WO2024192567A1 - 建立新型人肠道及结直肠肿瘤类器官培养体系 - Google Patents

建立新型人肠道及结直肠肿瘤类器官培养体系 Download PDF

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WO2024192567A1
WO2024192567A1 PCT/CN2023/082192 CN2023082192W WO2024192567A1 WO 2024192567 A1 WO2024192567 A1 WO 2024192567A1 CN 2023082192 W CN2023082192 W CN 2023082192W WO 2024192567 A1 WO2024192567 A1 WO 2024192567A1
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organoids
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intestinal
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邓宏魁
屈默龙
熊亮
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Peking University
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Priority to EP24774051.7A priority patent/EP4682248A1/en
Priority to PCT/CN2024/082075 priority patent/WO2024193486A1/zh
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    • C12N2501/72—Transferases [EC 2.]
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    • G01N2500/10—Screening for compounds of potential therapeutic value involving cells

Definitions

  • the present invention relates to organoids derived from human intestinal tissue and colorectal tumor tissue, and in particular to human intestinal and colorectal tumor organoids, their uses and methods for obtaining them.
  • Organoid technology allows stem cells to self-organize in vitro, simulating the complex structure and function of in vivo tissues.
  • Organoid culture technology has been developed on a variety of tissues and organs, which provides a valuable tool for studying differentiation and development and disease treatment.
  • traditional human intestinal organoid culture technology is difficult to capture the epithelial cell heterogeneity of in situ intestinal and tumor tissues, and the organoid expansion rate is slow. These defects severely limit the clinical application of human intestinal organoid technology.
  • traditional human intestinal organoid technology is difficult to capture the characteristics of intermediate plasticity states, making it difficult to reconstruct the characteristics of colorectal tumor development and resistance to treatment.
  • organoids have the ability to simulate tissue structure and function under physiological conditions, providing a valuable tool for in vitro modeling and disease research.
  • traditional human intestinal organoid culture technology has difficulty capturing the characteristics of in situ intestinal and colorectal tumor tissues.
  • we established a new human intestinal organoid culture system that can capture and maintain the key characteristics of in situ intestinal and colorectal tumor tissues.
  • organoids derived from human intestinal and colorectal tumor tissues are significantly enriched in epithelial cell heterogeneity, such as the activation of goblet cell lineage genes, and the proliferation and expansion capacity of organoids are significantly enhanced.
  • the present invention provides a composition and method for culturing and obtaining novel human intestinal organoids having in situ intestinal and colorectal tumor tissue characteristics. Specifically, the present invention provides the following technical solutions:
  • a composition characterized in that it includes (1) an ALK inhibitor, (2) a WNT agonist, (3) an EGF agonist and (4) an FGF agonist, preferably, the composition is used to obtain and/or culture human intestinal tissue-derived organoids or human colorectal tumor tissue-derived organoids.
  • composition according to item 1 wherein the (1) ALK inhibitor is selected from one or more of the group consisting of: LDN193189, LDN214117, LDN212854, ML347, K02288; preferably LDN214117.
  • composition according to item 1 wherein the (2) WNT agonist is selected from one or more of the group consisting of: CHIR-99021, CHIR-98014, CHIR-98023, CHIR-98024, GSK3 ⁇ inhibitor XV, TD114-2, BIO-acetoxim; preferably CHIR-99021.
  • composition according to item 1 wherein the (3) EGF agonist is selected from one or more of the group consisting of EGF, HB-EGF, AREG, EREG, NRG1, NRG2, NRG3, NRG4; preferably EGF.
  • composition according to item 1 wherein the (4) FGF agonist is selected from one or more of the group consisting of: FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, FGF23; preferably FGF2.
  • composition according to item 1 further comprising (5) a dual regulator of EBP1 and I ⁇ B;
  • the (5) EBP1 and I ⁇ B dual regulator is selected from one or more of the group consisting of: WS3, WS6, (+)-Usnic acid, Pim-1/2 kinase inhibitor 1, RMC-5552, Sauchinone, PS-1145; preferably WS6.
  • a method for obtaining and/or culturing organoids derived from human intestinal tissue or human colorectal tumor tissue characterized in that the culture is carried out in a culture medium, preferably normal human intestinal crypt tissue or human colorectal tumor tissue is cultured in a culture medium, wherein the culture medium contains the composition described in any one of items 1-6.
  • the basal culture medium is also supplemented with HEPES, GlutaMAX, B27, and 1 mM N-Acetylcysteine.
  • Organoids derived from human intestinal tissue or human colorectal tumor tissue obtained by the method described in Item 7.
  • the human intestinal tissue-derived organoids or human colorectal tumor tissue-derived organoids described in Item 9 are characterized in that they express one or more of the marker genes LGR5, OLFM4, CHGA, CHGB, MUC2, AQP8, and LYPD8.
  • the human colorectal tumor tissue-derived organoids described in Item 9 include cells with an "intermediate plasticity state", wherein the "intermediate plasticity state” cells increase the pan-cancer epithelial-mesenchymal plasticity marker genes and significantly increase the expression of one or more of the markers DPP4, EMP1, ITGA2, and ANXA1.
  • the human colorectal tumor tissue-derived organoids described in Item 9 are capable of maintaining cells in an “intermediate plasticity state” for a long time.
  • the human colorectal tumor tissue-derived organoids described in Item 9 are used to study tumor resistance to treatment, metastasis and invasion in vitro, to screen therapeutic compounds that are highly effective in inhibiting or killing tumor cells, or as an in vitro model for reconstructing the tumor development process.
  • a composition comprising (1) an ALK inhibitor, (2) a WNT agonist, (3) an EGF agonist, (4) a dual regulator of EBP1 and I ⁇ B and (5) a p38 inhibitor, preferably the composition is used to obtain and/or culture organoids and/or "intermediate plasticity state" cells having characteristics of proliferative intestinal crypt tissue.
  • composition described in item 14, wherein the (1) ALK inhibitor is selected from one or more of the group consisting of: LDN193189, LDN214117, LDN212854, ML347, K02288; preferably LDN214117.
  • composition of item 14, wherein the (2) WNT agonist is selected from one or more of the group consisting of: CHIR-99021, CHIR-98014, CHIR-98023, CHIR-98024, GSK3 ⁇ inhibitor XV, TD114-2, BIO-acetoxim; preferably CHIR-99021.
  • composition of item 14 wherein the (3) EGF agonist is selected from one or more of the group consisting of: EGF, HB-EGF, AREG, EREG, NRG1, NRG2, NRG3, NRG4; preferably EGF.
  • composition described in item 14 wherein the (4) dual regulator of EBP1 and I ⁇ B is selected from one or more of the group consisting of: WS3, WS6, (+)-Usnic acid, Pim-1/2 kinase inhibitor 1, RMC-5552, Sauchinone, PS-1145; preferably WS6.
  • composition of item 14, wherein the (5) p38 inhibitor is selected from one or more of the group consisting of: SB202190, Pexmetinib, SB203580, BIRB796, LY2228820, VX-702, PH-797804, VX-745, TAK-715, BMS-582949; preferably SB202190.
  • a method for obtaining and/or culturing human intestinal organoids and/or "intermediate plasticity state" cells having characteristics of hyperplastic intestinal crypt tissue characterized in that the cells are cultured in a culture medium, preferably human normal intestinal crypt tissue or hyperplastic intestinal crypt tissue or human intestinal tissue-derived organoids as described in Item 9 are cultured in a culture medium, wherein the culture medium contains the composition described in any one of Items 14-19.
  • the basal culture medium is also supplemented with HEPES, GlutaMAX, B27, and 1 mM N-Acetylcysteine.
  • the human intestinal organoids with hyperplastic intestinal crypt tissue characteristics described in Item 22 are characterized by significantly downregulating some differentiation lineage genes, such as one or more of CHGA, CHGB, MUC2, AQP8, and LYPD8.
  • the human intestinal organoids with the characteristics of proliferative intestinal crypt tissue described in Item 22 are characterized by the ability to maintain cells in an "intermediate plasticity state" for a long time.
  • the human intestinal organoids with the characteristics of proliferative intestinal crypt tissue as described in Item 22 are used for in vitro studies of tumor initiation, for screening therapeutic compounds that are highly effective in inhibiting or killing tumor cells, or as an in vitro model for studying tumor occurrence.
  • novel human intestinal organoid culture system of the present invention By using the novel human intestinal organoid culture system of the present invention, a novel human intestinal organoid system with similar characteristics to in situ intestinal and colorectal tumor tissues in vivo can be established.
  • the organoids In our organoid culture system, the organoids have epithelial cell heterogeneity similar to in situ intestinal and colorectal tumor tissues in vivo.
