Method for preparing bone and soft tissue sarcoma organoids and uses thereof

Abstract

The application provides a preparation method and application of a bone and soft tissue sarcoma organoid. A bone and soft tissue sarcoma tissue sample is placed in a modified organoid culture medium and placed in a CO2 incubator for culture at 37 DEG C. Half of the medium is replaced every two days, and a bone and soft tissue sarcoma organoid can be obtained within two weeks. The method is simple to operate, significantly improves the survival rate and quantity of organoids, and shortens the culture time of organoids. Eight different subtypes of bone and soft tissue sarcoma organoids can be obtained by using the method. The bone and soft tissue sarcoma organoids obtained by the application can be passaged and stored at low temperature, retain all cell types of the original tumor tissue block, more truly simulate the tumor microenvironment, and provide more clinically consistent drug sensitivity test results. The bone and soft tissue sarcoma organoids obtained by the application can be used for personalized CAR-T treatment evaluation, drug sensitivity detection of chemotherapeutic drugs and targeted drugs, and preparation of personalized tumor vaccines.

Description

Technical Field

[0001] This invention relates to the field of organoid culture technology, and more specifically, to a method for preparing and applying bone and soft tissue sarcoma organoids. Background Technology

[0002] Sarcoma is a type of malignant tumor, accounting for approximately 1% of all human malignant tumors. Sarcomas grow in mesenchymal tissues derived from the embryonic mesoderm, such as muscle, adipose tissue, and bone. Based on the appearance of the primary tissue, sarcomas can be classified into liposarcoma, fibrosarcoma, undifferentiated pleomorphic sarcoma (UPS), synovial sarcoma (SS), other pediatric sarcomas, osteosarcoma, chondrosarcoma, giant cell tumor of bone (GCTB), and Ewing sarcoma (ES). Clinically, surgery is usually the first-line treatment for sarcomas, followed by chemotherapy to prevent tumor metastasis and recurrence. Despite these treatment options, clinical outcomes for sarcomas remain unsatisfactory. Molecular heterogeneity, along with biological and clinical diversity, contributes to these poor outcomes. Therefore, establishing reliable models to reflect the molecular characteristics and heterogeneity of sarcomas for use in drug development and personalized medicine is crucial.

[0003] Traditional in vitro culture models require substantial resources and sustained suppression of fibroblast growth, inevitably leading to the gradual loss of molecular characteristics in tumors during successive passages. Patient-derived xenograft models can preserve components of the tumor environment, but their high maintenance costs make them unsuitable for large-scale drug screening. Artificial organoid systems, capable of representing tumor phenotypes and molecular characteristics, have been used to construct organoids for various tumor types, including intestinal, colorectal, liver, pancreas, prostate, breast, bladder, ovarian, and gastrointestinal tumors. However, most tumor organoid construction methods utilize single cells dissected from tumor tissue, lacking immune or connective tissue elements, and an organoid biobank for bone and soft tissue sarcoma (STBS) has not yet been established. Summary of the Invention

[0004] The purpose of this invention is to provide a method for preparing bone and soft tissue sarcoma organoids and their applications.

[0005] To achieve the objective of this invention, in a first aspect, this invention provides a method for preparing bone and soft tissue sarcoma organoids, comprising the following steps: 1) Obtain bone and soft tissue sarcoma tissue samples and preserve them in tissue preservation solution; 2) Remove the tissue sample from the tissue preservation solution, place it in a sterile container, add cell culture medium, and chop the tissue sample in the cell culture medium to obtain tissue clumps; 3) Pass the chopped tissue sample-cell culture medium suspension through a sterile filter; 4) Rinse the tissue clumps intercepted by the filter into a new sterile container with organoid culture medium, add organoid culture medium, and mix well; 5) Add to a low-adsorption culture dish, place in a CO2 incubator, and incubate at 37°C. Change the medium halfway every two days. After about two weeks of culture, bone and soft tissue sarcoma organoids are obtained.

[0006] Step 1) The tissue preservation solution used can be AQIX® Biopreservative Medium "ready-to-use".

[0007] The cell culture medium described in steps 2) and 3) is DMEM / F12 containing 100 μg / mL Normocin.

[0008] Step 4) describes an organoid culture medium containing 50% L-WRN cells, 1 mM HEPES, 2 mM L-alanyl-L-glutamine dipeptide, 10 mM nicotinamide, 1 mM N-acetylcysteine, 2 v / v % vitamin A-free B-27, and 0.5 μM A83-01 (molecular formula C). 25 H 19 N5S), 100 U / mL penicillin, 100 μg / mL streptomycin, 292 μg / mL L-glutamine, 10 nM gastrin, 10 μM SB-202190 (molecular formula C 20 H 14 DMEM / F12 medium containing FN3O and 50 ng / mL human epidermal growth factor (EGF).

