g., to delay or minimize infection and the sequelae of infection. A therapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease. Further, a therapeutically effective amount with respect to a therapeutic agent of the invention means the amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of a disease.

[0056] As used herein, the term “dosing regimen” refers to a set of unit doses (typically more than one) that are administered individually to a subject, typically separated by periods of time.In some embodiments, a given therapeutic agent has a recommended dosing regimen, which may involve one or more doses. In some embodiments, a dosing regimen comprises a plurality of doses each of which are separated from one another by a time period of the same length; in some embodiments, a dosing regimen comprises a plurality of doses and at least two different time periods separating individual doses. In some embodiments, all doses within a dosing regimen are of the same unit dose amount. In some embodiments, different doses within a dosing regimen are of different amounts. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount different from the first dose amount. In some embodiments, a dosing regimen comprises a first dose in a first dose amount, followed by one or more additional doses in a second dose amount same as the first dose amount. In some embodiments, a dosing regimen is correlated with a desired or beneficial outcome when administered across a relevant population (i.e. , is a therapeutic dosing regimen).

[0057] "In combination with", "combination therapy" and "combination products" refer, in certain embodiments, to the concurrent administration to a patient of one or more therapeutic agents; or to the screening of two or more agents in a culture. When administered in combination, each component can be administered at the same time or sequentially in any order at different points in time. Thus, each component can be administered separately but sufficiently closely in time so as to provide the desired therapeutic effect.

[0058] "Concomitant administration" means administration of one or more components at such time that the combination will have a therapeutic effect. Such concomitant administration may involve concurrent (i.e. at the same time), prior, or subsequent administration of components. A person of ordinary skill in the art would have no difficulty determining the appropriate timing, sequence and dosages of administration.

[0059] The use of the term "in combination" does not restrict the order in which prophylactic and / or therapeutic agents are administered to a subject with a disorder. A first prophylactic or therapeutic agent can be administered prior to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks 6 weeks, 8 weeks, or 12 weeks before), concomitantly with, or subsequent to (e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8 weeks, or 12 weeks after) the administration of a second prophylactic or therapeutic agent to a subject with a disorder.

[0060] As used herein, a "therapeutically effective amount" refers to that amount of the therapeutic agent sufficient to treat or manage a disease or disorder. A therapeutically effective amount may refer to the amount of therapeutic agent sufficient to delay or minimize the onset of disease, e.g., to delay or minimize the growth and spread of cancer. Atherapeutically effective amount may also refer to the amount of the therapeutic agent that provides a therapeutic benefit in the treatment or management of a disease. Further, a therapeutically effective amount with respect to a therapeutic agent of the invention means the amount of therapeutic agent alone, or in combination with other therapies, that provides a therapeutic benefit in the treatment or management of a disease.

[0001] As used herein, endpoints for treatment will be given a meaning as known in the art and as used by the Food and Drug Administration.

[0002] Overall survival is defined as the time from randomization until death from any cause, and is measured in the intent-to-treat population. Survival is considered the most reliable cancer endpoint, and when studies can be conducted to adequately assess survival, it is usually the preferred endpoint. This endpoint is precise and easy to measure, documented by the date of death. Bias is not a factor in endpoint measurement. Survival improvement should be analyzed as a risk-benefit analysis to assess clinical benefit. Overall survival can be evaluated in randomized controlled studies. Demonstration of a statistically significant improvement in overall survival can be considered to be clinically significant if the toxicity profile is acceptable, and has often supported new drug approval. A benefit of the methods of the invention can include increased overall survival of patients.

[0003] Endpoints that are based on tumor assessments include DFS, ORR, TTP, PFS, and time-to-treatment failure (TTF). The collection and analysis of data on these time-dependent endpoints are based on indirect assessments, calculations, and estimates (e.g., tumor measurements). Disease-Free Survival (DFS) is defined as the time from randomization until recurrence of tumor or death from any cause. The most frequent use of this endpoint is in the adjuvant setting after definitive surgery or radiotherapy. DFS also can be an important endpoint when a large percentage of patients achieve complete responses with chemotherapy.

[0004] Objective Response Rate . ORR is defined as the proportion of patients with tumor size reduction of a predefined amount and for a minimum time period. Response duration usually is measured from the time of initial response until documented tumor progression. Generally, the FDA has defined ORR as the sum of partial responses plus complete responses. When defined in this manner, ORR is a direct measure of drug antitumor activity, which can be evaluated in a single-arm study.

[0005] Time to Progression and Progression-Free Survival. TTP and PFS have served as primary endpoints for drug approval. TTP is defined as the time from randomization until objective tumor progression; TTP does not include deaths. PFS is defined as the time from randomization until objective tumor progression or death. The precise definition of tumor progression is important and should be carefully detailed in the protocol.

[0061] Cancer therapy includes chemotherapy, targeted therapy, immunotherapy and radiation sherapy.

[0062] Chemotherapy may include Abitrexate (Methotrexate Injection), Abraxane (Paclitaxel Injection), Adcetris (Brentuximab Vedotin Injection), Adriamycin (Doxorubicin), Adrucil Injection (5-FU (fluorouracil)), Afinitor (Everolimus) , Afinitor Disperz (Everolimus) , Alimta (PEMET EXED), Alkeran Injection (Melphalan Injection), Alkeran Tablets (Melphalan), Aredia (Pamidronate), Arimidex (Anastrozole), Aromasin (Exemestane), Arranon (Nelarabine), Arzerra (Ofatumumab Injection), Avastin (Bevacizumab), Bexxar (Tositumomab), BiCNU (Carmustine), Blenoxane (Bleomycin), Bosulif (Bosutinib), Busulfex Injection (Busulfan Injection), Campath (Alemtuzumab), Camptosar (Irinotecan), Caprelsa (Vandetanib), Casodex (Bicalutamide), CeeNU (Lomustine), CeeNU Dose Pack (Lomustine), Cerubidine (Daunorubicin), Clolar (Clofarabine Injection), Cometriq (Cabozantinib), Cosmegen (Dactinomycin), Cytosarll (Cytarabine), Cytoxan (Cytoxan), Cytoxan Injection (Cyclophosphamide Injection), Dacogen (Decitabine), DaunoXome (Daunorubicin Lipid Complex Injection), Decadron (Dexamethasone), DepoCyt (Cytarabine Lipid Complex Injection), Dexamethasone Intensol (Dexamethasone), Dexpak Taperpak (Dexamethasone), Docefrez (Docetaxel), Doxil (Doxorubicin Lipid Complex Injection), Droxia (Hydroxyurea), DTIC (Decarbazine), Eligard (Leuprolide), Ellence (Ellence (epirubicin)), Eloxatin (Eloxatin (oxaliplatin)), Elspar (Asparaginase), Emcyt (Estramustine), Erbitux (Cetuximab), Erivedge (Vismodegib), Erwinaze (Asparaginase Erwinia chrysanthemi), Ethyol (Amifostine), Etopophos (Etoposide Injection), Eulexin (Flutamide), Fareston (Toremifene), Faslodex (Fulvestrant), Femara (Letrozole), Firmagon (Degarelix Injection), Fludara (Fludarabine), Folex (Methotrexate Injection), Folotyn (Pralatrexate Injection), FUDR (FUDR (floxuridine)), Gemzar (Gemcitabine), Gilotrif (Afatinib), Gleevec (Imatinib Mesylate), Gliadel Wafer (Carmustine wafer), Halaven (Eribulin Injection), Herceptin (Trastuzumab), Hexalen (Altretamine), Hycamtin (Topotecan), Hycamtin (Topotecan), Hydrea (Hydroxyurea), Iclusig (Ponatinib), Idamycin PFS (Idarubicin), Ifex (Ifosfamide), Inlyta (Axitinib), Intron A alfab (Interferon alfa-2a), Iressa (Gefitinib), Istodax (Romidepsin Injection), Ixempra (Ixabepilone Injection), Jakafi (Ruxolitinib), Jevtana (Cabazitaxel Injection), Kadcyla (Ado-trastuzumab Emtansine), Kyprolis (Carfilzomib), Leukeran (Chlorambucil), Leukine (Sargramostim), Leustatin (Cladribine), Lupron (Leuprolide), Lupron Depot (Leuprolide), Lupron DepotPED (Leuprolide), Lysodren (Mitotane), Marqibo Kit (Vincristine Lipid Complex Injection), Matulane (Procarbazine), Megace (Megestrol), Mekinist (Trametinib), Mesnex (Mesna), Mesnex (Mesna Injection), Metastron (Strontium-89 Chloride), Mexate (Methotrexate Injection), Mustargen (Mechlorethamine), Mutamycin (Mitomycin), Myleran (Busulfan), Mylotarg (Gemtuzumab Ozogamicin), Navelbine (Vinorelbine), Neosar Injection (Cyclophosphamide Injection), Neulasta (filgrastim), Neulasta (pegfilgrastim), Neupogen (filgrastim), Nexavar (Sorafenib),Nilandron (Nilandron (nilutamide)), Nipent (Pentostatin), Nolvadex (Tamoxifen), Novantrone (Mitoxantrone), Oncaspar (Pegaspargase), Oncovin (Vincristine), Ontak (Denileukin Diftitox), Onxol (Paclitaxel Injection), Panretin (Alitretinoin), Paraplatin (Carboplatin), Perjeta (Pertuzumab Injection), Platinol (Cisplatin), Platinol (Cisplatin Injection), PlatinolAQ (Cisplatin), PlatinolAQ (Cisplatin Injection), Pomalyst (Pomalidomide), Prednisone Intensol (Prednisone), Proleukin (Aldesleukin), Purinethol (Mercaptopurine), Reclast (Zoledronic acid), Revlimid (Lenalidomide), Rheumatrex (Methotrexate), Rituxan (Rituximab), RoferonA alfaa (Interferon alfa-2a), Rubex (Doxorubicin), Sandostatin (Octreotide), Sandostatin LAR Depot (Octreotide), Soltamox (Tamoxifen), Sprycel (Dasatinib), Sterapred (Prednisone), Sterapred DS (Prednisone), Stivarga (Regorafenib), Supprelin LA (Histrelin Implant), Sutent (Sunitinib), Sylatron (Peginterferon Alfa-2b Injection (Sylatron)), Synribo (Omacetaxine Injection), Tabloid (Thioguanine), Taflinar (Dabrafenib), Tarceva (Erlotinib), Targretin Capsules (Bexarotene), Tasigna (Decarbazine), Taxol (Paclitaxel Injection), Taxotere (Docetaxel), Temodar (Temozolomide), Temodar (Temozolomide Injection), Tepadina (Thiotepa), Thalomid (Thalidomide), TheraCys BCG (BCG), Thioplex (Thiotepa), TICE BCG (BCG), Toposar (Etoposide Injection), Torisel (Temsirolimus), Treanda (Bendamustine hydrochloride), Trelstar (Triptorelin Injection), Trexall (Methotrexate), Trisenox (Arsenic trioxide), Tykerb (lapatinib), Valstar (Valrubicin Intravesical), Vantas (Histrelin Implant), Vectibix (Panitumumab), Velban (Vinblastine), Velcade (Bortezomib), Vepesid (Etoposide), Vepesid (Etoposide Injection), Vesanoid (Tretinoin), Vidaza (Azacitidine), Vincasar PFS (Vincristine), Vincrex (Vincristine), Votrient (Pazopanib), Vumon (Teniposide), Wellcovorin IV (Leucovorin Injection), Xalkori (Crizotinib), Xeloda (Capecitabine), Xtandi (Enzalutamide), Yervoy (Ipilimumab Injection), Zaltrap (Ziv-aflibercept Injection), Zanosar (Streptozocin), Zelboraf (Vemurafenib), Zevalin (Ibritumomab Tiuxetan), Zoladex (Goserelin), Zolinza (Vorinostat), Zometa (Zoledronic acid), Zortress (Everolimus), Zytiga (Abiraterone), Nimotuzumab and immune checkpoint inhibitors such as nivolumab, pembrolizumab / MK-3475, pidilizumab and AMP-224 targeting PD-1 ; and BMS-935559, MEDI4736, MPDL3280A and MSB0010718C targeting PD-L1 and those targeting CTLA-4 such as ipilimumab.