  • the intestinal organoids obtained by the culture system of the present invention can be used to simulate the complex structure and function of intestinal tissues in vivo, providing a valuable tool for studying differentiation development and the treatment of intestinal diseases.
  • novel colorectal tumor organoids obtained by the present invention can capture and maintain the "intermediate plasticity state" IPS cells that appear during the progression of patient-derived colorectal tumors, enabling them to acquire long-term drug response capabilities. It is worth noting that inducing the IPS program in the culture system of the present invention in intestinal organoids derived from normal tissues can obtain organoids with the characteristics of proliferative intestinal crypt tissue, because obtaining the IPS program plays an important role in inducing and capturing tumor initiation and progression characteristics.
  • Tumor tissues and adjacent normal tissues were obtained from Peking University Shougang Hospital, and all patients had signed informed consent and obtained approval from the ethics committee.
  • the clinical data of the patients in this study are shown in Table 1 .
  • Tumors and adjacent normal tissues were isolated from surgically resected tissues and washed at least 10 times with pre-cooled PBS containing 2% penicillin/streptomycin (GIBCO). Tumor cells and colonic crypts were isolated and cultured as follows. Briefly, adjacent normal colon was cut into 2-4 mm fragments, incubated in 5 mM EDTA at 4°C for 30 min with gentle shaking, and then vigorously shaken in pre-cooled PBS to release colonic crypts.
  • the culture medium contains ALK inhibitors (e.g., 0.2 ⁇ M LDN-214177), EGF agonists (e.g., 50ng/ml EGF), WNT agonists (e.g., 3 ⁇ M CHIR-99021) and FGF agonists (e.g., 100ng/ml FGF2), i.e., 4C conditions.
  • ALK inhibitors e.g., 0.2 ⁇ M LDN-214177
  • EGF agonists e.g., 50ng/ml EGF
  • WNT agonists e.g., 3 ⁇ M CHIR-99021
  • FGF agonists e.g., 100ng/ml FGF2
  • the culture medium contains ALK inhibitors (e.g., 0.2 ⁇ M LDN-214177), EGF agonists (e.g., 50 ng/ml EGF), WNT agonists (e.g., 3 ⁇ M CHIR-99021), dual regulators of EBP1 and I ⁇ B (e.g., 0.5 ⁇ M WS6), and p38 inhibitors (e.g., 3 ⁇ M SB202190).
  • the culture medium is refreshed every other day.
  • the organoids or tumoroids are removed from Matrigel and mixed with TrypLE Express (Gibco).
  • the organoids or tumoroids are dissociated into small pieces, which are then suspended in Matrigel and distributed into new 48-wells. Passaging is performed every 1-2 weeks, with a passaging ratio of 1:6 for conventional conditional organoids or tumoroids and a passaging ratio of 1:16 for intestinal tissue-derived organoids or colorectal tumor organoids.
  • the digestion time is extended to 20-30 minutes, and then the cells are filtered with a 40 ⁇ m cell strainer and suspended in Matrigel at a concentration of 4000 cells/20 ⁇ L Matrigel. It is recommended to use 10 ⁇ M Y-27632 for the first 2 days after single-cell passaging.
  • the reagent details for organoid culture are shown in Table 2.
  • organoids were released from Matrigel with cell recovery solution (Corning) and fixed with 4% paraformaldehyde (DingGuo) at room temperature for 15 min. After permeabilization with PBS containing 0.1% Triton X-100 (Sigma-Aldrich), primary antibodies were added and incubated overnight at 4°C, followed by incubation with secondary antibodies for 1 h at 37°C. Nuclei were stained with DAPI (Roche Life Science). Immunohistochemical staining was performed on 8 ⁇ M paraffin-embedded tissue sections after embedding using standard histological protocols.
  • Heat-induced antigen retrieval was performed with Tris-EDTA (pH 9.0) buffer, and slides were blocked with blocking solution (PBS, 0.1% Triton X-100, 5% serum homologous to the secondary antibody) for 1 h at room temperature. Slides were incubated with primary antibodies overnight at 4°C, followed by incubation with secondary antibodies for 1 h at room temperature. Nuclei were stained with DAPI (Roche Life Science). Details of the antibodies used are shown in Table 3.
  • the cell culture medium was removed and TrypLE Express (Gibico) was added. After incubation at 37°C for 10 min, the organoids were digested into isolated single cells, stained with PE anti-human DPP4 (BioLegend) at 4°C for 30 min, and washed 3 times with PBS. The cells were filtered using a 40 ⁇ m cell strainer. Finally, flow cytometry experiments were performed using a BD LSRFortessa instrument. Data analysis was performed using FlowJo software (Ashland).
  • Tumor organoids were collected and separated into single cells according to the above-mentioned passaging procedure. 20 ⁇ L of Matrigel containing 4000 cells were inoculated in 48-well plates and cultured in different culture media.
  • the final concentration of each drug was adjusted to 0.3, 1, 3, 10, 20, and 50 ⁇ M.
  • the cells were exposed to single doses of 2, 4, 6, 8, 12, and 16 Gy using a GammaCell 40 irradiator.
  • the culture medium was then renewed every 2 to 3 days, and at the end of the treatment, the cell viability was assessed using the Cell Counter Kit-8 (CCK-8) assay, and the dose response curve was calculated by comparing the treated wells with the solvent control wells.
  • RNA sequencing count data were transferred to transcripts per million (TPM) and log-normalized, and the batch effect was corrected by the ComBat_seq function.
  • the samples were clustered and distances were calculated using hclust.
  • the differentially expressed genes (DE genes) were obtained using the DESeq2 package (v1.34.0) and displayed in volcano plots and heat maps, respectively.
  • GSEA Gene set enrichment analysis
  • GSEA Gene set variation analysis
  • GSVA was performed on 11 different EMT-related features, and the differential expression of these EMT-related features in the samples was found based on the log-TPM of these EMT-related features using the GSVA R package.
  • the GSVA scores were visualized using heat maps and box plots.
  • the MuSiC package (v1.0.0) was used to predict the cell state composition of tumor organoids cultured under different conditions.
  • Sequencing data were processed by DNBelab_C_Series_HT_scRNA-analysis-software. Cells were filtered based on the number of transcripts and the percentage of mitochondrial transcripts. Expression matrices were converted to Seurat objects using the Seurat package (v4.1.1). scRNA-seq count data of 49155 epithelial cells in the iCMS dataset were reanalyzed.
  • cNMF consensus non-negative matrix factorization
  • K was selected as 6 based on stability and error, and the density threshold was 0.1.
  • Marker genes for each state were found by FindAllMarkers, and the expression levels of marker genes related to pan-cancer epithelial-mesenchymal plasticity were determined by AddModuleScore_UCell.
  • UMAP analysis was performed using the UMAP-learn method based on cNMF.
  • Our scRNA-seq data from conventional tumor organoids and colorectal tumor tissue-derived organoids were merged after quality filtering and log normalization.
  • the combination of 12,718 cell data was processed by FindVariableFeatures, ScaleData, and RunPCA, all with default parameters. We then used the RunHarmony function to eliminate batch effects arising from the two different culture conditions, using First 50 dimensions of PCA.
  • Seurat collections were made from cells from in situ tumor tissue, traditional tumor organoids, and colorectal tumor tissue-derived organoids.
  • Ten cell types were annotated by comparing markers with known epithelial lineage markers.
  • the cell types and main markers are as follows: stem cells (LGR5, SMOC2, ASCL2, OLFM4); rapidly amplifying cells_1 (MKI67, TOP2A, HIST1H4C, and CENPF); rapidly amplifying cells_2 (HILPDA, LGALS1, and RBP1); enterocyte progenitor cells (PYCARD); intestinal epithelial cells (EPCAM, FABP1, PHGR1, and CA2); mature intestinal epithelial cells (SLC26A3, SLC26A3, and AQP8); secretory precursor cells (FCGBP and TFF3); goblet cells (FCGBP, TFF3, MUC2, and SPINK4); tuft cells (LRMP and BMX); enteroendocrine cells (CHGA, CHGB, SCGN, and FEV).
  • stem cells LGR5, SMOC2, ASCL2, OLFM4
  • rapidly amplifying cells_1 MKI67, TOP2A, HIST1H4C, and CENPF
  • CNV inference using single-cell transcriptome data was based on a previously published method modified by inferCNV software. Only genes with an average relative expression of more than 1.5 in a single cell and passing all quality controls were used for CNV inference.
  • the CNV score of a gene was reduced to the average expression level of its 100 neighboring genes. Then, the CNV scores were aggregated to zero by subtracting the average CNV score of all cells. Finally, the relative CNV score was calculated using the average CNV score of all genes within the 10M CNV window.