[0009] The method for preparing the L-WRN cell culture medium includes: mixing 1×10 6 -5×10 6 One L-WRN cell was evenly seeded in a 10 cm diameter cell culture dish. Using 10 mL of DMEM / F12 medium containing 10% FBS, the L-WRN cells were cultured at 37°C and 5% CO2 for 3-7 days. The medium was then collected. No medium change was required during the culture period.

[0010] Furthermore, L-WRN cells were obtained by transfecting L-Wnt3A cells with a co-expression vector of R-spondin 3 and noggin. This cell line is a stable, commercially available cell line from ATCC. During culture, these cells secrete Wnt3A, R-spondin 3, and noggin into the culture medium. This method is a classic approach for obtaining cells containing Wnt3A, R-spondin 3, and noggin.

[0011] In the aforementioned method, the bone and soft tissue sarcoma tissue in step 1) is derived from human tissue.

[0012] The bone and soft tissue sarcoma types mentioned in step 1) of the aforementioned method include liposarcoma, fibrosarcoma, pleomorphic undifferentiated sarcoma, synovial sarcoma, osteosarcoma, chondrosarcoma, giant cell tumor of bone, and Ewing sarcoma, etc.

[0013] In the aforementioned method, the volume of the tissue mass in step 2) is less than 0.5mm × 0.5mm, preferably 0.1mm × 0.1mm.

[0014] Furthermore, the tissue mass described in step 2) contains tumor cells, fibroblasts, mesenchymal cells, epithelial cells, macrophages, and natural killer cells, etc.

[0015] In the aforementioned method, the pore size of the filter screen in step 4) is preferably 100 μm.

[0016] Secondly, the present invention provides bone and soft tissue sarcoma organoids prepared according to the above method.

[0017] The bone and soft tissue sarcoma organoids described herein can be cut and recultured for passage.

[0018] The bone and soft tissue sarcoma organoids described above can be preserved for a long time under low temperature conditions (liquid nitrogen).

[0019] Thirdly, the present invention provides any of the following applications of bone and soft tissue sarcoma organoids prepared according to the above method: (1) Used for drug screening; (2) Used for CAR-T cell therapy evaluation; (3) Used for drug sensitivity testing of chemotherapy drugs and targeted drugs; (4) Used in the preparation of personalized tumor vaccines.

[0020] The applications include applying different types, concentrations, and combinations of antitumor drugs to bone and soft tissue sarcoma organs, and testing the therapeutic effects and drug resistance of the drugs on bone and soft tissue sarcoma organs after a period of time.

[0021] The specific steps are as follows: ① When bone and soft tissue sarcoma organoids are used for CAR-T therapy evaluation Bone and soft tissue sarcoma organoids were mixed with CAR-T cells and co-cultured. After 1-7 days, the survival rate of tumor cells, the degree of T cell infiltration, and the secretion of inflammatory factors in the tumor bone and soft tissue organoids were measured to obtain the therapeutic effect of CAR-T cells.

[0022] ② When bone and soft tissue sarcoma organoids are used for chemotherapy drug sensitivity testing The prepared bone and soft tissue sarcoma organoids were cultured with different types, concentrations and mixing methods of chemotherapy drugs. After 3-21 days, the status and activity of the bone and soft tissue sarcoma organoids were examined to rank the optimal chemotherapy drug regimen.

[0023] ③ When bone and soft tissue sarcoma organoids are used for targeted drug sensitivity testing The prepared bone and soft tissue sarcoma-like tumor tissues were cultured with targeted drugs of different types and concentrations. After 3-21 days, the status and activity of the sarcoma-like organs were examined, and the optimal targeted drug regimen was determined.

[0024] Fourthly, the present invention provides an organoid culture medium, which is a DMEM / F12 medium containing 50% L-WRN cell culture medium, 1 mM HEPES, 2 mM L-alanyl-L-glutamine dipeptide, 10 mM nicotinamide, 1 mM N-acetylcysteine, 2% (v / v) vitamin A-free B-27, 0.5 μM A83-01, 100 U / mL penicillin, 100 μg / mL streptomycin, 292 μg / mL L-glutamine, 10 nM gastrin, 10 μM SB-202190 and 50 ng / mL human epidermal growth factor (EGF).

[0025] The method for preparing the L-WRN cell culture medium includes: mixing 1×10 6 -5×10 6 One L-WRN cell was evenly seeded in a 10 cm diameter cell culture dish. Using 10 mL of DMEM / F12 medium containing 10% FBS, the L-WRN cells were cultured at 37°C and 5% CO2 for 3-7 days. The medium was then collected. No medium change was required during the culture period.