[0063] Antibiotics, e.g. antibiotics with the classes of aminoglycosides; carbapenems; and the like; penicillins, e.g. penicillin G, penicillin V, methicillin, oxacillin, carbenicillin, nafcillin, ampicillin, etc. penicillins in combination with p-lactamase inhibitors, cephalosporins, e.g. cefaclor, cefazolin, cefuroxime, moxalactam, etc:; tetracyclines; cephalosporins; quinolones; lincomycins; macrolides; sulfonamides; glycopeptides including the anti-infective antibiotics vancomycin, teicoplanin, telavancin, ramoplanin and decaplanin. Derivatives of vancomycin include, for example, oritavancin and dalbavancin (both lipoglycopeptides). Telavancin is a semi-synthetic lipoglycopeptide derivative of vancomycin (approved by FDA in 2009). Other vancomycin analogs are disclosed, for example, in WO 2015022335 A1 and Chen et al. (2003)PNAS 100(10): 5658-5663, each herein specifically incorporated by reference. Non-limiting examples of antibiotics include vancomycin, linezolid, azithromycin, daptomycin, colistin, eperezolid, fusidic acid, rifampicin, tetracyclin, fidaxomicin, clindamycin, lincomycin, rifalazil, and clarithromycin.

[0064] Radiotherapy means the use of radiation, usually X-rays, to treat illness. X-rays were discovered in 1895 and since then radiation has been used in medicine for diagnosis and investigation (X-rays) and treatment (radiotherapy). Radiotherapy may be from outside the body as external radiotherapy, using X-rays, cobalt irradiation, electrons, and more rarely other particles such as protons. It may also be from within the body as internal radiotherapy, which uses radioactive metals or liquids (isotopes) to treat cancer.

[0065] Immunotherapy is the use of the immune system to treat cancer. Immunotherapies can be categorized as active, passive or hybrid (active and passive). These approaches exploit the fact that cancer cells often have molecules on their surface that can be detected by the immune system, known as tumor-associated antigens (TAAs); they are often proteins or other macromolecules (e.g. carbohydrates).

[0066] Immune-checkpoint receptors for T cells include antibodies that interfere with binding of cytotoxic T-lymphocyte-associated antigen 4 (CTLA4; also known as CD152) and programmed cell death protein 1 (PD1 ; also known as CD279) with their counter-receptors. The clinical activity of antibodies that block either of these receptors implies that antitumor immunity can be enhanced at multiple levels and that combinatorial strategies can be intelligently designed, guided by mechanistic considerations and preclinical models.

[0067] CTLA4 is expressed exclusively on T cells where it primarily regulates the amplitude of the early stages of T cell activation. CTLA4 counteracts the activity of the T cell costimulatory receptor, CD28. CD28 and CTLA4 share identical ligands: CD80 (also known as B7.1 ) and CD86 (also known as B7.2). The major physiological roles of CTLA4 are downmodulation of helper T cell activity and enhancement of regulatory T (TReg) cell immunosuppressive activity. CTLA4 blockade results in a broad enhancement of immune responses. Two fully humanized CTLA4 antibodies, ipilimumab and tremelimumab, are in clinical testing and use. Clinically the response to immune-checkpoint blockers is slow and, in many patients, delayed up to 6 months after treatment initiation. In some cases, metastatic lesions actually increase in size on computed tomography (CT) or magnetic resonance imaging (MRI) scans before regressing. Anti-CTLA4 antibodies that antagonize this inhibitory immune function are very potent therapeutics but have significant side effects since this enables also T cell activity against the self that is usually inhibited through these inhibitory molecules and pathways.

[0068] Other immune-checkpoint proteins are PD1 and PDL1 . Antibodies in current clinical use against these targets include nivolumab and pembrolizumab. The major role of PD1 is tolimit the activity of T cells in peripheral tissues at the time of an inflammatory response to infection and to limit autoimmunity. PD1 expression is induced when T cells become activated. When engaged by one of its ligands, PD1 inhibits kinases that are involved in T cell activation. PD1 is highly expressed on T Regcells, where it may enhance their proliferation in the presence of ligand. Because many tumors are highly infiltrated with Tpeg cells, blockade of the PD1 pathway may also enhance antitumor immune responses by diminishing the number and / or suppressive activity of intratumoral Tpeg cells.

[0069] The two ligands for PD1 are PD1 ligand 1 (PDL1 ; also known as B7-H1 and CD274) and PDL2 (also known as B7-DC and CD273). The PD1 ligands are commonly upregulated on the tumor cell surface from many different human tumors. On cells from solid tumors, the major PD1 ligand that is expressed is PDL1 . PDL1 is expressed on cancer cells and through binding to it’s receptor PD1 on T cells it inhibits T cell activation / function. Therefore, PD1 and PDL1 blocking agents can overcome this inhibitory signaling and maintain or restore antitumor T cell function.

[0070] PDL1 is expressed on cancer cells and through binding to its receptor PD1 on T cells it inhibits T cell activation / function. Therefore, PD1 and PDL1 blocking agents can overcome this inhibitory signaling and maintain or restore anti-tumor T cell function. However, since PDL1 is expressed on tumor cells, antibodies that bind and block PDL1 can also enable ADCP, ADCC, and CDC of tumor cells. Anti-CD47 agents can synergize with targeted monoclonal antibodies and enhance their potency to stimulate ADCP and ADCC.

[0071] Lymphocyte activation gene 3 (LAG3; also known as CD223), 2B4 (also known as CD244), B and T lymphocyte attenuator (BTLA; also known as CD272), T cell membrane protein 3 (TIM3; also known as HAVcr2), adenosine A2a receptor (A2aR) and the family of killer inhibitory receptors have each been associated with the inhibition of lymphocyte activity and in some cases the induction of lymphocyte anergy. Antibody targeting of these receptors can be used in the methods of the invention. LAG3 is a CD4 homolog that enhances the function of TRegcells. LAG3 also inhibits CD8+effector T cell functions independently of its role on TReg cells. The only known ligand for LAG3 is MHC class II molecules, which are expressed on tumor-infiltrating macrophages and dendritic cells. LAG3 is one of various immune- checkpoint receptors that are coordinately upregulated on both Tpeg cells and anergic T cells, and simultaneous blockade of these receptors can result in enhanced reversal of this anergic state relative to blockade of one receptor alone. In particular, PD1 and LAG3 are commonly co-expressed on anergic or exhausted T cells. Dual blockade of LAG3 and PD1 synergistically reversed anergy among tumor-specific CD8+T cells and virus-specific CD8+T cells in the setting of chronic infection. LAG3 blocking agents can overcome this inhibitory signaling and maintain or restore anti-tumor T cell function.

[0072] TIM3 inhibits T helper 1 (TH1 ) cell responses, and TIM3 antibodies enhance antitumor immunity. TIM3 has also been reported to be co-expressed with PD1 on tumor-specific CD8+T cells. Tim3 blocking agents can overcome this inhibitory signaling and maintain or restore anti-tumor T cell function.

[0073] Other active cellular therapies that can be screened by the methods described herein may involve the removal of immune cells from the organoid culture. Those specific for the tumor are cultured and returned to the patient where they attack the tumor. Cell types that can be used in this way are natural killer cells, lymphokine-activated killer cells, cytotoxic T cells and dendritic cells.

[0074] Alternatively adoptive T-cell therapy can be screened, e.g. in a form of passive immunization by the transfer of T-cells. Multiple ways of producing and obtaining tumor targeted T-cells have been developed. T-cells specific to a tumor antigen can be removed from a tumor sample (TILs) or filtered from blood. Subsequent activation and culturing is performed ex vivo, with the results reinfused. Activation can take place through gene therapy, or by exposing the T cells to tumor antigens.

[0075] Cytokines that potentially modulate immune responses can also be screened. The tumor often employs them to allow it to grow and reduce the immune response. These immune-modulating effects allow them to be used as drugs to provoke an immune response. Two commonly used cytokines are interferons and interleukins.

[0076] Many immuno-oncology agents are antibodies. As used herein, "antibody" includes reference to an immunoglobulin molecule immunologically reactive with a particular antigen, and includes both polyclonal and monoclonal antibodies. The term also includes genetically engineered forms such as chimeric antibodies (e.g., humanized murine antibodies) and heteroconjugate antibodies. The term "antibody" also includes antigen binding forms of antibodies, including fragments with antigen-binding capability (e.g., Fab', F(ab')2, Fab, Fv and rlgG. The term also refers to recombinant single chain Fv fragments (scFv). The term antibody also includes bivalent or bispecific molecules, diabodies, triabodies, and tetrabodies.

[0077] Selection of antibodies may be based on a variety of criteria, including selectivity, affinity, cytotoxicity, etc. The phrase "specifically (or selectively) binds" to an antibody or "specifically (or selectively) immunoreactive with," when referring to a protein or peptide, refers to a binding reaction that is determinative of the presence of the protein, in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antibodies bind to a particular protein sequences at least two times the background and more typically more than 10 to 100 times background. In general, antibodies of interest bind antigens on the surface of target cells in the presence of effector. Fc receptors on effector cells recognize bound antibodies.

[0078] An antibody immunologically reactive with a particular antigen can be generated by recombinant methods such as selection of libraries of recombinant antibodies in phage or similar vectors, or by immunizing an animal with the antigen or with DNA encoding the antigen. Methods of preparing polyclonal antibodies are known to the skilled artisan. The antibodies may, alternatively, be monoclonal antibodies. Monoclonal antibodies may be prepared using hybridoma methods. In a hybridoma method, an appropriate host animal is typically immunized with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent. Alternatively, the lymphocytes may be immunized in vitro. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell.

[0079] Antibodies also exist as a number of well-characterized fragments produced by digestion with various peptidases. Thus pepsin digests an antibody below the disulfide linkages in the hinge region to produce F(ab)'2, a dimer of Fab which itself is a light chain joined to VH-CHI by a disulfide bond. The F(ab)'2may be reduced under mild conditions to break the disulfide linkage in the hinge region, thereby converting the F(ab)'2dimer into an Fab' monomer. The Fab' monomer is essentially Fab with part of the hinge region. While various antibody fragments are defined in terms of the digestion of an intact antibody, one of skill will appreciate that such fragments may be synthesized de novo either chemically or by using recombinant DNA methodology. Thus, the term antibody, as used herein, also includes antibody fragments either produced by the modification of whole antibodies, or those synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv) or those identified using phage display libraries.

[0080] Antibodies of interest may be tested for their ability to induce ADCC (antibodydependent cellular cytotoxicity) or ADCP (antibody dependent cellular phagocytosis). Antibody-associated ADCC activity can be monitored and quantified through detection of either the release of label or lactate dehydrogenase from the lysed cells, or detection of reduced target cell viability (e.g. annexin assay). Assays for apoptosis may be performed by terminal deoxynucleotidyl transferase-mediated digoxigenin-11 -dUTP nick end labeling (TUNEL) assay (Lazebnik et al., Nature: 371 , 346 (1994). Cytotoxicity may also be detected directly by detection kits known in the art, such as Cytotoxicity Detection Kit from Roche Applied Science (Indianapolis, Ind.).

[0081] Other types of immune modulators include non-antibody entities such as polypeptides, nucleic acid-based entities such as CpG or DNA or RNA aptamers, small molecule chemical compounds and the like. These all could be tested in the organoid system.

[0082] The terms “cancer,” “neoplasm,” and “tumor” are used interchangeably herein to refer to cells which exhibit autonomous, unregulated growth, such that they exhibit an aberrantgrowth phenotype characterized by a significant loss of control over cell proliferation. Cells of interest for detection, analysis, or treatment in the present application include precancerous (e.g., benign), malignant, pre-metastatic, metastatic, and non-metastatic cells. Cancers of virtually every tissue are known. The phrase “cancer burden” refers to the quantum of cancer cells or cancer volume in a subject. Reducing cancer burden accordingly refers to reducing the number of cancer cells or the cancer volume in a subject. The term “cancer cell” as used herein refers to any cell that is a cancer cell or is derived from a cancer cell e.g. clone of a cancer cell. Many types of cancers are known to those of skill in the art, including solid tumors such as carcinomas, sarcomas, glioblastomas, melanomas, lymphomas, myelomas, etc. Examples of cancer include but are not limited to, ovarian cancer, breast cancer, colon cancer, lung cancer, prostate cancer, hepatocellular cancer, gastric cancer, pancreatic cancer, cervical cancer, ovarian cancer, liver cancer, bladder cancer, cancer of the urinary tract, thyroid cancer, renal cancer, carcinoma, melanoma, head and neck cancer, and brain cancer.