  • inferCNV copy number from single-cell transcriptome data.
  • Inferred ecnv analysis identified 6 groups of cells with different copy number variation patterns in in vivo tumor tissues. Among the 6 groups, all chromosomes in group 6 had no copy number variation, and we considered it to be non-malignant cells.
  • group 6 in tumor organoids cultured under traditional conditions and group 8 in colorectal tumor tissue-derived organoids were considered to be non-malignant cells, and the remaining cells were considered to be malignant cells.
  • the purity of the tumor was then determined by calculating the ratio of the number of malignant cells to the total number of cells in each sample. Based on the above method, we finally found that the proportion of malignant cells to total cells in in vivo tumor tissues, traditional tumor organoids, and colorectal tumor tissue-derived organoids was 87%, 90%, and 94.2%, respectively.
  • Tumor tissue-derived organoids were implanted into the spleens of 6-8 week-old, 18-20 g male immunodeficient mice (NOD.Cg-PrkdcscidII2rgtm1 Sug/JicCrl) purchased from Beijing Weitonglihua Laboratory Animal Technology Co., Ltd. Mice were housed in a pathogen-free environment and used in accordance with institutional animal care guidelines. Tumor size was quantified by in vivo bioluminescence imaging using an in vivo imaging system (IVIS) Spectrum CT (PerkinElmer) and performed in a blinded manner.
  • IVIS in vivo imaging system
  • mice were imaged for 60 seconds, and regions of interest (ROIs) were created and measured as area flux, defined as average brightness (photons/second/cm2/stereo). Data were analyzed using Living Image V.4.0 software (Caliper Life Sciences).
  • ROIs regions of interest
  • BD Biosciences Matrigel
  • Tumor tissue-derived organoids were surgically injected into the spleen through an abdominal incision under anesthesia. After injection, the spleen was gently returned to the abdominal cavity to restore homeostasis. Mice were killed at week 6 after injection. The size of tumors in organs such as the spleen was measured within 20 minutes after the death of the mice.
  • the bar graph shows the proportion of each cell type in organoids and intestinal crypt tissues cultured under different conditions.
  • k Typical morphology of human intestinal tissue-derived organoids cultured under different ALK inhibitors. Imaged on day 10 after single-cell passaging.
  • Con. conventional intestinal organoid culture conditions
  • 4C novel intestinal organoid culture conditions.
  • GSEA analysis showed the enrichment of cancer-related gene markers in organoids derived from colorectal tumor tissue, with human intestinal tissue-derived organoids used as a control.
  • SNVs Single nucleotide variants
  • Con. traditional intestinal organoid culture conditions
  • 4C novel intestinal organoid culture conditions
  • CRC tumor in vivo colorectal tumor tissue.
  • the bar graph shows the proportion of each cell type in tumor organoids and orthotopic tumors of two patients (P19 and P18).
  • Pan-cancer epithelial-mesenchymal plasticity marker gene expression superimposed on the UMAP shown in panel b.
  • Con. conventional intestinal organoid culture conditions
  • 4C intestinal organoid culture conditions of the present invention.
  • Figure 5 WS6 enhances the “intermediate plasticity state” to enable colorectal tumor tissue-derived organoids to acquire epithelial-mesenchymal plasticity and increase metastatic potential
  • GSVA Gene set variation analysis
  • GSVA Gene set variation analysis
  • the bar graph shows the proportion of each cell population obtained by label transfer in organoids under different conditions.
  • Organoids cultured under different conditions at early (upper) and late (lower) stages. Organoids cultured at early stages can be cultured for a maximum of 1-2 weeks, while organoids cultured at late stages can be cultured for at least 2 months.
  • Con. traditional intestinal organoid culture conditions
  • 4C/5C intestinal organoid culture conditions of the present invention.
  • the term "intestinal organoid” refers to a three-dimensional cell tissue body formed by separating human intestinal crypt tissue and accumulating cells at a high density in a controlled space through three-dimensional culture.
  • the "intestinal organoid” includes the "intestinal tissue-derived organoid” obtained from the isolated human intestinal crypt tissue described below by the 4C culture system of the present invention and the "traditional intestinal organoid” obtained from the isolated human intestinal crypt tissue described below by the disclosed method of the prior art.
  • intestinal tissue-derived organoids refers to the crypt tissue of the human intestine isolated and cultured using the LDN214117, EGF, CHIR99021, FGF2 conditions (i.e., 4C conditions) described in the present invention. It is characterized by the production of certain crypt-budding structures in morphology, and has very strong amplification and cloning capabilities, significantly enriched epithelial cell heterogeneity, such as the activation of goblet cell lineage genes, and has a lineage composition that is more similar to the intestinal crypts in vivo.
  • traditional intestinal organoids refers to organoids obtained by isolating the crypt tissue of the human intestine and culturing them using published traditional conditions (see the “Experimental Materials and Procedures” section for details). They are characterized by being spherical in shape, with almost no crypt-budding structures, and can basically only amplify stem cells, lacking various differentiation lineage cells.
  • tumor organoid refers to an organoid cultured using colorectal tumor cells.
  • the "tumor organoid” includes the "colorectal tumor tissue-derived organoid” obtained from human colorectal tumor tissue by the 4C culture system of the present invention as described below and the "traditional tumor organoid” obtained from human colorectal tumor tissue by the disclosed method in the prior art as described below.
  • colonal tumor tissue-derived organoids refers to organoids obtained by isolating human colorectal tumor tissue and culturing using the 4C conditions of the present invention (e.g., LDN214117, EGF, CHIR99021, FGF2). It is characterized by having very strong expansion and cloning capabilities, and having epithelial cell heterogeneity similar to that of in situ tumor tissue.
  • organoids with characteristics of hyperplastic intestinal crypt tissue refers to crypt tissue isolated from the human intestine and cultured using the 5C conditions of the present invention (e.g., LDN214117, EGF, CHIR99021, WS6, SB202190). It is characterized by the central cyst cavity showing obvious signs of contraction, while the newly formed crypt-like budding structures are significantly increased, similar to the phenotype of hyperplastic polyp tissue. Compared with the organoids obtained by culture under traditional conditions, it significantly downregulates some differentiation lineage genes, such as CHGA, CHGB, MUC2, AQP8, LYPD8, etc.; and IPS characteristic genes, tumor-specific genes, etc. are significantly improved in organoids with characteristics of hyperplastic intestinal crypt tissue. Organoids with characteristics of hyperplastic intestinal crypt tissue provide an ideal in vitro model for studying a variety of human intestinal diseases and the initiation of colorectal tumors.
  • intermediate plasticity state refers to a state of high plasticity that occurs during the progression of colorectal tumors in patients. It is characterized by the enrichment of a variety of different programs, such as trophectoderm cells, glial cells, and astrocytes, with highly mixed transcriptional programs, and compared to organoids or cells cultured under traditional conditions, it also significantly increases the previously reported pan-cancer epithelial-mesenchymal plasticity marker genes (see, David P. Cook et al., "Transcriptional census of epithelial-mesenchymal plasticity in cancer", 2022), and significantly expressed one or more of the markers DPP4, EMP1, ITGA2, and ANXA1 at high levels. In addition, it plays an important role in the occurrence and development of tumors, the degree of malignancy, and resistance to radiotherapy and chemotherapy.
  • hyperplastic intestinal crypt tissue refers to abnormal proliferation of the intestinal mucosa due to long-term external stimulation, such as inflammation, infection, etc., which manifests as hypertrophy and hyperplasia of the mucosa, abnormal increase and growth of the crypt structure, and may further develop into adenoma or cancer.
  • matrigel also known as extracellular matrix (ECM)
  • ECM extracellular matrix
  • Matrigel TM BD Biosciences
  • laminin, entactin and collagen IV BD Biosciences
  • inhibitors bind to the ALK receptor and prevent cytokines from binding to the corresponding receptor.
  • Preferred ALK inhibitors include LDN214117, and the concentration thereof can be conventionally used, for example, 0.2 ⁇ M-20 ⁇ M, preferably 1 ⁇ M-4 ⁇ M, and most preferably 2 ⁇ M. Its structural formula is as follows:
  • ALK inhibitors that are known in the art and commercially available may also be used in the methods disclosed herein, including but not limited to: LDN193189, LDN214117, LDN212854, ML347, K02288.
  • EGF agonists include EGF, and conventional concentrations thereof may be used, for example, 10 ng/ml to 250 ng/ml, preferably 25 ng/ml to 100 ng/ml, and most preferably 50 ng/ml.