[0026] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects: (i) This invention employs a simple method to obtain bone and soft tissue sarcoma organoids, which is easy to operate, significantly improves the survival rate and quantity of organoids, and greatly shortens the organoid culture time. Furthermore, when bone and soft tissue sarcoma organoids obtained using the method of this invention are cultured in high-passage cultures, the organoids exhibit high proliferation capacity and good morphology.

[0027] (ii) Compared to enzymatic digestion, the first passage time for organoids is generally 1-3 weeks, and the number of tumor organoids obtained is small. Enzymatic digestion takes a long time and reduces the viability of organoids. However, the present invention obtains a large number of bone and soft tissue sarcoma organoids without damaging their viability.

[0028] (III) The present invention can obtain at least eight different subtypes of bone and soft tissue sarcoma organoids: liposarcoma, fibrosarcoma, pleomorphic undifferentiated sarcoma, synovial sarcoma, osteosarcoma, chondrosarcoma, giant cell tumor of bone and Ewing sarcoma, etc.

[0029] (iv) The bone and soft tissue sarcoma organoids obtained by this invention retain all cell types of the original tumor tissue block, which can more realistically simulate the tumor microenvironment and provide drug sensitivity test results that are more in line with clinical practice.

[0030] (v) The bone and soft tissue sarcoma organoids obtained by this invention can be passaged and stored at low temperatures.

[0031] (vi) The bone and soft tissue sarcoma organoids obtained by this invention can be used for personalized CAR-T therapy evaluation, and drug sensitivity testing of chemotherapy drugs and targeted drugs.

[0032] (vii) By comparing drug sensitivity tests on bone and soft tissue sarcoma organoids with clinical data, the drugs screened by this invention are consistent with the medications used by patients who have received effective treatment. This invention can provide effective personalized drug selection in the future, eliminating the pain, risks, and economic losses of patients trying drugs on themselves. Attached Figure Description

[0033] Figure 1 The structure of A83-01 and SB-202190 involved in this invention.

[0034] Figure 2 This is a preferred embodiment of the invention showing fragments of a patient's tumor tissue block before and after shearing.

[0035] Figure 3 The image shows a statistical diagram (a) of eight subtypes of bone and soft tissue sarcoma organoids cultured in a preferred embodiment of the present invention, and a microscopic photograph (b) of eight subtypes of bone and soft tissue sarcoma organoids cultured.

[0036] Figure 4 The cell type statistics of single-cell sequencing of fibrosarcoma tissue blocks and corresponding organoids in a preferred embodiment of the present invention are shown.

[0037] Figure 5 The cell type statistics of single-cell sequencing of osteosarcoma tissue blocks and corresponding organoids are shown in the preferred embodiment of the present invention.

[0038] Figure 6 These are photomicrographs of organoids (a), passaged organoids (b), and cryopreserved and revived organoids (c) in a preferred embodiment of the present invention.

[0039] Figure 7In a preferred embodiment of the present invention, bone and soft tissue organoids were used to perform sensitivity tests on five chemotherapeutic drugs; wherein, a~d represent the changes in morphology and size of four organoids—fibrosarcoma, low-grade malignant fibrosarcoma, dermatofibrosarcoma protuberans, and giant cell tumor of bone—when incubated with five chemotherapeutic drugs: ifosfamide (IFO), vincristine (VCR), doxorubicin (DOX), methotrexate (MTX), and carboplatin (CBP).

[0040] Figure 8 The results of CAR-T cell construction verification in a preferred embodiment of the present invention are shown below; where a represents the GD2 CAR or CD19 CAR structure containing GD2 or CD19scFv, CD28 transmembrane constituent region and CD3ζ intradomain sequence; b represents the CAR gene expression level in GD2-CAR T cells or CD19-CAR T cells detected by RT-qPCR; and c represents the CAR protein translation level in GD2-CAR T cells or CD19-CAR T cells detected by Western blotting.

[0041] Figure 9 The following are the results of the sensitivity test of bone and soft tissue sarcoma organoids to CAR-T cells in a preferred embodiment of the present invention; wherein, ab represents the flow cytometry and statistical results of GD2 expression in organoids (SO-2 and SO-31) after incubation with GD2-CAR T cells or CD19-CAR T cells for one day, two days, and three days; c represents the changes in the morphology of organoids (SO-2 and SO-31) and their surface after incubation with GD2-CAR T cells or CD19-CAR T cells for one day, two days, and three days; d represents the secretion levels of IFN-γ, TNF-α, and IL-2 in the culture supernatant of organoids (SO-2 and SO-31) after incubation with GD2-CAR T cells or CD19-CAR T cells for one day, two days, and three days; e and f represent the immunofluorescence results and statistical results of CD3 and CC3 signal intensity (CD3: T cell markers; DAPI: nuclear dye; CC3: cleaved caspase 3.