[0083] Hematologic cancers are of particular interest, which cancers are of hematopoietic cell origin, e.g. leukemias, lymphomas and myelomas. Leukemia is a malignant condition involving the excess production of immature or abnormal leukocytes, which eventually suppresses the production of normal blood cells and results in symptoms related to cytopenias. Malignant transformation usually occurs at the pluripotent stem cell level, although it sometimes involves a committed stem cell with more limited capacity for self-renewal. Abnormal proliferation, clonal expansion, aberrant differentiation, and diminished apoptosis (programmed cell death) lead to replacement of normal blood elements with malignant cells. The four most common leukemias are Acute myeloid leukemia (AML), Acute lymphoblastic leukemia (ALL); Chronic myeloid leukemia (CML); Chronic lymphocytic leukemia (CLL).

[0084] Myelodysplastic syndromes are a group of clonal hematopoietic stem cell disorders unified by the presence of distinct mutations of hematopoietic stem cells. They involve progressive bone marrow failure but with an insufficient proportion of blast cells (< 20%) for making a definite diagnosis of acute myeloid leukemia; 40 to 60% of cases evolve into acute myeloid leukemia.

[0085] Multiple myeloma is a cancer of plasma cells that produce monoclonal immunoglobulin and invade and destroy adjacent bone tissue. Common manifestations include lytic lesions in bones that cause pain and / or fractures, renal insufficiency, hypercalcemia, anemia, and recurrent infections. Diagnosis typically requires demonstration of M-protein (sometimes present in urine and not serum but rarely absent entirely) and / or light-chain proteinuria, and excessive plasma cells in the bone marrow. Specific treatment most often includes some combination of conventional chemotherapy, corticosteroids, and one or more additional medications such as proteasome inhibitors (eg, bortezomib, carfilzomib, ixazomib), immunomodulating agents (eg, lenalidomide, thalidomide, pomalidomide), or monoclonalantibodies (eg, daratumumab, isatuximab, elotuzumab). Antibody- and T-cell-based approaches to targeting of B-cell maturation antigen have shown efficacy. High-dose melphalan followed by autologous peripheral blood stem cell transplantation may also be used.

[0086] The “pathology” of cancer includes all phenomena that compromise the well-being of the patient. This includes, without limitation, abnormal or uncontrollable cell growth, metastasis, interference with the normal functioning of neighboring cells, release of cytokines or other secretory products at abnormal levels, suppression or aggravation of inflammatory or immunological response, neoplasia, premalignancy, malignancy, invasion of surrounding or distant tissues or organs, such as lymph nodes, etc.

[0087] The term “cancer” is not limited to any stage, grade, histomorphological feature, invasiveness, aggressiveness or malignancy of an affected tissue or cell aggregation. In particular stage 0 cancer, stage I cancer, stage II cancer, stage III cancer, stage IV cancer, grade I cancer, grade II cancer, grade III cancer, malignant cancer and primary carcinomas are included.

[0088] As used herein, the terms “cancer recurrence” and “tumor recurrence,” and grammatical variants thereof, refer to further growth of neoplastic or cancerous cells after diagnosis of cancer. Particularly, recurrence may occur when further cancerous cell growth occurs in the cancerous tissue. “Tumor spread,” similarly, occurs when the cells of a tumor disseminate into local or distant tissues and organs; therefore tumor spread encompasses tumor metastasis. “Tumor invasion” occurs when the tumor growth spread out locally to compromise the function of involved tissues by compression, destruction, or prevention of normal organ function.

[0089] As used herein, the term “metastasis” refers to the growth of a cancerous tumor in an organ or body part, which is not directly connected to the organ of the original cancerous tumor. Metastasis will be understood to include micrometastasis, which is the presence of an undetectable amount of cancerous cells in an organ or body part which is not directly connected to the organ of the original cancerous tumor. Metastasis can also be defined as several steps of a process, such as the departure of cancer cells from an original tumor site, and migration and / or invasion of cancer cells to other parts of the body.

[0090] The term “sample” with respect to a patient encompasses blood and other liquid samples of biological origin, solid tissue samples such as a biopsy specimen or tissue cultures or cells derived therefrom and the progeny thereof. The definition also includes samples that have been manipulated in any way after their procurement, such as by treatment with reagents; washed; or enrichment for certain cell populations, such as cancer cells. The definition also includes sample that have been enriched for particular types of molecules, e.g., nucleic acids, polypeptides, etc. The term “biological sample” encompasses a clinical sample,and also includes tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, and the like. A “biological sample” includes a sample obtained from a patient’s cancer cell, e.g., a sample comprising polynucleotides and / or polypeptides that is obtained from a patient’s cancer cell (e.g., a cell lysate or other cell extract comprising polynucleotides and / or polypeptides); and a sample comprising cancer cells from a patient. A biological sample comprising a cancer cell from a patient can also include non-cancerous cells.

[0091] The term “diagnosis” is used herein to refer to the identification of a molecular or pathological state, disease or condition, such as the identification of a molecular subtype of breast cancer, prostate cancer, or other type of cancer.

[0092] The term “prognosis” is used herein to refer to the prediction of the likelihood of cancer- attributable death or progression, including recurrence, metastatic spread, and drug resistance, of a neoplastic disease, such as ovarian cancer. The term “prediction” is used herein to refer to the act of foretelling or estimating, based on observation, experience, or scientific reasoning. In one example, a physician may predict the likelihood that a patient will survive, following surgical removal of a primary tumor and / or chemotherapy for a certain period of time without cancer recurrence. The present methods allow prediction of whether a patient will be responsive to a therapy of interest.Medium

[0093] In some embodiments, the culture medium comprises an effective dose of an activator of the FGF pathway, including, for example FGF10 protein. Activators of the FGF pathway include small molecule activators, small molecule inhibitors, peptide activators, peptide inhibitors, antibodies, nucleic acid activators, nucleic acid inhibitors, and the like that activate or inhibit at least one component of the FGF pathway resulting in a corresponding activation or inhibition in cellular FGF signaling. Components and downstream effectors of the FGF pathway include but are not limited to, e.g., akt1 (UniProtID P31749), beta-klotho (UniProtID Q86Z14), camkiia (UniProtID Q9UQM7), cbl (UniProtID P22681 ), cortactin (UniProtID Q14247), e-cadherin (UniProtID P12830), erk1 (UniProtID P27361 ), erk2 (UniProtID P28482), FGF1 (UniProtID P05230), FGF16 (UniProtID 060258), FGF17 (UniProtID 060258), FGF18 (UniProtID 076093), FGF19 (UniProtID 095750), FGF2 (UniProtID P09038), fgf23 (UniProtID Q9GZV9), FGF4 (UniProtID P08620), FGF6 (UniProtID P10767), FGF8 (UniProtID P55075), FGF9 (UniProtID P31371 ), fgfrl (UniProtID P1 1362), fgfr2 (UniProtID P21802), fgfr2b (UniProtID P21802-18), FGFR2c (UniProtID P21802-5), FGFR3c (UniProtID P22607-1 ), FGFR4 (UniProtID P22455), fos (UniProtID P01 100), frs2 (UniProtID Q8WU20), gab1 (UniProtID Q13480), grb2 (UniProtID P62993), hgf (UniProtID P14210), jun (UniProtID P05412), klotho (UniProtID Q9UEF7), mapk 14 (UniProtID Q16539), met (UniProtID P08581 ),mkp-3 (UniProtID Q16828), mmp9 (UniProtID P14780), n-cad-ctf1 (UniProtID P19022), n-cad- ctf2 (UniProtID P19022), n-cadherin (UniProtID P19022), ncam (UniProtID P13591), osteocalcin (UniProtID P02818), osteopontin (UniProtID P10451 ), p110-alpha (UniProtID P42336), p120ctn (UniProtID 060716), p90-rsk 1 (UniProtID Q15418), pak4 (UniProtID Q8WYL5), pak4 (UniProtID 096013), pdk1 (UniProtID 015530), pik3r1 (UniProtID P27986), plcgammal (UniProtID P19174), pro-e-cadherin (UniProtID P12830), pro-mmp9 (UniProtID P14780), ps1 (UniProtID gamma), pyk2 (UniProtID Q14289), runx2 (UniProtID Q13950), se- cad (UniProtID P12830), secad-ntf2 (UniProtID P12830), set (UniProtID Q8NFM7), she (UniProtID P29353), shp2 (UniProtID Q06124), sn-cad (UniProtID P19022), sos1 (UniProtID Q07889), sprouty2 (UniProtID 043597), sre (UniProtID P12931 ), statl (UniProtID P42224), stat3 (UniProtID P40763), stat5b (UniProtID P51692), syndecan-2 (UniProtID P34741 ), syndecan-4 (UniProtID P31431 ), upa (UniProtID P00749), upar (UniProtID Q03405), and the like.

[0094] Activators of the FGF pathway (FGF agents) include but are not limited to, FGF family ligands (e.g., FGF1 , FGF2, FGF-3, FGF-4, FGF-5, FGF-6, KGF / FGF-7, FGF-8, FGF-9, FGF- 10, FGF-1 1 , FGF-12, FGF-13, FGF-15, FGF-16, FGF-17, FGF-19, FGF-20, FGF-21 , FGF-22, FGF-23, etc.), SUN 1 1602 (4-[[4-[[2-[(4-Amino-2, 3,5,6- tetramethylphenyl)amino]acetyl]methylamino]-1 -piperidinyl]methyl]benzamide), t-Butylhydroquinone, U-46619, 02 Ceramide, Lactosyl Ceramide, Angiotensin II, Baicalin, and the like.

[0095] In some embodiments the culture medium comprises an effective dose of an activator of epidermal growth factor receptor (EGFR), e.g. an EGF agent. EGFR is a transmembrane receptor tyrosine kinase that plays a crucial role in regulating cell proliferation, differentiation, and survival. EGFR activation involves ligand binding to the receptor’s extracellular domain, leading to dimerization, autophosphorylation, and the activation of downstream signaling pathways such as the PI3K / AKT and RAS / RAF / MEK / ERK pathways. Activators of interest include, for example Epidermal Growth Factor (EGF) protein, which may be a preferred agent. Transforming Growth Factor-a (TGF-a) also binds to EGFR and triggers similar responses to EGF. Amphiregulin (AREG) alsol binds and activates EGFR, as do Betacellulin (BTC), Epiregulin (EREG), and Heparin-Binding EGF-like Growth Factor (HB-EGF). Other agents include phorbol esters such as PMA that can activate EGFR indirectly through protein kinase C (PKC)-mediated pathways.

[0096] In some embodiments the culture medium comprises an effective dose of an activator of thrombopoietin receptor, e.g. a TPO agent The thrombopoietin receptor (TPO-R), also known as MPL or CD1 10, is a critical receptor involved in the regulation of hematopoiesis, particularly in promoting the proliferation and differentiation of megakaryocytes and the production of platelets (thrombopoiesis). TPO-R is primarily activated by its ligand,thrombopoietin (TPO), but there are several factors in a cell culture setting that can modulate its activation or influence its signaling pathways. In some embodiments the activator is thrombopoietin, a glycoprotein produced by the liver and kidneys. TPO binds to TPO-R on the surface of hematopoietic stem cells (HSCs), megakaryocyte progenitors, and mature megakaryocytes, leading to receptor dimerization, activation, and downstream signaling. This includes the JAK / STAT, PI3K / AKT, and MAPK / ERK pathways. Recombinant TPO (rTPO) or pegylated TPO (PEG-TPO are often used to stimulate TPO-R activation, promoting the proliferation of megakaryocytes and platelet production in vitro. Small molecules or peptides that mimic the action of TPO can sometimes be used in cell culture to stimulate TPO-R, including Eltrombopag and Romiplostim. Eltrombopag is a small-molecule drug that binds to the transmembrane domain of TPO-R, while Romiplostim is a peptide mimetic that activates TPO-R by binding to its extracellular domain.

[0097] In some embodiments the culture medium comprises an SCF agent. SCF activators (i.e., SCF agents) will vary and may include small molecule activators, peptide activators, agonist antibodies, nucleic acid activators, and the like that activate a molecule that responds to SCF or promotes the expression or functional bioactivity of SCF. In some instances, activation of SCF may be achieved through repression of a SCF inhibitor, e.g., including but not limited to the use of an inhibitory nucleic acid targeting an inhibitor of SCF or an antibody or small molecule directed to a SCF inhibitor. SCF agents include but are not limited to, e.g., a SCF protein or polypeptide, an agonistic SCF peptide, a nucleic acid encoding a SCF protein or polypeptide, a nucleic acid encoding an agonistic SCF peptide, and the like. A stem cell factor includes SCF protein, or a small molecule SCF replacement, e.g. a PI3K activator (see, for example, Sakurai et al. Nature. 2023 Mar;615(7950) :127-133). The chemical agonist 740Y- P is also useful as a stem cell factor.