  • EGF agonists that can be used in the methods disclosed herein are known in the art and commercially available, including but not limited to: EGF, HB-EGF, AREG, EREG, NRG1, NRG2, NRG3, NRG4.
  • WNT agonists can be used to activate TCF/LEF-mediated transcription in cells.
  • the WNT agonist includes CHIR-99021, and its concentration can be conventionally used, for example, 0.3 ⁇ M-30 ⁇ M, preferably 1.5 ⁇ M-6 ⁇ M, and most preferably 3 ⁇ M.
  • concentration can be conventionally used, for example, 0.3 ⁇ M-30 ⁇ M, preferably 1.5 ⁇ M-6 ⁇ M, and most preferably 3 ⁇ M. Its structural formula is as follows:
  • WNT agonists that can be used in the methods disclosed herein are known in the art and commercially available, including but not limited to: CHIR-99021, CHIR-98014, CHIR-98023, CHIR-98024, GSK3 ⁇ inhibitor XV, TD114-2, BIO-acetoxim
  • FGF agonists include FGF2, and conventional concentrations thereof may be used, such as 10 ng/ml-1000 ng/ml, preferably 50 ng/ml-200 ng/ml, and most preferably 100 ng/ml.
  • FGF agonists may also be used in the methods disclosed herein, which are known in the art and commercially available, including but not limited to: FGF1, FGF2, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, FGF10, FGF11, FGF12, FGF13, FGF14, FGF15, FGF16, FGF17, FGF18, FGF19, FGF20, FGF21, and FGF23.
  • Preferred dual regulators of EBP1 and I ⁇ B include WS6, and its concentration can be conventionally used, for example, 0.05 ⁇ M-5 ⁇ M, preferably 0.25 ⁇ M-1 ⁇ M, and most preferably 0.5 ⁇ M. Its structural formula is as follows:
  • EBP1 and I ⁇ B may also be used in the methods disclosed herein, including but not limited to: WS3, (+)-Usnic acid, Pim-1/2 kinase inhibitor 1, RMC-5552, Sauchinone, PS-1145.
  • Preferred p38 inhibitors include SB202190, and the concentration thereof can be conventionally used, for example, 1 ⁇ M-100 ⁇ M, preferably 5 ⁇ M-20 ⁇ M, and most preferably 10 ⁇ M. Its structural formula is as follows:
  • p38 inhibitors known in the art and commercially available may also be used in the methods disclosed herein, including but not limited to: Pexmetinib, SB203580, BIRB796, LY2228820, VX-702, PH-797804, VX-745, TAK-715, BMS-582949.
  • the lineage composition of intestinal tissue-derived organoids was more similar to in situ intestinal crypt tissue than traditional intestinal organoids ( Figure 1, g, h).
  • Each cell population showed different gene expression patterns that were consistent with the corresponding lineages in the human colorectum ( Figure 1, i, j).
  • Figure 1, i, j The results indicate that intestinal tissue-derived organoids can capture and maintain a cell lineage composition comparable to in situ intestinal crypt tissue.
  • RNA-seq global transcriptome sequencing
  • colorectal tumor tissue-derived organoids To determine the genomic characteristics of colorectal tumor tissue-derived organoids, we performed whole exome sequencing (WES) and compared gene mutations in colorectal tumor tissue-derived organoids and corresponding in situ tumor tissues. We found that colorectal tumor tissue-derived organoids exhibited a point mutation pattern very similar to that of paired in situ tumors ( Figure 2, j). The heat map of gene mutation variations showed that the characteristic mutated genes of each patient were also reproduced in colorectal tumor tissue-derived organoids ( Figure 2, k). Overall, these results indicate that colorectal tumor tissue-derived organoids can faithfully reflect the histopathological and genomic characteristics of the primary tumor.
  • WES whole exome sequencing
  • stem cell populations with OLFM4 as marker genes, enteroendocrine cells with CHGA and CHGB as marker genes, goblet cells with MUC2 as marker genes, and intestinal epithelial cells with AQP8 and LYPD8 as marker genes were significantly increased in colorectal tumor tissue-derived organoids.
  • the lineage composition of colorectal tumor tissue-derived organoids was more similar to that of in situ tumor epithelial cells than that of traditional tumor organoids ( Figure 3, b).
  • Each cell population exhibited distinct gene expression patterns that were consistent with the corresponding lineages of human tissues ( Figure 3, c, d). Overall, these results suggest that colorectal tumor tissue-derived organoids can capture and maintain epithelial heterogeneity comparable to that of in situ tumors.
  • cell cluster 7 was enriched in previously reported pan-cancer epithelial-mesenchymal plasticity marker genes ( Figure 4b), indicating that cell cluster 7 is an intermediate plastic state that actively participates in and may promote significant phenotypic transitions, including cell lineage conversion, epithelial-mesenchymal transition, and oncofetal reprogramming processes. Therefore, we defined the state of these cells as an intermediate plastic state (IPS).
  • IPS intermediate plastic state
  • Example 4 Discovery of small molecules that enable colorectal tumor tissue-derived organoids to acquire epithelial-mesenchymal plasticity and increase metastatic potential by enhancing the “intermediate plasticity state”
  • WS6 a dual regulator of EBP1 and I ⁇ B, can significantly induce epithelial-mesenchymal transition in colorectal tumor tissue-derived organoids, which is manifested by a large number of tumor cells showing mesenchymal-like spindle morphology ( Figure 5a, b).
  • immunofluorescence analysis showed that WS6-treated colorectal tumor tissue-derived organoids lost apical-basal polarity and showed reduced cell-cell adhesion ( Figure 5a, b).
  • IPS characteristic genes are significantly more enriched than intestinal tissue-derived organoids and traditional intestinal organoids (Figure 6a), and multiple epithelial-mesenchymal plasticity marker genes are significantly increased ( Figure 6b). Consistent with this result, we performed single-cell transcriptional profiling and found that the proportion of IPS cells in organoids with proliferative intestinal crypt tissue characteristics was significantly increased compared with intestinal tissue-derived organoids and organoids cultured under traditional conditions ( Figure 6c, d). In summary, we have established a new small molecule combination that can significantly induce the expression of the IPS program in intestinal epithelial cells.
  • Example 6 Establishment of human intestinal organoids with hyperplastic intestinal crypt tissue characteristics
  • hyperplastic intestinal crypt characteristics induced by 5C conditions are not a transient stress response of organoids, but a stable state transition, which simulates the hyperplastic stage experienced by normal intestinal crypt tissue in the early stage of tumorigenesis in vivo. Therefore, direct culture of hyperplastic human intestinal crypt tissue using 5C conditions can also maintain the characteristics of proliferative intestinal crypt tissue well. And we found that compared with intestinal tissue-derived organoids and organoids cultured under traditional conditions, the expression of recently reported colorectal tumor characteristic genes was significantly enhanced in organoids with hyperplastic intestinal crypt tissue characteristics, although the degree was still lower than that in colitis-related cancer tissues (Fig. 6g).