[0042] Figure 10 This is a transmission electron microscope (TEM) image of a personalized tumor vaccine prepared according to a preferred embodiment of the present invention. In the image, a is a TEM image of PLGA nanoparticles; b is a TEM image of PLGA nanoparticles coated with organoid cell membranes, i.e., a TEM image of the personalized tumor vaccine.

[0043] Figure 11This is a graph showing the morphological and size changes of Ewing sarcoma organoids (SO-2) over time when the organoid culture medium composition is optimized in a preferred embodiment of the present invention. Detailed Implementation

[0044] This invention provides a strategy for preparing STBS organoids from clinical tumor tissues, eliminating the need for single-cell dissociation and enabling establishment within two weeks. The invention covers eight typical STBS organoid subtypes, all of which can be passaged and cryopreserved. The constructed STBS organoids can be used for chemotherapy susceptibility testing and to optimize chemotherapy strategies, reducing potential toxic side effects. Single-cell transcriptomics analysis confirms that STBS organoids retain significant heterogeneity in cell types within the tumor microenvironment (TME), including immune-infiltrating cells. We found a low number of tumor-infiltrating lymphocytes (TILs) in STBS organoids or tumors, which is a reason for poor clinical outcomes in immune checkpoint immunotherapy. Based on this, we used two types of chimeric antigen receptor T cells (CAR-T) to accurately predict the sensitivity of STBS organoids to CAR-T cells, providing effective guidance for personalized tumor immunotherapy.

[0045] The present invention adopts the following technical solution: This invention provides a method for preparing bone and soft tissue sarcoma organoids and their applications.

[0046] The method for preparing the organoids is as follows: Obtain bone and soft tissue sarcoma tissue samples and preserve them in a tissue preservation solution; remove the sample tissue from the preservation solution, place it in a sterile container, add cell culture medium, and chop the sample tissue in the cell culture medium to obtain tissue clumps; pass the chopped sample tissue-cell culture medium suspension through a sterile filter; rinse the tissue clumps intercepted by the filter with organoid culture medium into a new sterile container, add organoid culture medium, and mix well; add to a low-adsorption culture dish, place in a CO2 incubator, and incubate at 37°C, changing the medium halfway every two days. Bone and soft tissue sarcoma organoids can be obtained within two weeks.

[0047] The method for preparing bone and soft tissue sarcoma organoids of the present invention is simple to operate, significantly improves the survival rate and quantity of organoids, and greatly shortens the organoid culture time.

[0048] Using the method of this invention, eight different subtypes of bone and soft tissue sarcoma organoids can be obtained: liposarcoma, fibrosarcoma, pleomorphic undifferentiated sarcoma, synovial sarcoma, osteosarcoma, chondrosarcoma, giant cell tumor of bone, and Ewing sarcoma, etc.

[0049] The bone and soft tissue sarcoma organoids obtained by this invention can be passaged and cryopreserved, and retain all cell types of the original tumor tissue block, which can more realistically simulate the tumor microenvironment and provide drug sensitivity test results that are more in line with clinical practice.

[0050] The bone and soft tissue sarcoma organoids obtained by this invention can be used for personalized CAR-T therapy evaluation, and drug sensitivity testing of chemotherapy drugs and targeted drugs.

[0051] Specifically, the method for preparing bone and soft tissue sarcoma organoids includes the following steps: 1) Obtain bone and soft tissue sarcoma tissue samples and preserve them in tissue preservation solution; 2) Remove the sample tissue from the preservation solution, place it in a sterile container, add cell culture medium, and chop the sample tissue in the cell culture medium to obtain tissue clumps; 3) Pass the chopped sample tissue-cell culture medium suspension through a sterile filter; 4) Rinse the tissue clumps intercepted by the filter into a new sterile container with organoid culture medium, add organoid culture medium, and mix well; 5) Add to a low-adsorption culture dish, place in a CO2 incubator, incubate at 37°C, and change the medium halfway every two days. Bone and soft tissue sarcoma organoids can be obtained within two weeks.

[0052] In step 1), the bone and soft tissue sarcoma tissues are derived from humans.

[0053] The bone and soft tissue sarcoma types mentioned in step 1) include liposarcoma, fibrosarcoma, pleomorphic undifferentiated sarcoma, synovial sarcoma, osteosarcoma, chondrosarcoma, giant cell tumor of bone, and Ewing sarcoma, etc.