[0098] In some embodiments the culture medium comprises an effective dose of a pyrimido- [4,5-b]-indole derivative, for example UM729, which enhances the self-renewal of human hematopoietic stem cells in vitro. UM729 does not inhibit the aryl hydrocarbon receptor (AHR) pathway.

[0099] In some embodiments the culture medium comprises an erythropoietin agent. In some embodiments the agonist is erythropoietin (EPO), EPO derivatives, and EPO-stimulating compounds. Suitable examples include but are not limited to: EPO alpha, EPO beta, EPO delta, EPO omega, EPO zeta, Darbepoetin alfa (Aranesp), Epoetin alfa (Procrit), Epocept (Lupin pharma), Nanokine (Nanogen Pharmaceutical biotechnology, Vietnam), Epofit (Intas pharma), Epogen (Amgen), Epogin, Eprex, (Janssen-Cilag), NeoRecormon (Hoffmann-La Roche), Recormon, Methoxy polyethylene glycol-epoetin beta (Mircera)(Roche), Dynepo, Epomax, Silapo (Stada), Retacrit (Hospira), Epocept (Lupin Pharmaceuticals), EPOTrust (Panacea Biotec Ltd.), Erypro Safe (Biocon Ltd.), Repoitin (Serum Institute of IndiaLimited), Vintor (Emcure Pharmaceuticals), Epofit (Intas pharma), Erykine (Intas Biopharmaceutica), Wepox (Wockhardt Biotech), Espogen (LG life sciences), ReliPoietin (Reliance Life Sciences), Shanpoietin (Shantha Biotechnics Ltd.), Zyrop Cadila (Healthcare Ltd.), EPIAO (rHuEPO), and (Shenyang Sunshine Pharmaceutical Co.. LTD. China).

[0100] The culture medium may further comprise a FLT3 agonist or FLT3 agent, e.g. FLT3 ligand. FLT3 (Fms-like tyrosine kinase 3) is a receptor tyrosine kinase that plays a vital role in the survival, proliferation, and differentiation of hematopoietic stem cells (HSCs) and progenitor cells, particularly in early myeloid and lymphoid lineages. In cell culture, several factors can activate FLT3, enhancing its signaling through downstream pathways like PI3K / AKT, RAS / RAF / MEK / ERK, and JAK / STAT. FLT3 ligand (FLT3-L) is the primary and most direct activator of the FLT3 receptor. FLT3-L is a membrane-bound or soluble cytokine that binds to FLT3, inducing receptor dimerization and activation. FLT3-L is crucial for the expansion of hematopoietic stem cells and early progenitors, as well as for the development of dendritic cells and natural killer cells. In cell culture, recombinant FLT3-L is commonly used to stimulate FLT3 signaling in HSCs, progenitor cells, and leukemia cell lines. SCF, which signals through the c-KIT receptor, can work synergistically with FLT3-L to enhance the proliferation of hematopoietic progenitor cells. Small-molecule FLT3 agonists may induce paradoxical activation, where low concentrations of FLT3 inhibitors such as midostaurin, quizartinib, gilteritinib can lead to paradoxical activation of the receptor, when the inhibitor binding induces a partial conformational change in the receptor, activating signaling pathways.

[0101] The culture medium may further comprise an IL-3 agent. The interleukin-3 receptor (IL-3R) is a receptor complex that plays a crucial role in the regulation of hematopoietic cell proliferation, differentiation, and survival. It is particularly important for the development of myeloid progenitor cells, such as granulocytes, macrophages, and megakaryocytes. The IL-3 receptor is a heterodimer composed of an alpha chain (IL-3Ra, CD123), which binds IL-3 specifically, and a beta chain (Pc, CD131 ), which is shared with the receptors for GM-CSF and IL-5. Activation of IL-3R triggers downstream signaling through pathways such as JAK / STAT, PI3K / AKT, and MAPK / ERK. The primary and most direct activator of IL-3R is interleukin-3 (IL- 3), a cytokine that binds specifically to the IL-3Ra chain. Upon binding, the receptor complex recruits the pc chain, leading to receptor dimerization and the activation of intracellular signaling cascades. IL-3 promotes the proliferation, survival, and differentiation of hematopoietic progenitors, including those that give rise to granulocytes, monocytes, and erythroid cells. In cell culture, recombinant human or mouse IL-3 is commonly used to activate the IL-3R on hematopoietic cells, driving their proliferation and differentiation.

[0102] The culture medium may further comprise an IL-7 agent. The interleukin-7 receptor (IL-7R) is a key player in the survival, proliferation, and development of T cells and B cells, particularly during lymphopoiesis. It is composed of two subunits: the IL-7Ra chain (CD127),which binds specifically to IL-7, and the common gamma chain (yc or CD132), shared with other cytokine receptors like those for IL-2, IL-4, IL-9, IL-15, and IL-21 . Activation of the IL-7R primarily triggers the JAK / STAT, PI3K / AKT, and MAPK / ERK signaling pathways, which are critical for lymphocyte survival and proliferation. The most direct activator of the IL-7 receptor is interleukin-7 (IL-7), a cytokine crucial for T cell development in the thymus, survival of naive and memory T cells, and B cell development during early stages of hematopoiesis. IL-7 binds to the IL-7Ra chain, which then associates with the common gamma chain, leading to receptor dimerization and activation of downstream signaling pathways. In cell culture, recombinant IL- 7 is used to activate the IL-7R, promoting the proliferation and survival of lymphoid progenitors, T cells, and B cell precursors. Thymic Stromal Lymphopoietin (TSLP) is structurally similar to IL-7, and can also activate IL-7R signaling, although through a different receptor complex involving the TSLP receptor (TSLPR) and IL-7Ra. In some cell culture systems, TSLP is used to promote the growth of lymphoid progenitors, especially in settings where mucosal immunity or skin immunity is being modeled.Methods

[0103] Culture systems and methods are provided for culture of bone marrow, including stromal and immune cells. The cultures can be maintained for up to 5 days, up to 7 days, up to 10 days, up to 15 days, up to 21 days, up to 28 days, up to 42 days, or more. In some embodiments, tissue, i.e. bone tissue is obtained from an individual with a hematologic cancer, e.g. a leukemia, lymphoma or myeloma. The tissue may be from any mammalian species, e.g. human, equine, bovine, porcine, canine, feline, rodent, e.g. mice, rats, hamster, primate, etc.

[0104] Bone tissue may be obtained by any convenient method, e.g. by biopsy, e.g. during endoscopy, during surgery, by needle, etc., and is typically obtained as aseptically as possible. Upon removal, the tissue is immersed in ice-cold buffered solution, e.g. PBS, Ham’s F12, MEM, culture medium, etc. Pieces of tissue may be minced to a size less than about 1 mm3, and may be less than about 0.5 mm3, or less than about 0.1 mm3. The minced tissue is mixed with a gel substrate, e.g. a collagen gel solution, e.g. Cellmatrix type l-A collagen (Nitta Gelatin Inc.); a matrigel solution, etc. Subsequently, the tissue-containing gel substrate is layered over a layer of gel (a “foundation layer”) in a container with a lower semi-permeable support, e.g. a membrane, supporting the foundation gel layer, and the tissue-containing gel substrate is allowed to solidify. This container is placed into an outer container containing a suitable medium.

[0105] The arrangement described above allows nutrients to travel from the bottom, through the membrane and the foundation gel layer to the gel layer containing the tissue. The level of the medium is maintained such that the top part of the gel, i.e. the gel layer containing theexplants, is not submerged in liquid but is exposed to air. Thus the tissue is grown in a gel with an air-liquid interface. A description of an example of an air-liquid interface culture system is provided in Ootani et al. in Nat Med. 2009 Jun;15(6):701 -6, the disclosure of which is incorporated herein in its entirety by reference. The air-liquid interface organoid cultures could be moved into other formats such as multi-wells for screening or in submerged 2D or 3D geometries where the cells are placed underneath the tissue culture medium.

[0106] The continued growth of the PDO may be confirmed by any convenient method, e.g. phase contrast microscopy, stereomicroscopy, histology, immunohistochemistry, electron microscopy, etc. In some instances, cellular ultrastructure and multi-lineage differentiation may be assessed. Ultrastructure of the intestinal explants in culture can be determined by performing Hematoxylin-eosin staining, PCNA staining, electron microscopy, and the like using methods known in the art.

[0107] Experimental modifications may be made by any method known in the art, for example, as described below with regard to methods for providing candidate agents that are nucleic acids, polypeptides, small molecules, viruses, etc. to explants and the cells thereof for screening purposes.

[0108] For culturing bone marrow, the medium, e.g. IDDM, DMEM, etc., is supplemented with factors, including an effective dose of an SCF agent, an EGF agonist, an FGF agonist, e.g. FGF-10; a TPO-receptor agonist, e.g. Butyzamide, TPO, etc; and a pyrimidoindole derivative, e.g. UM729. A collagenase inhibitor may be included. Optionally an EPO agent is included. Optionally one or more of IL-7, IL-3 and a FLT3 ligand are included. Optionally PCL-PVAc- PEG is included. The factors are chosen to be suitable for the species of the tissue, e.g. human factors for human bone marrow, mouse factors for mouse bone marrow, etc.

[0109] The effective concentration of a particular factor will vary and will depend on the agent. In addition, in some instances, the effective concentration may also depend on the cells being induced, the culture condition of the cells, other induction agents co-present in the culture media, etc. As such, the effective concentration of induction agents will vary and may range from 1 ng / mL to 10 pg / mL or more, including but not limited to, e.g., 1 ng / mL, 2 ng / mL, 3 ng / mL, 4 ng / mL, 5 ng / mL, 6 ng / mL, 7 ng / mL, 8 ng / mL, 9 ng / mL, 10 ng / mL, 11 ng / mL, 12 ng / mL, 13 ng / mL, 14 ng / mL, 15 ng / mL, 16 ng / mL, 17 ng / mL, 18 ng / mL, 19 ng / mL, 20 ng / mL, 21 ng / mL, 22 ng / mL, 23 ng / mL, 24 ng / mL, 25 ng / mL, 26 ng / mL, 27 ng / mL, 28 ng / mL, 29 ng / mL, 30 ng / mL, 31 ng / mL, 32 ng / mL, 33 ng / mL, 34 ng / mL, 35 ng / mL, 36 ng / mL, 37 ng / mL, 38 ng / mL, 39 ng / mL, 40 ng / mL, 41 ng / mL, 42 ng / mL, 43 ng / mL, 44 ng / mL, 45 ng / mL, 46 ng / mL, 47 ng / mL, 48 ng / mL, 49 ng / mL, 50 ng / mL, 1 -5 ng / mL, 1 -10 ng / mL, 1 -20 ng / mL , 1-30 ng / mL, 1 -40 ng / mL, 1 -50 ng / mL, 5-10 ng / mL, 5-20 ng / mL, 10-20 ng / mL, 10-30 ng / mL, 10-40 ng / mL, 10-50 ng / mL, 20-30 ng / mL, 20-40 ng / mL, 20-50 ng / mL, 30-40 ng / mL, 30-50 ng / mL, 40-50 ng / mL, 1-100 ng / mL, 50-100 ng / mL, 60-100 ng / mL, 70-100 ng / mL, 80-100 ng / mL, 90-100 ng / mL, 10-100 ng / mL, 50-200 ng / mL, 100-200 ng / mL, 50-300 ng / mL, 100-300 ng / mL, 200-300 ng / mL, 50-400 ng / mL, 100-400 ng / mL, 200-400 ng / mL, 300-400 ng / mL, 50-500 ng / mL, 100-500 ng / mL, 200-500 ng / mL, 300-500 ng / mL, 400 to 500 ng / mL, 0.001 -1 pg / mL, 0.001 -2 pg / mL, 0.001 -3 pg / mL, 0.001-4 pg / mL, 0.001 -5 pg / mL, 0.001 -6 pg / mL, 0.001 -7 pg / mL, 0.001 -8 pg / mL, 0.001 -9 pg / mL, 0.001 -10 pg / mL, 0.01 -1 pg / mL, 0.01 -2 pg / mL, 0.01 -3 pg / mL, 0.01 -4 pg / mL, 0.01 -5 pg / mL, 0.01 -6 pg / mL, 0.01 -7 pg / mL, 0.01 -8 pg / mL, 0.01 -9 pg / mL, 0.01 -10 pg / mL, 0.1 -1 pg / mL, 0.1 -2 pg / mL, 0.1 -3 pg / mL, 0.1 -4 pg / mL, 0.1 -5 pg / mL, 0.1 - 6 pg / mL, 0.1-7 pg / mL, 0.1 -8 pg / mL, 0.1 -9 pg / mL, 0.1 -10 pg / mL, 0.5-1 pg / mL, 0.5-2 pg / mL, 0.5-3 pg / mL, 0.5-4 pg / mL, 0.5-5 pg / mL, 0.5-6 pg / mL, 0.5-7 pg / mL, 0.5-8 pg / mL, 0.5-9 pg / mL, 0.5-10 pg / mL, and the like.Screening Methods

[0110] Methods and culture systems are provided for screening candidate agents or cells for an activity of interest. In these methods, candidate agents or cells are screened for their effect on cells in the PDO of the disclosure.