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Abstract

提供了一种用于获得和/或培养人肠道组织来源类器官或人结直肠肿瘤组织来源类器官的组合物，其包括ALK抑制剂、WNT激动剂、EGF激动剂和FGF激动剂。还提供了一种用于获得和/或培养具有增生性肠道隐窝组织特征的类器官和/或"中间可塑性状态"细胞的组合物，其包括ALK抑制剂、WNT激动剂、EGF激动剂、EBP1及IκB双重调节剂和p38抑制剂。

Description

建立新型人肠道及结直肠肿瘤类器官培养体系 技术领域
本发明涉及人类肠道组织及结直肠肿瘤组织来源的类器官，特别涉及人肠道及结直肠肿瘤类器官，其用途以及获得它们的方法。
背景技术
类器官技术的发展允许干细胞在体外进行自组织，模拟了体内组织的复杂结构和功能。目前已经在多种组织器官上开发了类器官培养技术，这为研究分化发育和疾病治疗提供了宝贵的工具。然而，传统人肠道类器官培养技术难以捕获原位肠道及肿瘤组织的上皮细胞异质性，同时类器官扩增速率慢。这些缺陷严重限制了人肠道类器官技术的临床应用。除此之外，传统人肠道类器官技术难以捕获中间可塑性状态特征，导致其难以重建结直肠肿瘤发生发展以及抵抗治疗等方面的特征。为了弥补传统人肠道类器官技术的不足，在此，我们建立了一种新的人体肠道类器官技术，能够在体外捕获和维持与原位肠道及结直肠肿瘤组织特征相似的类器官；同时，我们能够在新型人肠道类器官体系中捕获中间可塑性状态，从而获得结直肠肿瘤发生发展以及抵抗治疗等方面的特征。
发明内容
三维类器官具有模拟生理状态下组织结构和功能的能力，为体外建模和疾病研究提供了宝贵的工具。然而，传统人肠道类器官培养技术难以捕获原位肠道及结直肠肿瘤组织特征。在此，我们建立了一种新的人体肠道类器官培养系统，能够捕获并维持原位肠道及结直肠肿瘤组织的关键特征。与传统肠道类器官相比，人肠道及结直肠肿瘤组织来源的类器官显著富集了上皮细胞异质性，如杯状细胞谱系基因的启动，并且类器官的增殖及扩增能力有了显著的增强。进一步地，单细胞分析显示，在本发明所述条件下培养的结直肠肿瘤类器官中存在一种在结直肠病人肿瘤进展过程中出现的“中间可塑性状态”，简称为IPS。在肿瘤类器 官中捕获IPS，可以获得长期药物反应能力。此外，在肠道类器官中捕获IPS可以诱导获得人增生性肠道隐窝特征，同时启动肿瘤特异性基因表达。综上，我们的研究建立了一个新的人肠道及结直肠肿瘤组织来源类器官的体外模型，为探索人体肠道组织发育和肿瘤发生发展开辟了新的途径。
具体地，本发明提供了用于培养获得具有原位肠道及结直肠肿瘤组织特征的新型人肠道类器官的组合物和方法。具体地，本发明提供了以下技术方案：
1.一种组合物，其特征在于，包括(1)ALK抑制剂，(2)WNT激动剂，(3)EGF激动剂和(4)FGF激动剂，优选地所述组合物用于获得和/或培养人肠道组织来源类器官或人结直肠肿瘤组织来源类器官。
2.根据项目1所述的组合物，其中，所述(1)ALK抑制剂选自由以下各项组成的组中的一种或多种：LDN193189，LDN214117，LDN212854，ML347，K02288；优选为LDN214117。
3.根据项目1所述的组合物，其中，所述(2)WNT激动剂选自由以下各项组成的组中的一种或多种：CHIR-99021，CHIR-98014，CHIR-98023，CHIR-98024，GSK3β inhibitor XV，TD114-2，BIO-acetoxim；优选为CHIR-99021。
4.根据项目1所述的组合物，其中，所述(3)EGF激动剂选自由以下各项组成的组中的一种或多种：EGF，HB-EGF，AREG，EREG，NRG1，NRG2，NRG3，NRG4；优选为EGF。
5.根据项目1所述的组合物，其中，所述(4)FGF激动剂选自由以下各项组成的组中的一种或多种：FGF1，FGF2，FGF3，FGF4，FGF5，FGF6，FGF7，FGF8，FGF9，FGF10，FGF11，FGF12，FGF13，FGF14，FGF15，FGF16，FGF17，FGF18，FGF19，FGF20，FGF21，FGF23；优选为FGF2。
6.根据项目1所述的组合物，其还包含(5)EBP1及IκB双重调节剂；
任选地，所述(5)EBP1及IκB双重调节剂选自由以下各项组成的组中的一种或多种：WS3，WS6，(+)-Usnic acid，Pim-1/2激酶抑制剂1，RMC-5552，Sauchinone，PS-1145；优选为WS6。
7.获得和/或培养人肠道组织来源类器官或人结直肠肿瘤组织来源类器官的方法，其特征在于，在培养基中进行培养，优选地在培养基中将正常人肠道隐窝组织或人结直肠肿瘤组织进行培养，其中所述培养基包含项目1-6任一项所述的组合物。
8.根据项目7所述的方法，其中，使用DMEM/F12作为基础培养基；
优选地，所述基础培养基中还添加有HEPES、GlutaMAX、B27、1mM N-Acetylcysteine。
9.项目7所述的方法获得的人肠道组织来源类器官或人结直肠肿瘤组织来源类器官。
10.项目9所述的人肠道组织来源类器官或人结直肠肿瘤组织来源类器官，其特征在于，其表达标志基因LGR5、OLFM4、CHGA、CHGB、MUC2、AQP8、LYPD8中的一种或多种。
11.项目9所述的人结直肠肿瘤组织来源类器官，其包括具有“中间可塑性状态”细胞，所述“中间可塑性状态”细胞提高泛癌上皮-间充质可塑性标志基因，并显著提高标志物DPP4，EMP1，ITGA2，ANXA1中的一种或多种的表达。
12.项目9所述的人结直肠肿瘤组织来源类器官，其能够长期维持“中间可塑性状态”细胞。
13.项目9所述的人结直肠肿瘤组织来源类器官，其用于体外研究肿瘤抗治疗能力、转移侵袭等能力，用于筛选高效抑制或杀伤肿瘤细胞的治疗化合物，或用作重建肿瘤发展过程的体外模型。
14.组合物，其包括(1)ALK抑制剂，(2)WNT激动剂，(3)EGF激动剂，(4)EBP1及IκB双重调节剂和(5)p38抑制剂，优选地所述组合物用于获得和/或培养具有增生性肠道隐窝组织特征的类器官和/或“中间可塑性状态”细胞。
15.项目14所述的组合物，其中，所述(1)ALK抑制剂选自由以下各项组成的组中的一种或多种：LDN193189，LDN214117，LDN212854，ML347，K02288；优选为LDN214117。
16.项目14所述的组合物，其中，所述(2)WNT激动剂选自由以下各项组成的组中的一种或多种：CHIR-99021，CHIR-98014，CHIR-98023，CHIR-98024，GSK3β inhibitor XV，TD114-2，BIO-acetoxim；优选为CHIR-99021。
17.项目14所述的组合物，其中，所述(3)EGF激动剂选自由以下各项组成的组中的一种或多种：EGF，HB-EGF，AREG，EREG，NRG1，NRG2，NRG3，NRG4；优选为EGF。
18.项目14所述的组合物，其中，所述(4)EBP1及IκB双重调节剂选自由以下各项组成的组中的一种或多种：WS3，WS6，(+)-Usnic acid，Pim-1/2 kinase inhibitor 1，RMC-5552，Sauchinone，PS-1145；优选为WS6。
19.项目14所述的组合物，其中，所述(5)p38抑制剂选自由以下各项组成的组中的一种或多种：SB202190，Pexmetinib，SB203580，BIRB796，LY2228820，VX-702，PH-797804，VX-745，TAK-715，BMS-582949；优选为SB202190。
20.获得和/或培养人具有增生性肠道隐窝组织特征的肠道类器官和/或“中间可塑性状态”细胞的方法，其特征在于，在培养基中进行培养，优选地在培养基中将人正常肠道隐窝组织或增生性肠道隐窝组织或项目9所述的人肠道组织来源类器官进行培养，其中所述培养基包含项目14-19中任一项所述的组合物。
21.项目20所述的方法，其中，使用DMEM/F12作为基础培养基；
优选地，所述基础培养基中还添加有HEPES、GlutaMAX、B27、1mM N-Acetylcysteine。
22.项目21所述的方法获得的人具有增生性肠道隐窝组织特征的肠道类器官和/或“中间可塑性状态”细胞。
23.项目22所述的人具有增生性肠道隐窝组织特征的肠道类器官，其特征在于，显著下调部分分化谱系基因，如CHGA、CHGB、MUC2、AQP8、LYPD8中的一种或多种。