[0054] In step 2), the volume of the tissue mass is less than 0.5mm × 0.5mm, with the optimal volume being 0.1mm × 0.1mm.

[0055] The tissue mass in step 2) contains tumor cells, fibroblasts, mesenchymal cells, epithelial cells, macrophages, and natural killer cells.

[0056] The pore size of the filter screen in step 4) is 100μm.

[0057] The cell culture medium in steps 2) and 3) is DMEM / F12 containing 100 μg / mL Normocin.

[0058] The organoid culture medium formulation in step 4) is as follows: Basic culture medium: DMEM / F12; Supplements: 50% L-WRN cell culture medium (DMEM / F12, containing Wnt3a, RSPO1 and noggin), 1 mM MEPES, 1× Glutamax, 10 mM nicotinamide, 1 mM N-acetylcysteine, 1× B-27 (without vitamin A), 0.5 μM A83-01, 1× Pen-Strep Glutamine, 10 nM Gastrin, 10 μM SB-202190 and 50 ng / mL EGF.

[0059] The L-WRN cell culture medium is the medium collected after culturing L-WRN cells in DMEM / F12 for 3-7 days. The specific steps are as follows: 1×10 6 -5×10 6 One L-WRN cell was evenly seeded in a 10 cm diameter cell culture dish. Using 10 mL of DMEM / F12 medium containing 10% FBS, the L-WRN cells were cultured at 37°C and 5% CO2 for 3-7 days. The medium was then collected. No medium change was required during the culture period.

[0060] This invention provides bone and soft tissue sarcoma organoids that can be passaged and stored at low temperatures, prepared according to the above method.

[0061] The bone and soft tissue sarcoma organoids retain the cell types found in the original tumor tissue, including tumor cells, fibroblasts, mesenchymal cells, epithelial cells, macrophages, and natural killer cells.

[0062] The bone and soft tissue sarcoma organoids can be cut and recultured for passage.

[0063] The bone and soft tissue sarcoma organoids can be preserved in liquid nitrogen for extended periods.

[0064] The present invention also provides the use of the bone and soft tissue sarcoma organoids in drug screening.

[0065] The applications include, but are not limited to, CAR-T therapy evaluation, chemotherapy drug and targeted drug sensitivity testing.

[0066] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0067] The DMEM / F12 culture medium used in this invention was purchased from Gbico; B-27, Glutamax, Pen-StrepGlutamine and EGF were purchased from Invitrogen; Normocin, Gastrino and SB-202190 were purchased from Sigma, and A83-01 was purchased from Tocris.

[0068] The structures of A83-01 and SB-202190 are shown below. Figure 1 .

[0069] Example 1: Culture method for fibrosarcoma 1. Obtain fibrosarcoma tissue samples from the patient and preserve them in tissue preservation solution.

[0070] 2. Remove the sample tissue from the preservation solution and place it in a 10 cm culture dish. Add DMEM / F12 cell culture medium containing 100 μg / mL Normocin. Mince the sample tissue in the cell culture medium to obtain tissue fragments approximately 0.1 mm × 0.1 mm in size. See [link to relevant documentation]. Figure 2 .

[0071] 3. Pass the chopped sample tissue-cell culture medium suspension through a 100 μm sterile filter.

[0072] 4. Rinse the tissue clumps intercepted by the filter into a new sterile container with organoid culture medium, add organoid culture medium, and mix well.

[0073] Organoid culture medium: DMEM / F12 medium containing 50% L-WRN cell culture medium, 1 mM HEPES, 2 mM L-alanyl-L-glutamine dipeptide, 10 mM nicotinamide, 1 mM N-acetylcysteine, 2% volume of vitamin A-free B-27, 0.5 μM A83-01, 100 U / mL penicillin, 100 μg / mL streptomycin, 292 μg / mL L-glutamine, 10 nM gastrin, 10 μM SB-202190 and 50 ng / mL human epidermal growth factor (EGF).

[0074] The method for preparing the L-WRN cell culture medium includes: mixing 2×10 6 One L-WRN cell was evenly seeded in a 10 cm diameter cell culture dish. The L-WRN cells were cultured for 5 days at 37°C and 5% CO2 using 10 mL of DMEM / F12 medium containing 10% FBS. The medium was then collected. No medium change was required during the culture period.

[0075] 5. Add to a low-adsorption culture dish, place in a CO2 incubator, and incubate at 37°C. Change the medium halfway every two days. Fibrosarcoma organoids can be obtained within two weeks.