[0111] The effect of an agent or cells is determined by adding the agent or cells to the cells of the cultured explants as described herein, usually in conjunction with a control culture of cells lacking the agent or cells. The effect of the candidate agent or cell is then assessed by monitoring one or more output parameters. Parameters are quantifiable components of explants or the cells thereof, particularly components that can be accurately measured, in some instances in a high throughput system. For example, a parameter of the explant may be the growth, differentiation, survival, gene expression, proteome, phenotype with respect to markers etc. of the explant or the cells thereof, e.g. any cell component or cell product including cell surface determinant, receptor, protein or conformational or posttranslational modification thereof, lipid, carbohydrate, organic or inorganic molecule, nucleic acid, e.g. mRNA, DNA, etc. or a portion derived from such a cell component or combinations thereof. While most parameters will provide a quantitative readout, in some instances a semi- quantitative or qualitative result will be acceptable. Readouts may include a single determined value, or may include mean, median value or the variance, etc. Characteristically a range of parameter readout values will be obtained for each parameter from a multiplicity of the same assays. Variability is expected and a range of values for each of the set of test parameters will be obtained using standard statistical methods with a common statistical method used to provide single values.

[0112] Candidate agent or cells can be added to the cells within the intact organoid. In other embodiments, the organoids are dissociated, and candidate agent or cells is added to the dissociated cells. The cells may be freshly isolated, cultured, genetically altered as described above; or the like. The cells may be environmentally induced variants of clonal cultures: e.g.split into independent cultures and grown into organoids under distinct conditions, for example with or without pathogen; in the presence or absence of other cytokines or combinations thereof. The manner in which cells respond to an agent, particularly a pharmacologic agent, including the timing of responses, is an important reflection of the physiologic state of the cell.

[0113] Candidate agents of interest for screening include known and unknown compounds that encompass numerous chemical classes, primarily organic molecules, for example antibodies, cytokines, etc. genetic sequences, etc. An important aspect of the invention is to evaluate candidate agents to predict patient responsiveness to immune-oncology agents.

[0114] In some cases, the candidate polypeptide agents to be screened are antibodies. The term “antibody” or “antibody moiety” is intended to include any polypeptide chain-containing molecular structure with a specific shape that fits to and recognizes an epitope, where one or more non-covalent binding interactions stabilize the complex between the molecular structure and the epitope. The specific or selective fit of a given structure and its specific epitope is sometimes referred to as a “lock and key” fit. The archetypal antibody molecule is the immunoglobulin, and all types of immunoglobulins, IgG, IgM, IgA, IgE, IgD, etc., from all sources, e.g. human, rodent, rabbit, cow, sheep, pig, dog, other mammal, chicken, other avians, etc., are considered to be “antibodies.” Antibodies utilized in the present invention may be either polyclonal antibodies or monoclonal antibodies. Antibodies are typically provided in the media in which the cells are cultured.

[0115] Candidate agents may be obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds, including biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs.

[0116] Candidate agents are screened for biological activity by adding the agent to at least one and usually a plurality of explant or cell samples, usually in conjunction with explants not contacted with the agent. The change in parameters in response to the test agent is measured, and the result evaluated by comparison to reference cultures, e.g. in the presence and absence of the agent, obtained with other agents, etc.

[0117] The agents are conveniently added in solution, or readily soluble form, to the medium of cells in culture. The agents may be added in a flow-through system, as a stream, intermittent or continuous, or alternatively, adding a bolus of the compound, singly orincrementally, to an otherwise static solution. In a flow-through system, two fluids are used, where one is a physiologically neutral solution, and the other is the same solution with the test compound added. The first fluid is passed over the cells, followed by the second. In a single solution method, a bolus of the test compound is added to the volume of medium surrounding the cells. The overall concentrations of the components of the culture medium should not change significantly with the addition of the bolus, or between the two solutions in a flow- through method. Alternatively, the agents can be injected into the explant, e.g. into the lumen of the explant, and their effect compared to injection of controls.

[0118] Preferred agent formulations do not include additional components, such as preservatives, that may have a significant effect on the overall formulation. Thus preferred formulations consist essentially of a biologically active compound and a physiologically acceptable carrier, e.g. water, ethanol, DMSO, etc. However, if a compound is liquid without a solvent, the formulation may consist essentially of the compound itself.

[0119] A plurality of assays may be run in parallel with different agent concentrations to obtain a differential response to the various concentrations. As known in the art, determining the effective concentration of an agent typically uses a range of concentrations resulting from 1 :10, or other log scale, dilutions. The concentrations may be further refined with a second series of dilutions, if necessary. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the level of detection of the agent or at or below the concentration of agent that does not give a detectable change in the growth rate.

[0120] In some embodiments, a candidate agent is screened for activity that is anti- tumorigenic (i.e. inhibiting cancer initiation) or anti-tumoral (i.e. inhibiting cancer progression, e.g. proliferation, invasion, metastasis). In such embodiments, the explant culture includes cancer cells, including cells suspected of being cancer stem cells. Assessment of anti-tumor activity may include measurements of one or more parameters including explant growth, the rate or extent of cell proliferation, the rate or extent of cell death, etc. Assessment of antitumor activity may also include analysis of markers of immune cell activation (which include but are not limited to IFN-y, granzyme, perforin, etc), expansion or alteration of immune cell populations (T, B, NK, monocyte / myeloid, dendritic cells, myeloid-derived suppressor cells), tumor cell death, tumor phagocytosis and the like. Immune cells could be isolated and / or analyzed by any number of means including FACS, CyTOF, MIBI, multiplexed immunohistochemistry, quantitative RT-PCR, Luminex or others.

[0121] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the subject invention, and are not intended to limit the scope of what is regarded as the invention.EXPERIMENTAL

[0122] The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.EXAMPLE 1Adult bone marrow organoids enable sustained physiologic and malignant hematopoiesis within a native niche microenvironment

[0123] Provided herein is a bone marrow organoid (BMO) system that grows intact fragments of mouse and human bone marrow in an air-liquid interface (ALI) with optimized medium. The resulting BMO robustly co-preserve HSC, progenitors and stroma within their native microenvironment, including bone, vasculature, and mesenchymal stem cells. Crucially, BMO allow long-term, multi-lineage physiologic hematopoiesis, and also propagation of leukemic blasts.

[0124] We first created mouse bone marrow organoids (mBMO). Naive 10-12-week-old C57 / BL6 mouse femur heads, tibial plateau and sternum were processed to intact fragments of trabecular bone marrow, plated in collagen I gel and cultured in ALI. We extensively optimized culture conditions and included additives to support hematopoietic and stromal components including endothelium.

[0125] Bone marrow-rich regions of mouse bones including femoral head, proximal tibia and sternum were minced into small (~1 mm) bone fragments and plated into type I collagen and / or matrigel into a transwell, air liquid interface culture system. Bone marrow organoid cultures are grown in IMDM-based, serum free media containing polyvinyl alcohol, UM729, TPO (1-20 ng / ml), SCF (10 ng / ml), EPO (20 ng / ml), EGF (50 ng / ml), FGF10 (100 ng / ml) and various supplements (N2, B27, ITS) under hypoxic conditions (5% 02). Media was generally changed every 3-4 days until endpoint analysis.

[0126] The initial mouse femur trabecular bone fragments reproducibly showed extensive stromal and hematopoietic outgrowth into the surrounding collagen (Fig. 1b, c). Mouse BMO at culture day 20 exhibited a stromakhematopoietic ratio of 10:90, comparable to fresh bone trabecular bone fragments. Flow cytometry of day 47 mBMO revealed HSC (CD45+, lineage , CD34 , cKit+, Sca1+, CD150+, CD48 ) and progenitor cells (CD45+, lineage , CD34 , cKit+,Sca1+,CD48+) and continuous differentiation to erythroid (CD71 / Ter1 19+), megakaryocytic (CD41+) and myeloid (Gr-1+) lineages (Fig. 1 d). Histology (Giemsa) indicated multilineage mBMO differentiation to stem / progenitor cells, megakaryocytes, erythroblasts and mature neutrophils (Fig. 1d). At day 20, BMO showed 33-fold expansion of total live cell numbers vs. fresh input bone marrow-derived single cells (p<0.0001 ) (Fig. 1 e).

[0127] Colony forming unit (CFU) assays were performed from fresh trabecular bone marrow fragments vs. cognate mBMO to confirm functional hematopoietic stem and progenitor cells (HSPC). Crucially, we confirmed clonogenic CFUs even after 92 days of BMO growth (Fig. 1f) with a >5-fold enrichment of HPSC during the 92 day mBMO culture (p<0.05, Fig. 1 f), attributable to significant increased granulocyte (-G) and monocyte (-M) progenitors (Fig. 1 g). Thus, ALI BMO maintain and expand hematopoietic stem and progenitor cells and allow multilineage hematopoietic differentiation and proliferation within the same holistic culture.

[0018] Next, we examined if the stromal compartment of BMOs recapitulated an adult bone marrow niche. Flow cytometry of mBMO at day 47 showed endothelium (CD45 ,CD31+), mesenchymal stem / stromal cells (MSC) (CD45 , LEPR+and / or CD105+), fibroblasts (CD45 , PDGFRA+), and osteoblasts (CD45 , CD51+) (Fig. 2a). BMO-derived MSC at day 42 exhibited stem cell differentiation to adipocytes, osteocytes, and chondrocytes (Fig. 2b). Day 35 BMO showed 3D architecture with vimentin+ stroma, CD45+ hematopoietic cells, and notably, CD31 + endothelial capillaries (Fig. 2c). Further, the BMO niche autonomously produces diverse growth factors such as IL-6 and vascular endothelial growth factor (VEGF), and chemokines known to be important in hematopoiesis (e.g. CXCL1 , CXCL5, CXCL10, CCL2- 5) (Fig. 2d). Combined, it is shown that ALI BMO are a self-sustainable entity with vascular structures, MSC, stromal cells, osteoblasts, and hematopoietic cells with autonomous secretion of essential niche factors that have been previously implicated in HSC maintenance and lineage differentiation.

[0129] To rigorously confirm HSC functionality and multilineage potential of mBMO, we transplanted single cells from Ly5.2(+) 3-week-old mBMO into lethally irradiated Ly5.1 (+) recipient animals (n=5) (Fig. 3a) of which 4 mice survived at 12 weeks with multilineage hematopoiesis with blood hemoglobulin (Hb) 10-12 g / dl, platelets 150-400 x 103 / ml and WBC 3-9 x 103 / ml (Fig. 3b). Lineage contribution of Ly5.2(+) WBC in peripheral blood revealed both lymphopoiesis and myelopoiesis from BMO progenitors in 3 / 5 mice at week 12 after transplant consistent with the presence of multipotent progenitors and short-term HSCs within BMOs (Fig. 3c). 4 mice continue to survive at day 1 12 with multilineage hematopoiesis (hemoglobulin (Hb) 1 1-12 g / dl, platelets 200-400 x 103 / ul, WBC 2-10 x 103 / jxl); 3-week-old mBMO with EPO exhibit identical rescue. Lineage contribution of Ly5.2(+) WBC in peripheral blood revealed lymphopoiesis and myelopoiesis at day 1 12 consistent with multipotent progenitors and shortterm HSCs in BMOs.