24.项目22所述的人具有增生性肠道隐窝组织特征的肠道类器官，其特征在于可以长期维持“中间可塑性状态”细胞。
25.项目22所述的人具有增生性肠道隐窝组织特征的肠道类器官，其用于体外研究肿瘤起始发生，用于筛选高效抑制或杀伤肿瘤细胞的治疗化合物，或用作研究肿瘤发生的体外模型。
本发明的技术效果
利用本发明中的新型人肠道类器官培养体系，能够建立一个与体内原位肠道及结直肠肿瘤组织有相似特征的新型人肠道类器官系统。在我们的类器官培养系统中，类器官具有与体内原位肠道及结直肠肿瘤组织相类似的上皮细胞异质性。本发明培养体系所获得的肠道类器官可用于模拟体内肠道组织的复杂结构和功能，为研究分化发育和肠道疾病治疗提供了宝贵的工具。
进一步还发现了，本发明所获得的新型结直肠肿瘤类器官能够捕获并维持病人来源的结直肠肿瘤进展过程中出现的“中间可塑性状态”IPS细胞，使其获得长期的药物反应能力。值得注意的是，在正常组织来源的肠道类器官中通过本发明的培养体系诱导IPS程序可以获得具有增生性肠道隐窝组织特征的类器官，因为获得IPS程序对于诱导及捕获肿瘤起始和进展特征发挥了重要的作用。
实验材料和操作方法
1.肿瘤及癌旁组织获取
肿瘤组织和邻近的正常组织从北京大学首钢医院获得，均已签署知情同意书并获得伦理委员会批准。本研究中患者的临床资料见表1。
表1患者临床治疗

2.组织分离与类器官培养
从手术切除组织中分离肿瘤及邻近正常组织，用含2％青霉素/链霉素(GIBCO)的预冷PBS冲洗至少10次。肿瘤细胞和结肠隐窝分离培养方式如下。简单地说，将相邻的正常结肠切成2～4mm的片段，置于5mM EDTA中4℃孵育30分钟，期间轻轻摇晃，之后在预冷的PBS中剧烈摇晃以释放结肠隐窝。将肿瘤组织切碎成0.5mm3的小碎片，放入10cm无菌皿中，加入含有500U/mL胶原酶IV(Sigma-aldrich，C9407)、1.5mg/mL胶原酶II(Solarbio，C8150)、20mg/mL透明质酸酶(Solarbio，h8030)、0.1mg/mL diase II(Sigma-aldrich，D4693)、10μM Y27632和1％胎牛血清的5ml消化液，37℃消化30-60分钟。并500g离心5min采集肿瘤细胞。将结肠隐窝和肿瘤细胞悬浮在Matrigel中，以20μL的液滴分布在预热48孔板上，在37℃，5％CO2培养箱中培养10-15分钟。待 Matrigel聚合后，加入500μL Advanced DMEM/F12(Gibco)培养基，其中含10mM HEPES(Gibco)、1X GlutaMAX(Gibco)、1％盘尼西林/链霉素(Gibco)、1X N2 Supplement(Gibco)、1X B27 Supplement(Gibco)和1.25mM n-乙酰半胱氨酸(Sigma-Aldrich)。传统条件人结直肠正常组织来源类器官培养条件：50％Wnt条件培养基、20％R-Spondin条件培养基、100ng/ml Noggin、50ng/ml EGF、10mM Niacinamide、10nM Gastrin、500nM A83-01、3μM SB202190和10nM前列腺素E2的培养基。传统条件人结直肠肿瘤组织来源类器官培养条件：20％R-Spondin条件培养基、100ng/ml Noggin、50ng/ml EGF、10mM Niacinamide、10nM Gastrin、500nM A83-01、3μM SB202190和10nM前列腺素E2的培养基。对于肠道组织来源类器官和结直肠肿瘤组织来源类器官，培养基中含有ALK抑制剂(例如0.2μM LDN-214177)、EGF激动剂(例如50ng/ml EGF)、WNT激动剂(例如3μM CHIR-99021)和FGF激动剂(例如100ng/ml FGF2)，即4C条件。对于5C增生性人肠道类器官培养，培养基中含有ALK抑制剂(例如0.2μM LDN-214177)、EGF激动剂(例如50ng/ml EGF)、WNT激动剂(例如3μM CHIR-99021)、EBP1及IκB双重调节剂(例如0.5μM WS6)和p38抑制剂(例如3μM SB202190)。培养基每隔一天更新一次。传代时，从Matrigel中去除类器官或类肿瘤，并与TrypLE Express(Gibco)混合。在37℃孵育5分钟后，类器官或类肿瘤解离成小块，然后悬浮在Matrigel中并分配到新的48孔中。传代每1-2周进行一次，传统条件类器官或类肿瘤的传代比例为1∶6，肠道组织来源类器官或结直肠肿瘤类器官的传代比例为1∶16。单细胞传代时，将消化时间延长至20～30分钟，然后用40μm细胞过滤器过滤细胞，以4000个细胞/20μL Matrigel的浓度悬浮在Matrigel中，单细胞传代后的前2天推荐使用10μm Y-27632。类器官培养的试剂细节见表2。
表2试剂来源

3.免疫组化及免疫荧光染色
免疫荧光染色时，用细胞恢复液(Corning)从Matrigel中释放类器官，并用4％多聚甲醛(DingGuo)室温固定15分钟。用含0.1％Triton X-100(Sigma-Aldrich)的PBS进行细胞通透后，加入一抗并在4℃孵育过夜，然后用二抗在37℃孵育1小时。细胞核用DAPI(Roche Life Science)染色。免疫组化染色采用标准组织学方案包埋切片后，对8μM石蜡包埋组织切片进行处理。用Tris-EDTA(pH 9.0)缓冲液热诱导抗原提取，用封闭液(PBS、0.1％Triton X-100、5％二抗同源血清)在室温下封闭载片1h。将载玻片在4℃下用一抗孵育一夜，然后在室温下用二抗孵育1小时。用DAPI(Roche Life Science)染色细胞核。使用抗体细节见表3。
表3抗体信息

4.荧光定量PCR分析
使用RNeasy Mini试剂盒(QIAGEN，74106)从培养类器官的整个孔中提取总RNA。使用Trans-Script First-Strand cDNA Synthesis SuperMix(全式金)将RNA反转录为cDNA。qPCR使用KAPA SYBR_FAST qPCR Kit Master Mix试剂(KAPA Biosystems)和CFX ConnectTM Real-Time System(Bio-Rad)仪器。数据采用delta-delta Ct法进行分析。β-actin被用作归一化基因表达的内参。本研究中使用的qPCR引物序列见表4。
表4 qRT-PCR引物

5.流式分析
除去细胞培养基，加入TrypLE Express(Gibico)。在37℃孵育10分钟后，类器官被消化成分离的单细胞，用PE anti-human DPP4(BioLegend)在4℃染色30分钟，PBS洗涤3次。细胞使用40μm细胞过滤器过滤。最后，使用BD LSRFortessa仪器进行流式分析实验。使用FlowJo软件(Ashland)进行数据分析。
6.放化疗处理
收集肿瘤类器官，并按照上述传代程序分离成单细胞。将含有4000个细胞的20μL Matrigel接种于48孔板中，在不同的培养基中培养。使用FOLFOXIRI方案(其中5-Fu∶伊立替康∶奥沙利铂＝1∶1∶1)进行化疗治疗时，将各药物终浓度调整为0.3、1、3、10、20、50μM。对于放射治疗，使用GammaCell 40照射器，将细胞暴露在单剂量2，4，6，8，12，16Gy的照射下。之后每2～3天更新培养基，并在处理结束时，使用Cell Counter Kit-8(CCK-8)试验评估细胞活力，通过将处理孔与溶剂对照孔进行比较计算剂量响应曲线。
7.肾包囊移植
将100万个肿瘤细胞用50μl的Matrigel重悬，并注射至6周龄雄性NPG小鼠(购买自北京维通达生物技术有限公司)的肾下极(肾包膜下方)。待观察到水泡形成，并且肿瘤细胞悬浮液几乎不再渗漏后，将针取出。研究中使用的动物在注射胶囊时形成了明显可见的水泡，且漏液量非常小。移植6周后处死小鼠。室温下用10％和20％的福尔马林固定整 个肾脏24h。石蜡包埋后，切5μm切片贴于载玻片上进行免疫组化染色。并采用苏木精-伊红(H&E)染色法进行组织和细胞鉴定。
8.整体RNA测序分析