[0076] This method can obtain eight different types of sarcoma organoids ( Figure 3 a) Liposarcoma, fibrosarcoma, undifferentiated pleomorphic sarcoma (UPS), synovial sarcoma (SS), osteosarcoma, chondrosarcoma, giant cell tumor of bone (GCTB), and Ewing sarcoma (ES), etc., organoid morphology is shown in [see section 1]. Figure 3 b.

[0077] Example 2: Comparison of cell types between tumor tissue of fibrosarcoma and corresponding organoids Single-cell sequencing of fibrosarcoma tumor tissue and cultured organoids revealed that patient-derived fibrosarcoma and cultured fibrosarcoma organoids contained the same cell types, namely: smooth muscle cells, fibroblast activation protein-positive cancer-associated fibroblasts, FAP-expressing cancer-associated fibroblasts (FAP-CAFs), myofibroblasts, natural killer cells, endothelial cells, macrophages, and mesenchymal stromal cells. Statistical results are shown below. Figure 4 .

[0078] Example 3: Comparison of cell types between osteosarcoma tumor tissue and corresponding organoids Single-cell sequencing of osteosarcoma tumor tissue and cultured organoids revealed that patient-derived osteosarcoma tissue blocks and cultured osteosarcoma organoids contained the same cell types: osteoblasts, fibroblasts, mesenchymal cells, proliferating osteoblasts, macrophages, endothelial cells, epithelial cells, and natural killer (NK) cells. Statistical results are shown below. Figure 5 .

[0079] Example 4: Passage and Cryopreservation Status Detection of Bone and Soft Tissue Sarcoma Organoids Bone and soft tissue sarcoma organoids maintained high viability after passage and cryopreservation. Both passaged organoids and organoids cryopreserved in liquid nitrogen for six months maintained good viability after thawing. Organoid morphology is shown in [Figure 1]. Figure 6 (a~c).

[0080] Example 5: Chemotherapy drug sensitivity testing of bone and soft tissue sarcoma organoids Four types of bone and soft tissue sarcoma (Fibrosarcoma, DFSP, GCTB, and ES) organoids were added to 24-well low-adsorption cell culture plates at a density of 20 ± 5 organoids per well, with 300 μl of organoid culture medium per well. Five replicates were prepared for each type of bone and soft tissue sarcoma to add different chemotherapeutic drugs. IFO, VCR, DOX, CBP, and MTX were then added to the four types of bone and soft tissue sarcoma to achieve final concentrations of 10 μM, 0.5 μM, 2 μM, 10 μM, and 10 μM, respectively. Microscopic observation and photography were performed every seven days to record the effects of different drugs on different types of organoids. Statistical results are shown below. Figure 7 (a~d).

[0081] Example 6: Evaluation of CAR-T efficacy using organoids Construction of CD19-CAR-T and GD2-CAR-T: Both CAR structures consist of GD2 or CD19 scFv, CD28 transmembrane constituent regions, and CD3ζ intramural sequences. Figure 8a) These CD19 or GD2-CAR structures were cloned into lentiviral vectors. Lentiviral particles were generated by co-transfection of the CAR vector and packaging plasmid using Lipofectamine 3000 (Invitrogen). The lentiviral supernatant was collected at 48 h and 72 h, followed by centrifugation at 50,000 × g for 2 h at 4 °C. The supernatant was resuspended in cold PBS, aliquoted, and stored at -80 °C. Peripheral blood mononuclear cells were isolated from healthy donors by Ficoll density gradient centrifugation. T cells were harvested by classification using CD3 magnetic beads (Miltenyi Biotec) and stimulated with human T activator CD3 / CD28 immunomagnetic beads (Thermo Fisher Scientific). The T cells were cultured in RPMI 1640 medium supplemented with 10% FBS and 100 μg / mL penicillin-streptomycin and transfected with lentiviral vectors encoding the CD19 or GD2 gene. Before use, culture CAR-T cells for 7-10 days in ImmunoCult-XF T cell expansion medium (purchased from StemCell Technologies) containing 100 U / mL recombinant human IL-2 (purchased from R&D).

[0082] At the RNA level, qPCR results confirmed the expression of the CAR gene in CD19-CAR-T and GD2-CAR-T. Figure 8 b); At the protein level, Western blot results confirmed the translation of the CAR protein ( Figure 8 c).