[0130] The BMO method was then extended to normal human bone marrow, as shown in FIG. 4A-4D. In a direct culture of of normal human bone marrow biopsies in ALI with schematic and implementation, where (B) normal human ALI BMO from a core biopsy of a healthy individual shows outgrowth of hematopoietic cells and phenotypic HSCs and HPCs (culture day 34). Flow cytometry analysis shows the presence of stromal cells. The hematopoietic component includes HSC, and progenitor cells (HPC). C. Schematic illustration of strategy 2, where human HSPCs are cultures in mouse bone marrow. D. Results of strategy 1 human bone cultures at days 1 and 27, showing D27 retention of erythroid, myeloid, lymphoid, progenitor and stem cell components.

[0131] Healthy human bone marrow were collected from femur head samples obtained from patient undergoing hip replacement surgery. Bone marrow organoids were then generated using the same protocol as described with mouse samples, with the addition of additional cytokines FLT3L (20 ng / ml) and IL-3 (20 ng / ml).

[0132] We evaluated the use of human BMO to culture primary leukemic blasts. The current lack of methods to routinely culture primary human AML blasts has impaired both biological investigations and therapeutics discovery. In vitro propagation of primary AML blasts has been historically difficult as they rapidly apoptosis in conventional suspension cultures. AML blast culture has been attempted ex vivo by mimicking bone marrow niche components, feeder-free growth factor manipulations, co-culture with stroma such as MSCs, or artificial scaffolds. Yet, these efforts lack the stromal complexity, 3D architecture and niches of native bone marrow, and also lack long-term propagation, perhaps underlying the current absence of widely implemented, reliable AML culture.

[0133] We thus created BMO from the femoral bone marrow of diverse human AML PDX models. BMO from a mouse PDX from an adult AML patient with NPM1, DNMT3A, TET2, and FLT3 mutations showed outgrowth at culture day 32 (Fig. 5a). Flow cytometry revealed concordant CD45+CD33+CD13+leukemia-associated immune phenotype (LAIP) of the organoid and the patient’s original AML. Bulk sequencing of BMO hCD45+ cells confirmed 4 / 5 patient mutations (DNMT3A, TET2, FLT3 and NPM1), while a subclone with PTPN11 mutation (Fig. 5a) was lost during PDX formation and / or BMO culture. A day 57 BMO from a PDX from a distinct pediatric AML retained viable blasts by flow immunophenotype and histology. After 50 days culture AML BMO transplanted to immunodeficient NSG mice revealed full-blown hCD45+ human AML after an additional 3 months, affirming that BMO contain functional engraftment-competent blasts. AML PDX BMO blasts also strongly recapitulate the original leukemia copy number variation profile (Fig. 5b). Successful AML hBMO growth for 90 days (longest evaluated) (Fig. 5) may be attributed to the holistic BMO microenvironment.

[0134] BMO were also directly generated from intact fragments of iliac crest bone marrow core biopsies of patients with leukemia. BMO from a pediatric AML patient core bone marrowbiopsy with outgrowth of stroma and hematopoietic cells (Fig. 6). Flow cytometry (d18) revealed a distinct blast population (LAIP: CD45+CD3 CD19 CD38+CD11 b-partial) with normal T (CD45+CD3+) and B cells (CD45+CD19+) (Fig. 6b) and blast histology (Fig. 6c) matching the original AML. I mmunostaining showed hematopoietic cells, capillary structures and stroma in 3D architecture (Fig. 6d). We also successfully generated BMO from core biopsies of two different pediatric B-ALL patients, with outgrowth (Fig. 6e), CD19+CD24+ immunophenotype (Fig. 6f), ALL blast histology at d24 (Fig. 6g), pathogenic KRASA14eTmutation (Fig. 6g) and copy number variation (Fig. 6h), all consistent with the original fresh core biopsies. We have thus successfully created BMO from patient core biopsies, maintaining histology, immunophenotype, mutation, copy number landscape and niche architecture of the original leukemia.

[0135] Leukemic drug response is crucially influenced by the tumor microenvironment (TME). However, in vitro AML therapeutics testing is not typically performed in a holistic TME, given the lack of native BM culture systems. Current AML drug screening typically uses short-term cultures that actively degenerate even without therapeutic challenge. If long-term AML culture were routinely possible, an aspirational goal for drug testing would then overlay a BM microenvironmental niche to study therapy response and resistance. Thus, we directly applied human AML BMO, with stromal niche recapitulation to drug testing and precision medicine.

[0136] The organotypic fragment nature of BMO cultures are well-suited to focused small- scale evaluation of therapeutic agents, for instance to guide clinical decision-making. From AML PDX models (Fig. 7), we miniaturized ALI BMO from 6 well (30 mm insert) to a smaller 24 well (12 mm insert) format, allowing multiple drug testing in AML BMO derived from only one PDX animal. First, we tested venetoclax (FDA-approved BCL2 inhibitor for AML and other leukemias) in PDX BMO from an adult NPM1 and FLT3 mutated AML. As venetoclax resistance in FLT3-ITD mutated AML can be overcome by JAK inhibition, we tested venetoclax + / - ruxolitinib (JAK1 / 2 inhibitor). At 14 days in vitro single ruxolitinib or venetoclax treatment, human AML BMO blasts (hCD45+) were reduced or unchanged, respectively. However, the ruxolitinib + venetoclax combination elicited almost quantitative killing of BMO human CD45+ blasts (BMO from n=3 biological replicate mice (Fig. 7a). Second, we grew AML BMO from a distinct pediatric AML PDX mouse model. The AML BMO blasts were unaffected by ruxolitinib, consistent with this AML lacking an activating JAK / STAT pathway mutation. In contrast, venetoclax ablated BMO AML blasts at clinically relevant concentrations (BMO from n=3 biological replicate mice, (Fig. 7b).

[0137] Media optimization to regulate BMO hematopoietic and stromal cell-type composition, shown in FIG. 8. Murine BMO (mBMO) were created from trabecular bone from the sternum, femur and tibia of wild-type C57BL / 6 mice. The bones are minced to intact bone marrow fragments and embedded in collagen I gel in an inner transwell at an air-liquid interface (ALI)allowing direct non-submerged air exposure. An outer dish surrounding the transwell contains serum-free HSC medium (including thrombopoietin (TPO), murine stem cell factor (SCF), and the pyromido-indole derivative UM729, with further growth factors to simultaneously support both HSC and the BMO stromal compartment). The microenvironment of ALI BMO successfully preserves both HSC and multilineage differentiation. Studies omitting TPO strongly decreased HSCs and progenitors (HSPC) suggested that some cytokines produced extramedullary are essential to maintain BMO. Accordingly, 6-week-old BMO cultures require EPO to successfully rescue a lethally irradiated host. Adding EPO increased erythroid progenitors and HSPCs (CFU-GEMM).

[0138] Our overall innovation is the successful generation of a holistic bone marrow organoid (BMO) system for normal and malignant hematopoiesis, preserving the native bone marrow microenvironment en bloc without reconstitution, while supporting HSC preservation, progenitor expansion, differentiation and leukemic blasts. In marked contrast to other 2D, 3D or hybrid models, BMO preserve the architecture and ECM of intact trabecular bone marrow fragments within an air-liquid interface (ALI), yielding a complex niche with endothelial networks, MSC and osteoid. This vastly surpasses current systems where scaffolds or ECM are reconstituted to recreate bone marrow architecture and are often seeded with a single stromal population.

[0139] BMOs enable prolonged culture of HSCs, progenitors and continuous trilineage differentiation (>80 days), with autonomous niche cytokine production, and can be derived from the primary tissue, and hence represent interpersonal variability of the bone marrow niche. In contrast, iPSC bone marrow systems require initial differentiation to HSCs, niche cells and mature blood cells (12 days), have limited subsequent culture duration (~12 days), need defined cytokine cocktails to mimic the microenvironment, lack of osteoid, adipocytes and lymphoid cells.

[0140] ALI BMO also enable human acute leukemia culture within the native BM microenvironment, from both PDX (>90d, longest examined) and patient-derived bone marrow core biopsies (20-50d). This is in marked distinction to conventional AML culture, typically measured in days.

[0141] The long-term culture of ALI BMO of both normal hematopoiesis and leukemic blasts is achieved without adding supraphysiological levels of hematopoietic cytokines, many of which are produced endogenously in BMO to foster paracrine / autocrine signaling and self- sustaining growth.

[0142] This platform fills a long-standing need for evaluating normal hematopoiesis, hematopoiesis-stimulating factors, pathogenic injury and toxicology. AML BMO uniquely enables profiling AML drug response in a fully native BM microenvironment over weeks,allowing readout of therapeutics that gradually induce effects or differentiation (e.g. menin and IDH2 inhibitors, ATRA, hypomethylating agents) and that are poorly suited to conventional short-term cultures. The leukemia BMO also provides a novel native bone marrow microenvironment organoid drug testing platform that is tailored to clinical mutations and subtypes, correlated to available clinical or PDX responses.Example 2Patient-derived leukemia bone marrow organoids

[0143] Currently, no ex vivo model can represent the complex microenvironment of the adult stem cell bone marrow niche. Hence there is no good way to long-term culture normal or malignant hematopoietic cells in vitro. \Ne developed an adult stem cell bone marrow niche organoid model containing not only the stem cells of the hematopoietic lineage, but also the stem cells of the stromal lineage and the bone cells and scaffold. We use the native bone architecture including the native stromal and hematopoietic cells as the basis of our model. These bone fragments maintain mesenchymal and hematopoietic stem cells long-term and provide an adequate niche for continuous hematopoietic differentiation of normal blood progenitor cells (into e.g. erythrocytes, megakaryocytes and mature myeloid cells, like neutrophils). The same method can also be used to long-term culture leukemic blasts ex vivo, both from patient-derived xenograft models as well as from patient-derived core biopsies.

[0144] Blood stem cells are nurtured and protected in the local microenvironment of the bone marrow, and 3D cellular interactions within the bone marrow are essential for an adequate blood cell production. This microenvironment is a complex niche containing not only hematopoietic stem cells, but also mesenchymal stem cells, their progeny, and endothelial cells. Within this bone marrow niche hematopoietic stem- and progenitor cells can self-renew and differentiate into the megakaryocyte-erytoid lineage, the myeloid lineage, and the lymphoid lineage. The bone marrow niche serves as a holistic entity that provides cytokines and chemokines for this process to occur. Only a few cytokines are produced outside the bone marrow niche, e.g. thrombopoietin (TPO) and erythropoeitin (EPO).

[0145] Leukemia cells hijack the bone marrow niche and can eliminate normal hematopoiesis completely. Leukemia cells are nurtured and protected within this niche and need interplay with the stromal compartment of the bone marrow niche to survive. Ex vivo leukemic cells are vulnerable and many primary leukemias cannot be culture for more than a few days in vitro. A bona fide ex vivo bone marrow niche would enable the study and targeting of leukemic cells for an extended period of time within their natural microenvironment. There is currently no such system available.

[0146] Shown in FIG. 1 , trabecular bone fragments from C57 / BL6 mice (femur heads, tibia plateau and sternum combined) were cultured in porcine-derived collagen type 1 A in an air-liquid-interface under hypoxic conditions. Bone marrow organoids of 5 different mice were initiated. Brightfield imaging of the entire insert (30mm) was performed with a Keyance brightfield microscope at day 1 , day 10, day 19, and day 25 after initiation of cultures. After preparation of bone fragments, 25% of the fragments were immediately dissociated and digested with collagenase and Liberase, after which live cells were counted by trypan blue assay. The total number of cells was recalculated for the entire mouse (at day 0: x4). The rest of the bone fragments (75%) were divided into 8 different 30 mm inserts for organoid formation with two different types of media: bone marrow organoid medium (mouse BMOM) and bone marrow organoid medium supplemented with EPO (1 U / ml) and IL7 (10ng / ml). After 20 days of culture, one of the wells was sacrificed and dissociated and digested into single cells. Live cells were counted by trypan blue assay and numbers were recalculated for the entire mouse (x10.7).

[0147] Trabecular bone fragments from C57 / BL6 mice (femur heads, tibia plateau and sternum combined) were cultured in porcine-derived collagen type 1 A in an air-liquid-interface under hypoxic conditions. Brightfield imaging of the entire insert (30mm) was performed with a Keyance brightfield microscope at day 1 , day 23, day 45, and day 92 after initiation of cultures. Trabecular bone fragments from C57 / BL6 mice (femur heads, tibia plateau and sternum combined) were cultured in porcine-derived collagen type 1 A in an air-liquid-interface under hypoxic conditions for 47 days. Single cells were harvested at day 47 of culture from the bone marrow organoids and stained with FVS780, CD45, CD1 17 (cKit), Seal , CD48, CD150, CD71 (erytroid progenitors), Ter1 19 (erytroid cells), CD19 (B cells), B220 (B cells), CD3 (T cells), Gr-1 (myeloid cells) within multiple panels to stain both hematopoietic stem- and progenitor cells and differentiated hematopoietic cells. After 47 days of culture, flow cytometric analysis showed the maintenance of hematopoietic stem- and progenitor cells in these cultures. Also multilineage differentiation was confirmed by surface marker expression. Before staining, a cell smear was created of part of the single cell suspension and stained with May-Grunwald Giemsa to show phenotypic multilineage differentiation within the bone marrow organoids.