将整体RNA测序计数数据转移到transcriptsper Million(TPM)并对数归一化，通过ComBat_seq函数对批次效应进行校正。利用hclust对样本进行聚类和距离计算。利用DESeq2程序包(v1.34.0)获得差异表达基因(DE基因)，分别在火山图和热图中显示。对DE基因进行基因集富集分析(GSEA)。对11个不同的EMT相关特征进行基因集变异分析(Gene Set Variation Analysis，GSVA)，通过GSVA R包，根据这些EMT相关特征的log-TPM，寻找这些EMT相关特征在样本中的差异表达。GSVA评分用热图和箱线图进行可视化。基于iCMS scRNA-seq数据，MuSiC包(v1.0.0)用于预测不同条件培养下的肿瘤类器官细胞状态组成。
9.单细胞RNA测序分析
测序数据经DNBelab_C_Series_HT_scRNA-analysis-software处理。根据转录本的数量和线粒体转录本的百分比对细胞进行过滤。使用Seurat包(v4.1.1)将表达式矩阵转换为Seurat对象。对iCMS数据集中49155个上皮细胞的scRNA-seq计数数据进行了重新分析。我们使用cNMF包进行了共识非负矩阵分解(cNMF)，使用了6000个高变量基因。根据稳定性和误差选择K为6，密度阈值为0.1。使用这6个cNMF分量进行无监督聚类(分辨率＝0.2)，得到10个细胞状态。通过FindAllMarkers找到每个状态的标记基因，通过AddModuleScore_UCell确定泛癌上皮-间充质可塑性相关的标记基因表达水平。采用UMAP-learn方法在cNMF的基础上进行UMAP分析。我们传统肿瘤类器官与结直肠肿瘤组织来源类器官的scRNA-seq数据经过质量过滤和对数归一化后合并在一起。12,718个细胞数据的组合由FindVariableFeatures、ScaleData和RunPCA处理，所有参数均为默认参数。然后我们使用RunHarmony函数来消除两种不同培养条件下产生的批次效应，使用 PCA的前50个维度。我们使用带有默认参数的FindTransferArchors和TransferData，将iCMS scRNA-seq数据(还原＝cNMF，分辨率＝0.2)中的细胞状态标签转移到传统肿瘤类器官与结直肠肿瘤组织来源类器官中。通过FindAllMarkers检测各细胞状态的标记基因。我们还检测了泛癌上皮-间充质可塑性相关特征在这些不同细胞状态下的表达，使用AddModuleScore_UCell计算每个细胞的UCell评分，并对每个状态的伪体进行基因集变异分析(Gene Set Variation Analysis，GSVA)(伪体是通过计算细胞状态的平均归一化表达得到的)。然后根据和谐约简的结果和之前识别的细胞状态进行线性判别分析(LDA)，然后使用所有9个LDA分量和UMAP-learn方法进行UMAP降维。
10.细胞谱系划分
将原位肿瘤组织、传统肿瘤类器官与结直肠肿瘤组织来源类器官中细胞制成Seurat集合体。我们使用package Harmony的RunHarmony函数来消除批次效应，然后以1∶20维度的Harmony进行RunUMAP和FindNeighbors分析。利用scGate(v1.4.1)包的gating_model函数，首先根据上皮细胞的已知特征将细胞分为上皮细胞和非上皮细胞。我们进一步聚类20，223个上皮细胞，分析具有FindAllMarkers功能的差异表达基因。通过将标记物与已知的上皮谱系标记物进行比较，对10种细胞类型进行了注释。细胞类型及主要标志物如下：干细胞(LGR5、SMOC2、ASCL2、OLFM4)；快速扩增细胞_1(MKI67，TOP2A，HIST1H4C，和CENPF)；快速扩增细胞_2(HILPDA，LGALS1和RBP1)；肠细胞祖细胞(PYCARD)；肠上皮细胞(EPCAM，FABP1，PHGR1和CA2)；成熟肠上皮细胞(SLC26A3，SLC26A3和AQP8)；分泌前体细胞(FCGBP和TFF3)；杯状细胞(FCGBP，TFF3，MUC2和SPINK4)；簇细胞(LRMP和BMX)；肠内分泌细胞(CHGA，CHGB，SCGN和FEV)。
11.全外显子测序分析
采用最大精确匹配比对(BWA-MEM)v0.7.8-r455进行测序，由人类参考基因组GRCh37进行比对。根据最佳实践指南，我们使用Genome Analysis Toolkit(GATK)v3.8.0来处理bam文件，包括标记重复项、重新调整索引和基础重新校准。我们将参考数据和肿瘤或类器官测序数据提供给MuTect2(涉及GATK v3.8.0)，并使用默认参数识别体细胞突变。通过使用anno var，我们添加了效果预测和注释。为了高质量地检测体细胞CNAs，通过Control-FREEC v11.4对BAM文件的读取深度变化进行解释，并将肿瘤或类器官与参考细胞进行比较。利用Mutational Patterns R包v3.4分析突变特征，确定所有体细胞snv的基因组，并确定“人类癌症突变过程的特征”在原位肿瘤与肿瘤类器官样本中的贡献。
12.CNV分析
使用单细胞转录组数据的CNV推断是基于先前发表的经过inferCNV软件修改的方法。只有在单个细胞中平均相对表达超过1.5并通过所有质量控制的基因才被用于CNV推断。首先，将一个基因的CNV评分降低到其100个邻近基因的平均表达水平。然后，通过减去所有细胞的平均CNV分数，将CNV分数聚合到零。最后，利用平均10M CNV窗口内所有基因的CNV得分计算相对CNV得分。
13.肿瘤纯度预测
为了计算肿瘤纯度，我们应用inferCNV从单细胞转录组数据中估计拷贝数。推断的ecnv分析在体内肿瘤组织中鉴定了6组具有不同拷贝数变异模式的细胞。6组中，第6组所有染色体均无拷贝数变异，我们认为其为非恶性细胞。按照同样的策略，传统条件培养的肿瘤类器官中的第6组和结直肠肿瘤组织来源类器官条中的第8组被认为是非恶性细胞，其余的细胞被认为是恶性细胞。然后通过计算每个样本中恶性细胞数量与总细胞数量的比例来确定肿瘤的纯度。基于上述方法，我们最终发现体内肿瘤组织、传统肿瘤类器官与结直肠肿瘤组织来源类器官中恶性细胞占总细胞的比例分别为87％、90％和94.2％。
14.肿瘤组织来源类器官脾脏异种移植
将肿瘤组织来源类器官植入6-8周龄、18-20g雄性免疫缺陷小鼠(NOD.Cg-PrkdcscidII2rgtm1 Sug/JicCrl)脾脏，小鼠购自北京维通利华实验动物科技公司。小鼠被安置在无病原体的环境中，并按照机构动物护理指南使用。使用活体成像系统(IVIS)光谱ct(PerkinElmer)在体内生物发光成像定量肿瘤大小，并采用盲法进行。小鼠成像60秒，创建感兴趣区域(roi)，并测量为面积通量，定义为平均亮度(光子/秒/cm2/立体)。使用Living Image V.4.0软件(Caliper Life Sciences)分析数据。在异种移植实验中，将约2×106个细胞量的肿瘤组织来源类器官悬浮于培养基中，与Matrigel(BD Biosciences)1∶1混合，以50μL的最终体积注射于脾脏囊下。免疫缺陷小鼠使用Avertin(JT 0781，北京吉田生物科技有限公司)麻醉。肿瘤组织来源类器官在麻醉下通过腹部切口手术注入脾脏。注射后，将脾脏轻轻放回腹腔，恢复体内平衡。注射后第6周处死小鼠。小鼠死后20分钟内测定脾脏等器官肿瘤大小。
15.统计分析
所有值均用均数±SEM表示。统计参数，包括统计分析方法，统计显著性阈值，n值在图的图例和补充图的图例中都有说明。
16.数据可用性
当前研究中产生的数据均可以从GEO数据库中获取。本研究中使用的所有其他数据均在本文中作为源数据提供。
附图说明
图1建立肠道组织来源类器官
a.不同条件下培养的人肠道组织来源类器官的典型形态。在单细胞传代后第10天进行成像。
b.量化不同条件下类器官的数量和大小(n＝4)。
c.不同条件下类器官在不同传代代数下的数量统计(n＝3)。
d.长期传代(20次传代)后人肠道组织来源类器官代表性图像。
e.人肠道组织来源类器官培养超过100d的核型分析。
f.肠道组织来源类器官与传统肠道类器官对各谱系细胞标志物的免疫组织化学和免疫荧光染色。
g.不同条件培养的类器官及肠道隐窝组织的单细胞转录组数据的umap可视化。左边，颜色表示无监督集分群。右，颜色表示不同样品来源。
h.柱状图展示不同条件培养的类器官及肠道隐窝组织中各细胞类型的比例。
i.不同谱系细胞特征标记物的表达情况。
j.各细胞类型的代表性基因表达叠加展示在g图所示的UMAP上。
k.不同ALK抑制剂下培养的人肠道组织来源类器官的典型形态。在单细胞传代后第10天进行成像。