[0083] Organoids with high GD2 expression (ganglioside, PUBCHEM CID 6450346) (synovial sarcoma organoid constructed in Example 1, numbered SO-31) were selected to evaluate the therapeutic effect of GD2-CAR-T. Organoids with low GD2 expression (Ewing sarcoma organoid constructed in Example 1, numbered SO-2) were selected as controls, and CD19-CAR-T cells were selected as a control for GD2-CAR-T. Both organoids were co-incubated with 50,000 CD19-CAR-T or GD2-CAR-T cells, respectively, in ImmunoCult-XF T cell expansion medium in a low-adsorption 24-well plate. The cells were cultured at 37°C in a CO2 incubator. Flow cytometry analysis was performed on days 0, 1, 2, or 3, and the results are shown below. Figure 9 (a and b) After GD2-CAR-T was applied to GD2-high expression organoids, the GD2 signal decreased over time; microscopic observation of the organoid state revealed the adsorption and growth inhibition of GD2-CAR-T on GD2-high expression organoids. Figure 9c; The levels of IFN-γ, TNF-α, and IL-2 in the culture medium of the co-incubation system were detected by ELISA. With the extension of incubation time, the levels of cytotoxic factors (IFN-γ and TNF-α) and T cell proliferation signal (IL-2) in the supernatant of the culture medium of the GD2-CAR-T and SO-31 co-incubation groups all increased significantly. Statistical results are shown in […]. Figure 9 d; Immunofluorescence imaging of organoids after three days of co-incubation using CD3 (T cell marker), cleaved-caspase 3 (CC3, apoptosis marker), and DAPI (nuclear dye) showed T cell infiltration and organoid apoptosis in the GD2-CAR-T and SO-31 co-incubation groups. Laser confocal imaging results and fluorescence intensity statistics are shown in [reference needed]. Figure 9 (e and f).

[0084] Example 7: Preparation of Personalized Tumor Vaccines Using Organoids Synthesis of PLGA nanoparticles: 10 mg of PLGA was dissolved in 1 ml of dichloromethane, and 200 μl of ultrapure water was added. After sonication with a probe (15℃, 100 W, 5 min), the PLGA nanoparticles were dispersed in 2 ml of 2% sodium cholate aqueous solution. The solution was then sonicated again with a probe (15℃, 115 W, 5 min), and dispersed in 10 ml of 0.6% sodium cholate solution. The mixture was stirred (300 rpm, 25℃) for 15 min. After centrifugation at 12000 rpm for 10 min, the precipitate was collected, resuspended in pure water, and washed twice under the same conditions to obtain PLGA nanoparticles. Transmission electron microscopy characterization is shown in the figure. Figure 10 a.

[0085] Organoid digestion solution was prepared as follows: DMEM / F12 medium, 10% FBS, 200 U / mL type I collagenase (purchased from Worthington), and 100 μg / mL DNase I (purchased from Sigma).

[0086] Remove the organoid culture supernatant, add 1 mL of digestion solution, and digest at 37°C for 30 minutes to obtain single cells. Centrifuge the obtained single cells, remove the supernatant, and resuspend in 3 mL of hypotonic solution (1 mM PBS). Repeat the freeze-thaw cycle three times using liquid nitrogen and a 37°C water bath, centrifuge at 1000 rpm for 10 min to remove the precipitate, and then centrifuge at 12000 rpm for 30 min to obtain the cell membrane. Resuspend the cell membrane precipitate in 1 mM PBS, and mix with PLGA nanoparticles at a mass ratio of 10:1 to membrane protein. After passing through a liposome delivery system, PLGA nanoparticles coating the organoid cell membrane are obtained, which is the personalized tumor vaccine. Transmission electron microscopy characterization is shown in [see figure]. Figure 10 b.

[0087] Example 8: Optimization of Organoid Culture Medium Components Taking Ewing sarcoma (ES) organoid culture as an example, this invention optimizes the composition of three culture medium additives (L-WRN cell culture medium, gastrin, and human epidermal growth factor).

[0088] Four different organoid culture media were prepared: L-WRN cell culture medium (-), gastrin (-), human epidermal growth factor (-), and organoid culture medium containing all four components (i.e., the organoid culture medium of Example 1).

[0089] L-WRN cell culture medium (-): DMEM / F12 medium containing 1 mM HEPES, 2 mM L-alanyl-L-glutamine dipeptide, 10 mM nicotinamide, 1 mM N-acetylcysteine, 2% volume of vitamin A-free B-27, 0.5 μM A83-01, 100 U / mL penicillin, 100 μg / mL streptomycin, 292 μg / mL L-glutamine, 10 nM gastrin, 10 μM SB-202190 and 50 ng / mL human epidermal growth factor (EGF).

[0090] Gastrin-negative: DMEM / F12 medium containing 50% L-WRN cell culture medium, 1 mM HEPES, 2 mM L-alanyl-L-glutamine dipeptide, 10 mM nicotinamide, 1 mM N-acetylcysteine, 2% volume of vitamin A-free B-27, 0.5 μM A83-01, 100 U / mL penicillin, 100 μg / mL streptomycin, 292 μg / mL L-glutamine, 10 μM SB-202190 and 50 ng / mL human epidermal growth factor (EGF).