[0148] Shown in FIG. 9, trabecular bone fragments from C57 / BL6 mice (femur heads, tibia plateau and sternum combined) were cultured in porcine-derived collagen type 1 A in an airliquid-interface under hypoxic conditions for 47 days. Single cells were harvested at day 47 of culture from the bone marrow organoids and stained with FVS780, lineage cocktail, CD45, CD1 17 (cKit), Seal , CD48, CD150 to be able to quantify hematopoietic stem- and progenitor cells. After 47 days of culture, flow cytometric analysis show the maintenance of hematopoietic stem- and progenitor cells in these cultures. B. Single cells suspensions derived from bone marrow organoid cultures were plated in methylcellulose to start a colony-forming unit assay (according to manufacturers protocol). In this assay, every progenitor cell or stem cell makesone colony. 50.000 single cells were plated in the CFU assay and colony forming units were counted after 10-14 days of growth. C. Quantification of CFUs was performed by both manual count and by Image J software and plotted per CFU type. CFU-G, granulocyte colony-forming unit; CFU-M, monocyte colony-forming unit; CFU-GM, granulocyte-monocyte colony-forming unit; CFU-GEMM, granulocyte-erythrocyte-monocyte-megakaryocyte colony-forming unit; BFU-E, burst forming unit erythrocyte. D. Quantification of total CFU per timepoint of culture shows a significant enrichment of stem- and progenitor cells during bone marrow organoid culture. Statistical differences between groups were tested with a two-way ANOVA, * p<0.05.

[0149] Trabecular bone fragments from C57 / BL6 mice (femur heads, tibia plateau and sternum combined) were cultured in porcine-derived collagen type 1 A in an air-liquid-interface under hypoxic conditions for 24 and 47 days, as shown in FIG. 10. Single cells were harvested at day 47 of culture from the bone marrow organoids and stained with FVS780, CD45, CD31 , CD105, LEPR, CD51 , PDGFRa, and CD146 to differentiate different stromal subsets within the bone marrow organoids. After 47 days of culture, flow cytometric analysis show the maintenance of endothelial cells, mesenchymal cells, fibroblasts, and osteoblasts. After harvest from bone marrow organoids, single cell suspensions were plated in plastic flasks in MSC expansion medium. After expansion, MSCs were differentiated towards adipocyte, osteocyte, and chondrocyte lineages and stained with Oil Red O and Alcian Blue to confirm adipocyte and chondrocyte differentiation respectively. After 42 days of culture, bone marrow organoids still contain mesenchymal stem cells harboring multilineage differentiation potential. Bone marrow organoids were fixed with 4% paraformaldehyde and permeabilized within their collagen matrix and subsequently stained with primary antibodies against CD45 (hematopoietic cells), Vimentin (stromal cells) and CD31 (endothelial cells). After 35 days, we confirm the presence of hematopoietic cells, vascular structures and stromal cells and their 3- dimensional architecture.

[0150] Bone marrow organoids were stained with live surface marker-directed antibodies (designed for flow cytometry) overnight within their collagen matrix and subsequently washed with culture medium for >3 times by letting medium pass through the collagen matrix. Bone marrow organoids were stained with CD31 (endothelial cells), CD45 (hematopoietic cells), CD105 (subpopulation of MSCs), CD1 1 b (myeloid cells), and / or CD90 (subpopulation of MSCs and fibroblasts, but also hematopoietic progenitors). Organoids were subsequently imaged using a Zeiss LSM900 confocal microscope after which Z-stacked images were analyzed using Imaris software. Images are representative images of multiple organoids from multiple mice on day 8, day 21 and day 28 of organoid culture, shown in FIG. 1 1 . Bone marrow organoids were stained as described above after which Z-stacked imaging was performed overnight in an environmental chamber with 5% CO2 at 37 degrees Celsius. Representativelive imaging shows living vascular structures and interaction between stromal cells and hematopoietic cells within bone marrow organoids.

[0151] Patient-derived leukemic blasts from an adult patient with acute myeloid leukemia (AML) were transplanted and engrafted in immunocompromised mice (NSG). After engraftment, mice were held until disease symptoms appeared in order to mimick the equivalent of a full blown leukemia. Trabecular bone fragments (femur heads, tibia plateau and sternum) from these mice were cultured in BME2 in an air-liquid-interface under hypoxic conditions. Brightfield imaging of the entire insert (12mm) was performed with a Keyance brightfield microscope at day 32 after initiation of cultures. Flow cytometric analysis of the single cells harvested from the leukemia bone marrow organoids were stained with human CD45, human CD33 and human CD13 antibodies. We confirmed that the leukemia-associated immune phenotype (LAIP) of the patient was similar to the LAIP of the organoid-derived cells. Bulk sequencing of the CD45+ cells from the organoid confirmed the presence of 4 / 5 mutations (DNMT3A, TET2, FLT3 and NPM1 mutations) present in the organoid-derived leukemia cells. A subclone with a PTPN1 1 mutation was lost during PDX formation or organoid culture. Trabecular bone fragments of n = 3 PDXs were cultured in a 24 wells format using 12 mm inserts. This allowed the study of targeted therapy effects in multiple different conditions. We decided to test the drug venetoclax (a BCL2 inhibitor used in multiple phase 1 / 2 trials for primary, refractory or relapsed AML). Since it is known that patients with a FLT3-ITD of FLT3 mutation might need another targeted therapy to enhance the effect of venetoclax, we also tested ruxolitinib (a JAK1 / 2 inhibitor) and combinations of these drugs. After 14 days of monotreatment with either ruxolitinib or venetoclax, the human CD45+ population was reduced or unchanged, respectively. When these drugs were given as combination treatment almost all human CD45+ blasts were irradicated. Statistical differences between groups were tested with a two-way ANOVA, * p<0.05

[0152] Patient-derived leukemic blasts from a pediatric patient with an AML with a Evil rearrangement (t2;3) were transplanted and engrafted in immunocompromised mice (NSG), shown in FIG. 12. After engraftment, mice were held until disease symptoms appeared in order to mimick the equivalent of a full blown leukemia. Trabecular bone fragments (femur heads, tibia plateau and sternum) from these mice were cultured in porcine-derived collagen 1 A in an air-liquid-interface under hypoxic conditions. Brightfield imaging of the entire insert (12mm) was performed with a Keyance brightfield microscope at day 57 after initiation of cultures. B. Single cells harvested from the leukemia bone marrow organoids were used to make a cell smear on a glass slide. Cells were fixed with ice-cold 100% methanol and subsequently stained with May-Grunwald and Giemsa to show phenotypic blasts within the leukemia bone marrow organoids. C. Flow cytometric analysis of the single cells harvested from the leukemia bone marrow organoids were stained with moiuse CD45, human CD45,and human CD34 antibodies. We confirmed that the leukemia-associated immune phenotype (LAIP) of the patient was similar to the LAIP of the organoid-derived cells. D. Single cells derived from 50 day-old leukemia bone marrow organoids were used to inject a sublethally irradiated NSG mouse to show leukemic potential after culture. We show the engraftment and outgrowth of leukemia within this mouse. E. We studied the LAIP described in (c) over time, and show long-term persistence of phenotypical leukemic blasts in multiple animals (n=4) in leukemic niche organoids, even after engraftment in a new animal. F. Trabecular bone fragments of n = 3 PDXs were cultured in a 24 wells format using 12 mm inserts. This allowed the study of targeted therapy. We treated organoids for 14 days with the drugs venetoclax (a BCL2 inhibitor used in multiple phase 1 / 2 trials for primary, refractory or relapsed AML) and Ruxolitinib (a JAK1 / 2 inhibitor). Human CD45+ population was unaffected by two concentrations of Ruxolitinib, which was expected since this leukemia does not have an activating mutation in the JAK / STAT pathway. In contrast, Venetoclax irradicated human leukemic blasts in clinically relevant concentrations (serum levels of Venetoclax in patients can reach 1000 nM). G. Relative amount of leukemic blasts per all events compared to the DMSO control condition. Statistical differences between groups were tested with a two-way ANOVA, *“ p<0.001.

[0153] A trabecular bone biopsy (core) was obtained from a pediatric patient with an inversion- 16 (inv16) positive AML at diagnosis, shown in FIG. 13. The core biopsy was cut into small trabecular bone fragments and cultured in the above described ALI system in porcine-derived collagen 1A in under hypoxic conditions. Brightfield imaging of the entire insert (12mm) was performed with a Keyance brightfield microscope at day 10 and day 31 after initiation of cultures. B. After 18 days one of the inserts was sacrificed to perform flow cytometry. Single cells harvested from the leukemia bone marrow organoids were stained with FVS780 (live / dead), CD45, CD3, CD19, CD38, and CD34 antibodies. We show presence of both stromal and hematopoietic cells and show that the hematopoietic population mainly consists of CD3 negative, CD19 negative, CD38 positive, partially CD34 positive leukemic blasts. Next to this leukemic population, we also see the presence of healthy B cells and T cells. D. After 31 days of culture, bone marrow organoids were fixed with 4% paraformaldehyde and permeabilized within their collagen matrix and subsequently stained with primary antibodies against CD45 (hematopoietic cells), Vimentin (stromal cells) and CD31 (endothelial cells). Nuclei were stained with DAPI. After 35 days of organoid culture, we confirm the presence of leukemic cells, vascular structures and stromal cells and their intact 3-dimensional architecture. 9E. A trabecular bone biopsy (core) was obtained from a pediatric patient with a B cell acute lymphoblastic leukemia (B-ALL) at diagnosis. The core biopsy was cut into small trabecular bone fragments and cultured in the above described ALI system in porcine-derived collagen 1A in under hypoxic conditions. Brightfield imaging of the entire insert (12mm) wasperformed with a Keyance brightfield microscope at day 11 and day 24 after initiation of cultures. F. After 24 days single cells harvested from the primary leukemia bone marrow organoids and stained with FVS780 (live / dead), CD45, CD19, and CD34 antibodies. This patient’s blasts were CD45 negative, CD19 positive, CD34 positive at diagnosis. We show the preservation of this LAIP in our organoid cultures. G. The same cell suspension (as f.) was used to make a cell smear on a glass slide. Cells were fixed with ice-cold 100% methanol and subsequently stained with May-Grunwald and Giemsa to show phenotypic lymphoid blasts within the leukemia bone marrow organoids.

[0154] A trabecular bone biopsy (core) was obtained from adult patient with multiple myeloma (MM) at diagnosis, shown in FIG. 14. The core biopsy was cut into small trabecular bone fragments and cultured in the above described ALI system in porcine-derived collagen 1 A in under hypoxic conditions. Brightfield imaging of the entire insert (12mm) was performed with a Keyance brightfield microscope at day 6 and day 20 after initiation of cultures and zoomed in for increased detail. B. After 26 days single cells harvested from the primary MM bone marrow organoids and stained with FVS780 (live / dead), CD45, CD19, CD3, CD38, and CD138 antibodies. This patient’s blasts were CD45, CD19, CD3 negative, and CD18 CD138 positive at diagnosis. We show the preservation of this LAIP in our organoid cultures. C. A trabecular bone biopsy (core) was obtained from adult patient with Waldenstrom Macroglobulinemia (WM) at diagnosis. The core biopsy was cut into small trabecular bone fragments and cultured in the above described ALI system in BME2 under hypoxic conditions. Brightfield imaging of the entire insert (12mm) was performed with a Keyance brightfield microscope at day 1 , day 8, and day 36 after initiation of cultures and zoomed in for increased detail. D. After 36 days single cells harvested from the primary WM bone marrow organoids and stained with FVS780 (live / dead), CD45, CD19, CD10, CD38, and IgM antibodies. This patient's blasts were CD45, CD19, CD38, IgM positive at diagnosis. We show the preservation of this LAIP in our organoid cultures. E. Patient-derived MM bone marrow organoids were stained with live surface marker- directed antibodies (designed for flow cytometry) overnight within their collagen matrix and subsequently washed with culture medium for >3 times by letting medium pass through the collagen matrix. Bone marrow organoids were stained with CD38 (MM marker), CD45 (hematopoietic cells), and CD105 (subpopulation of MSCs). Organoids were subsequently imaged using a Zeiss LSM900 confocal microscope after which Z-stacked images were analyzed using Imaris software, e. shows a 360 degrees turn of the Z stack to show the 3D structure of the organoid. F. Live imaging of the MM blasts within their microenvironment overnight. G. Patient-derived WM bone marrow organoids were stained with TMRM (a dye accumulating within mitochondria without affecting cell survival and only fluorescing when a cell is alive) and NucDeadRed-647 (a dye staining DNA and hence dead cells). These dyes were added to the medium 3 hours before start op imaging with a LSM900 confocalmicroscope. H. Live imaging of living WM blasts (Z-stacked) and other living cells within the WM microenvironment overnight.