[0091] Human epidermal growth factor (-): DMEM / F12 medium containing 50% L-WRN cell culture medium, 1 mM HEPES, 2 mM L-alanyl-L-glutamine dipeptide, 10 mM nicotinamide, 1 mM N-acetylcysteine, 2% volume of vitamin A-free B-27, 0.5 μM MA83-01, 100 U / mL penicillin, 100 μg / mL streptomycin, 292 μg / mL L-glutamine, 10 nM gastrin, and 10 μMSB-202190.

[0092] Organoid culture medium: DMEM / F12 medium containing 50% L-WRN cell culture medium, 1 mM HEPES, 2 mM L-alanyl-L-glutamine dipeptide, 10 mM nicotinamide, 1 mM N-acetylcysteine, 2% volume of vitamin A-free B-27, 0.5 μM A83-01, 100 U / mL penicillin, 100 μg / mL streptomycin, 292 μg / mL L-glutamine, 10 nM gastrin, 10 μM SB-202190 and 50 ng / mL human epidermal growth factor (EGF).

[0093] The L-WRN cell culture medium was the supernatant collected after culturing L-WRN cells in DMEM / F12 for 5 days. The specific steps were as follows: 3 × 10⁻⁶ cells were cultured in DMEM / F12. 6 One L-WRN cell was evenly seeded in a 10 cm diameter cell culture dish. The L-WRN cells were cultured for 5 days at 37°C and 5% CO2 using 10 mL of DMEM / F12 medium containing 10% FBS. The medium was then collected. No medium change was required during the culture period.

[0094] Ewing sarcoma organoids were cultured using the four culture media described above. Morphological images were taken on days 1, 7, and 14. Results are shown below. Figure 11 The results showed that the absence of any of these three components restricted the growth of Ewing sarcoma organoids. Ultimately, this invention employed an optimized culture medium formulation for bone and soft tissue sarcoma organoids.

[0095] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A method for preparing bone and soft tissue sarcoma organoids, characterized in that, Includes the following steps: 1) Obtain bone and soft tissue sarcoma tissue samples and preserve them in tissue preservation solution; 2) Remove the tissue sample from the tissue preservation solution, place it in a sterile container, add cell culture medium, and chop the tissue sample in the cell culture medium to obtain tissue clumps; 3) Pass the chopped tissue sample-cell culture medium suspension through a sterile filter; 4) Rinse the tissue clumps intercepted by the filter into a new sterile container with organoid culture medium, add organoid culture medium, and mix well; 5) Add to a low-adsorption culture dish, place in a CO2 incubator, incubate at 37°C, change the medium halfway every two days, and culture for two weeks to obtain bone and soft tissue sarcoma organoids; The cell culture medium described in steps 2) and 3) is DMEM / F12 containing 100 μg / mL Normocin; Step 4) describes an organoid culture medium containing 50% L-WRN cell culture medium, 1 mM HEPES, 2 mM L-alanyl-L-glutamine dipeptide, 10 mM nicotinamide, 1 mM N-acetylcysteine, 2 v / v% vitamin A-free B-27, 0.5 μM MA83-01, 100 U / mL penicillin, 100 μg / mL streptomycin, 292 μg / mL L-glutamine, 10 nM gastrin, 10 μMSB-202190, and 50 ng / mL human epidermal growth factor in DMEM / F12 medium. The molecular formula of A83-01 is C 25 H 19 The molecular formula of N5S, SB-202190 is C 20 H 14 FN3O; The method for preparing the L-WRN cell culture medium includes: adding 1×10 6 -5×10 6 One L-WRN cell was evenly seeded in a 10 cm diameter cell culture dish. Using 10 mL of DMEM / F12 medium containing 10% FBS, the L-WRN cells were cultured at 37°C and 5% CO2 for 3-7 days. The medium was then collected. No medium change was required during the culture period. Step 1) The bone and soft tissue sarcoma types mentioned are liposarcoma, fibrosarcoma, pleomorphic undifferentiated sarcoma, synovial sarcoma, osteosarcoma, chondrosarcoma, giant cell tumor of bone, and Ewing sarcoma; The bone and soft tissue sarcoma tissue described in step 1) is derived from human tissue.

2. The method according to claim 1, characterized in that, Step 2) The tissue mass contains tumor cells, fibroblasts, mesenchymal cells, epithelial cells, macrophages and natural killer cells.

3. The method according to claim 1, characterized in that, Step 4) The filter screen has a pore size of 100 μm.

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