[0155] FIG. 15 depicts a culture from a normal human trabecular bone fragments were obtained during a diagnostic procedure from a child suffering from lymphoma without any bone marrow involvement. B. T rabecular bone fragments were cultured in porcine-derived collagen type 1 A in an air-liquid-interface under hypoxic conditions for 34 days. Single cells were harvested at day 34 of culture from the bone marrow organoids and stained with FVS780, CD45, lineage cocktail, CD38 and CD34. After 34 days of culture, flow cytometric analysis show the maintenance of hematopoietic stem (CD34+ CD38 -) and progenitor cells (CD34+, CD38+) in these cultures.Materials and methodsExperimental Model and Subject Details

[0156] Human specimens. Primary patient bone biopsies were obtained during the diagnostic procedure for new patients with leukemia diagnosed at Stanford University. All experiments utilizing human material were approved by the SUMC Institutional Review Board and performed under protocols #61001 . Written informed consent for research was obtained from donors and / or their parents prior to tissue acquisition. Samples were obtained from adult and / or pediatric male or female patients who were pre-treatment. We collected age, gender, and diagnostic information, which were anonymized. Bone biopsies were directly placed in HypoThermosol FRS preservation medium (Stem Cell Technologies) containing 1 X Normocin (InvivoGen) on ice and stored at 4 degrees Celsius before use. Samples were prepared at the same day of the biopsy.

[0157] Mouse models. Female C57BL / 6 mice were used for generation of normal murine bone marrow organoids. Mice were housed in pairs and used for experimentation at 8-10 weeks of age. Animals were maintained on a 12-hour light / dark cycle, in a temperature- and humidity- controlled room with food and water.

[0158] NOD.Cg-Prkd(^c'dH2rgtm1wj' / Sz (NSG) mice were used to generate patient- derived xenograft models of human leukemia. Primary leukemic cells from patients were CD3-depleted and subsequently injected 4-10-week-old NSG mice. Engraftment was confirmed by peripheral blood sampling and flow cytometry of human CD45 positive cells. Sampling was performed every month and when disease symptoms occurred.Method Details.

[0159] Preparing bone fragments from mice. Murine femur, tibia, and sternum were harvested from C57BL / 6 or engrafted NSG mice. All tissue was placed on ice immediately during the entire procedure. Muscle, tendons and cartilage were removed from the bones. The femoral heads were cut from the shaft with a sharp hardened fine scissors (Fine Science Tools) and handled with Fine Precision Medium Tipped Tweezers (Fisher). Likewise, the tibia plateau and shaft were separated. The shafts containing yellow bone marrow were discarded. Ribs, muscle and subcutaneous fat were removed from the sternum. Sternum, and the trabecular bone parts of the femur heads and the tibia plateau were cut into small fragments of intact bone with a sharp hardened fine scissors. Leukemic bone was cut less extensively compared to normal bone to prevent complete destruction of the bone marrow architecture. All fragments were harvested using Hank’s Balanced Salt Solution (HBSS, Thermo Fisher) and single cells were separated from the bone fragments using a 70 pm cell strainer (Greiner Bio-One). Fifty percent of the single cell suspension (flow through) was used as an internal co-culture control condition. The other 50% of the single cell suspension was viably frozen in Biobanker freeze medium (Wako Chemicals USA) to serve as a day 0 control at time of analysis.

[0160] Preparing bone fragments from patient-derived bone biopsies. Bone biopsies were minced finely on ice with sharp hardened fine scissors, keeping trabecular bone architecture intact. Fragments were harvested using Hank’s Balanced Salt Solution (HBSS, Thermo Fisher) and single cells were separated from the bone fragments using a 70 pm cell strainer (Greiner Bio-One). Fifty percent of the single cell suspension (flow through) was used as an internal co-culture control condition. The other 50% of the single cell suspension was viably frozen in Biobanker freeze medium (Wako Chemicals USA) to serve as a day 0 control at time of analysis.

[0161] AU organoid plates. Inserts containing a permeable, membranous bottom (PICM03050, Millicell-CM, Millipore) were inserted into tissue culture dishes as described (Li et al., 2014, Ootani et al., 2009). Matrices were prepared for transwell inserts by mixing collagen matrix (Cellmatrix type I-, Rat Collagen I), 10 X concentrated sterile Ham’s F-12, and sterile reconstitution buffer (2.2 g NaHCOs in 100 mL of 0.05 N NaOH and 200 mM HEPES) on ice at a ratio of 8:1 :1 until use. After mixing collagen matrix and concentrated culture medium, reconstitution buffer was added and mixed again, avoiding bubbles. This reconstituted collagen solution was kept on ice (4°C) to prevent gel formation until added to the insert. Base Membrane Extract 2 (BME2) was used without reconstitution. For a 6 well plate format, 1 mL of reconstituted collagen solution was added to the insert under sterile conditions, serving as a bottom layer gel without tissue. The bottom layer was left to solidify for 30 min in a 37°C incubator. The upper layer, containing bone fragments in 1 ml of ice-cold matrix was added subsequently on top of the bottom layer.

[0162] ALI cultures. Prepared bone fragments were resuspended in 1 mL of collagen or BME2 (as indicated in figure legends), and layered on top of pre- solidified 1 mL collagen gel within a 30 mm, 0.4 pm inner transwell to form the double dish air-liquid culture system as described above. The transwell containing tumor tissue and collagen was placed into an outer 60 mm cell culture dish containing 1 .2 mL of medium. For a 24 well format culture system, we used 12 mm 0.4 pm inner transwell with 100 pl of matrix as a first layer and 200 pl of matrix as organoid containing layer with 500 pl of culture medium.

[0163] Media for human leukemia bone marrow organoids. For culturing human leukemic organoids (derived from patients or PDX models), we used IMDM supplemented with GlutaMAX supplement, HEPES (1 mM, Invitrogen), B-27 without vitamin A (1 X, Invitrogen), N2 supplement (1 x Invitrogen), ITS-X (1%, Invitrogen), Pen-Strep (1X, Invitrogen), Fungin (InVivoGen), Normocin (InVivoGen), EGF (50 ng / mL, Invitrogen), FGF-10 (100 ng / ml, Invitrogen), Butyzamide (0,1 pM, a TH PO- receptor agonist used clinically as lusutrombopag), 0.1 % PCL-PVAc-PEG (Soluplus; BASF), and UM729 (500 nM, a pyrimidoindole derivative). As indicated primary samples were cultured in presence of Collagenase Inhibitor I (Calbiochem) to prevent degradation of collagen matrices. Media was changed three times a week by manually removing conditioned medium and replacing pre-warmed medium. Media was freshly prepared every 2-4 weeks.

[0164] Media for murine bone marrow organoids. For culturing normal murine bone marrow organoids, we used IMDM supplemented with GlutaMAX supplement, HEPES (1 mM, Invitrogen), B-27 without vitamin A (1 X, Invitrogen), N2 supplement (1 x Invitrogen), ITS-X (1 %, Invitrogen), Pen-Strep (1 X, Invitrogen), Fungin (InVivoGen), Normocin (InVivoGen), EGF (50 ng / mL, Invitrogen), FGF-10 (100 ng / ml, Invitrogen), murine TPO (20 ng / ml Peprotech), murine SCF (10 ng / ml, Peprotech) 0.1 % polyvinylalcohol (PVA), and UM729 (500 nM, a pyrimidoindole derivative). Additionally, in some cases, media was supplemented with recombinant murine IL-7 (10 ng / ml, Peprotech) and EPO (1 lU / ml, Peprotech) as indicated. Media was changed three times a week by manually removing conditioned medium and replacing pre-warmed medium. Media was freshly prepared every 2-4 weeks.

[0165] Harvesting single cells from bone marrow organoids. ALI cultures were harvested in a 15 ml Falcon tube and single cells were prepared by dissociation with 300 units mF collagenase IV (Worthington) at 37°C for 30 min, followed by digestion in Liberase-TL (Roche; 50 pg / mL final concentration) at 37°C for 30 min. Digestion was quenched by 100% FBS and cells were washed 2x with PBS. Single cells were separated from the empty trabecular bone fragments with a 70 pm strainer and used in downstream analysis.

[0166] FACS. FACS staining cocktails for murine cells contained per sample 10 pL Brilliant Stain Buffer (BD).

[0167] The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of the present invention is embodied by the appended claims.

Claims

THAT WHICH is CLAIMED IS:

1. An method for producing an in vitro adult stem cell bone marrow niche organoid model, the method comprising: placing a bone and bone marrow fragment with intact trabecular bone architecture in an air liquid interface transwell system, in a medium that allows long term maintenance and functionality of stromal cells, endothelial cells, mesenchymal stem cells and hematopoietic stem cells.

2. The method of claim 1 , wherein the organoid model maintains functional bone marrow populations for up to 30 days, up to 45 days, up to 60 days, up to 90 days, or more.

3. The method of claim 1 or claim 2, wherein the bone and bone marrow fragment is from a normal human individual.

4. The method of claim 1 or claim 2, wherein the bone and bone marrow fragment is from a human individual with a hematologic cancer.

5. The method of claim 1 or claim 2, wherein the bone and bone marrow fragment is from a normal mouse.

6. The method of claim 1 or claim 2, wherein the bone and bone marrow fragment is from a mouse with a human cancer xenograft.

7. The method of any of claims 1 -6, further comprising the step of contacting the organoid model with a candidate therapeutic agent; and determining the effect of the agent on cells in the organoid model.

8. The method of claim 7, wherein the candidate therapeutic agent is a targeted anticancer agent.

10. The method of claim 9, wherein the cells are sorted or analyzed by flow cytometry or live imaging to determine the effect of the agent.11 . The method of any of claims 7-10, further comprising the step of administering to the patient an therapeutic agent that the patient is determined to be responsive to.

12. The method of any of claims 1 -11 , wherein the organoid model maintains and produces functional stem cells, and progenitor cells.

13. The method of any of claims 1-12, wherein stem and progenitor cells differentiate de novo to effector cells, including neutrophils.

14. The method of any of claims 1 -13, wherein the medium comprises an effective dose of an epidermal growth factor (EGF) agent, an effective dose of a fibroblast growth factor (FGF) agent, an effective dose of a thrombopoietin (TPO) receptor agonist; an effective dose of a stem ceil factor (SCF) agent, and an effective dose of a pyrimidoindole derivative.

15. The method of claim 14, wherein the pyrimidoindole derivative is UM729.

16. The method of claim 14 or 15, wherein the medium further comprises an effective dose of an erythropoietin (EPO) agent.

17. The method of any of claims 14-16, further comprising one or more of an effective dose of a fms-like tyrosine kinase 3 (FLT3) ligand, an effective dose of an interleukin 3 (IL-3) agent, an effective dose of an interleukin 7 (IL-7) agent.

18. An organoid model produced by the method of any of claims 1 -17.

19. A medium for use in the organoid model of any of claims 1 -14, comprising: an effective dose of an epidermal growth factor (EGF) agent, an effective dose of a fibroblast growth factor (FGF) agent, an effective dose of a thrombopoietin (TPO) receptor agonist; an effective dose of a stem cell factor (SCF) agent, and an effective dose of a pyrimidoindole derivative.

20. The medium of claim 19, wherein the pyrimidoindole derivative is UM729.21 . The medium of claim 19 or 20, wherein the medium further comprises an effective dose of an erythropoietin (EPO) agent.

22. The medium of any of claims 19-21 , further comprising one or more of an effective dose of a fms-like tyrosine kinase 3 (FLT3) ligand, an effective dose of an interleukin 3 (IL-3) agent, an effective dose of an interleukin 7 (IL-7) agent