l.量化不同ALK抑制剂培养的类器官的数量(n＝3)。
缩写：Con.：传统肠道类器官培养条件；4C：新型肠道类器官培养条件。
***P＜0.001；**P＜0.01；**P＜0.05。标尺100μm。
图2建立结直肠肿瘤组织来源类器官
a.不同条件下培养的人结直肠肿瘤组织来源类器官的典型形态。在单细胞传代后第10天进行成像。
b.量化不同条件下肿瘤类器官的数量和大小(n＝4)。
c.不同条件下肿瘤类器官在不同传代代数下的数量统计(n＝3)。
d.长期传代(20次传代)后人结直肠肿瘤组织来源类器官代表性图像。
e.结直肠肿瘤组织来源类器官肾包囊移植物及相应结直肠肿瘤患者原发肿瘤切片的免疫组织化学和免疫荧光染色。
f.GSEA分析显示结直肠肿瘤组织来源类器官中癌症相关基因标记的富集，人肠道组织来源类器官作为对照。
g.热图显示不同患者的结直肠肿瘤组织来源类器官，对最近报道的肿瘤特异性基因的富集情况。患者编号显示在右侧。
h.肠道组织来源类器官和结直肠肿瘤组织来源类器官对整体基因表达谱进行无监督分层聚类分析。
i.肠道组织来源类器官和结直肠肿瘤组织来源类器官表达谱的PCA分析。
j.三名结直肠肿瘤患者(P12，P16，P17)的点突变类型在结直肠肿瘤组织来源类器官与原位肿瘤中所占比例的柱状统计图。
k.单核苷酸突变(SNVs)热图，显示结直肠肿瘤组织来源类器官和原位肿瘤中患者特异性的突变基因。
缩写：Con.：传统肠道类器官培养条件；4C：新型肠道类器官培养条件；CRC tumor：体内结直肠肿瘤组织。
***P＜0.001；**P＜0.01；**P＜0.05。标尺100μm。
图3结直肠肿瘤组织来源类器官上皮细胞异质性与原位肿瘤相似
a.来自2名患者(P19和P18)的肿瘤类器官及原位肿瘤细胞(共20223个细胞)单细胞转录组数据的umap可视化。左边，表示无监督集群。右，表示细胞来源。
b.柱状图展示2例患者(P19和P18)的肿瘤类器官及原位肿瘤中各细胞类型的比例。
c.各细胞类型的代表性基因表达叠加展示在a图所示的UMAP上。
d.不同谱系细胞特征标记物的表达情况。
e.热图显示不同条件培养的肿瘤类器官与原位肿瘤的大规模CNVs。每组细胞共鉴定出8个簇。
缩写：Con.：传统肠道类器官培养条件；4C：本发明的肠道类器官培养条件；tumor：体内结直肠肿瘤组织。
***P＜0.001；**P＜0.01；**P＜0.05。标尺100μm。
图4新型人肿瘤类器官维持中间可塑性状态及长期药物反应能力
a.63例结直肠肿瘤患者上皮细胞(共49155个细胞)单细胞转录组数据的umap可视化，由10个无监督聚类获得的簇着色。
b.泛癌上皮-间充质可塑性标志基因表达情况叠加在b图所示的UMAP上。
c.不同条件培养的肿瘤类器官(共12,718个细胞)的单细胞转录组数据umap可视化。
d.细胞分群7标志基因表达情况叠加在d图所示的UMAP上。
e.左图，UMAP可视化展示通过标签转移获得的肿瘤类器官分群情况(共12,718个细胞)。右，柱状图显示每种细胞分群的比例。
f.小提琴图显示传统肿瘤类器官和结直肠肿瘤组织来源类器官中泛癌上皮-间充质可塑性标志基因的表达情况。
g.通过标签转移获得的肿瘤类器官中不同细胞群对细胞分群7标志基因的表达热图。
h.结直肠肿瘤组织来源类器官与传统肿瘤类器官中IPS代表基因表达情况(n＝2)。
i.不同条件及不同传代代数下肿瘤类器官在放疗或FOLFOXIRI治疗下的相应曲线。在细胞接种后第2天对细胞进行放疗或FOLFOXIRI处理，在处理后第9天测量细胞活力。
缩写：Con.：传统肠道类器官培养条件；4C：本发明的肠道类器官培养条件。
***P＜0.001；**P＜0.01；**P＜0.05。
图5 WS6通过增强“中间可塑性状态”使结直肠肿瘤组织来源类器官获得上皮-间充质可塑性并增加转移潜力
a.在指定条件下培养的结直肠肿瘤组织来源类器官的代表图像和EPCAM的免疫荧光染色。
b.不同处理条件下结直肠肿瘤组织来源类器官中3种典型形态特征的类器官数量定量(n＝4)。
c.不同处理条件下结直肠肿瘤组织来源类器官中上皮-间充质转变标记基因表达的QPCR分析。
d.基因集变异分析(GSVA)结果，(左)热图与(右)箱线图显示不同处理条件下结直肠肿瘤组织来源类器官中上皮-间充质转变相关基因标记的富集。
e.来自含有和不含WS6的结直肠肿瘤组织来源类器官的上皮细胞(共15,279个细胞)单细胞转录谱的UMAP可视化。左为“中间可塑性状态”标记基因的表达叠加。右，通过标记转移获得的细胞类群着色。
f.显示通过标签传输获得的不同处理条件下结直肠肿瘤组织来源类器官中每个c细胞类群比例的堆栈条形图。
g.不经过(左)和经过WS6处理(右)的结直肠肿瘤组织来源类器官原位移植到免疫缺陷小鼠脾脏。取脾脏(上)和生殖系统(右)，在移植6周后通过生物荧光成像监测肿瘤生长和转移扩散。
h.(左)通过体内成像量化形成的转移瘤数量(n＝3只小鼠)，(右)通过特定
内脏器官的生物发光成像监测转移灶的平均亮度(n＝3只小鼠)。
***P＜0.001；**P＜0.01；**P＜0.05。标尺100μm。
图6建立具有增生性肠道隐窝组织特征的类器官
a.IPS标志基因在不同类型类器官中的表达热图(n＝3)。
b.基因集变异分析(GSVA)结果，热图(左)和箱线图(右)显示不同条件中上皮-间充质可塑性标志基因的富集。
c.来自不同类型类器官和肿瘤类器官的上皮细胞(共24279个细胞)的单细胞转录谱数据的umap可视化。左，展示细胞类型。右，由标记转移获得的细胞分群着色。
d.柱状图显示不同条件下类器官通过标签转移获得的各细胞分群比例。
e.不同条件培养的类器官在早期(上)和晚期(下)的代表性图像。传代早期类器官最多培养1-2周，传代晚期类器官至少培养2个月。
f.统计不同条件培养的类器官中隐窝样出芽结构域的数量(n＝6)。
g.热图显示不同条件培养的类器官中结直肠肿瘤特异性标记基因的表达(n＝3)，以结肠炎相关癌症样品作为对照。
h.QPCR检测不同条件及不同培养代数下各细胞谱系标记基因表达情况。
缩写：Con.：传统肠道类器官培养条件；4C/5C：本发明的肠道类器官培养条件。
***P＜0.001；**P＜0.01；**P＜0.05。标尺100μm。
具体实施方式
为使本发明的目的、技术方案和优点更加清楚明白，以下结合具体实施例，并参照附图，对本发明作进一步的详细说明。
定义
如本文所用的术语“肠道类器官”是指分离人肠道隐窝组织，通过三维培养使细胞在受控的空间内高密度地积聚而自组织成的立体的细胞组织体。在本发明中，所述“肠道类器官”包括下文所述的来自分离的人肠道隐窝组织的通过本发明的4C培养体系获得的“肠道组织来源类器官”和下文所述的来自分离的人肠道隐窝组织的通过现有技术已公开方法获得的“传统肠道类器官”。
如本文所用的术语“肠道组织来源类器官”是指分离人肠道的隐窝组织，并使用本发明所述的LDN214117，EGF，CHIR99021，FGF2条件(即，4C条件)培养获得的类器官。其特点为形态上具有一定隐窝-出芽结构产生，同时具有非常强的扩增能力与成克隆能力，显著富集了上皮细胞异质性，如杯状细胞谱系基因的启动，具有与体内肠道隐窝更加相似的谱系组成，相比于传统肠道类器官，其表达或显著高水平表达原位肠道组织的干性及分化功能细胞标志基因LGR5、OLFM4、CHGA、CHGB、MUC2、AQP8、LYPD8中的一种或多种。
如本文所用的术语“传统肠道类器官”是指分离人肠道的隐窝组织，并使用已发表的传统条件(具体请参见“实验材料和操作方法”部分)培养获得的类器官。其特点为形态上呈圆球状，几乎无任何隐窝-出芽结构产生，同时也基本只能扩增干祖细胞，缺少各种分化谱系细胞。
如本文所用的术语“肿瘤类器官”是指使用结直肠肿瘤细胞培养而成的类器官。在本发明中，所述“肿瘤类器官”包括下文所述的来自人结直肠肿瘤组织的通过本发明的4C培养体系获得的“结直肠肿瘤组织来源类器官”和下文所述的来自人结直肠肿瘤组织的通过现有技术已公开方法获得的“传统肿瘤类器官”。
如本文所用的术语“传统肿瘤类器官”是指分离人结直肠肿瘤组织，并使用已发表的传统条件(具体请参见“实验材料和操作方法”部分)培养获得的类器官。
如本文所用的术语“结直肠肿瘤组织来源类器官”是指分离人结直肠肿瘤组织，并使用本发明的4C条件(例如，LDN214117，EGF，CHIR99021，FGF2)培养获得的类器官。其特点为具有非常强的扩增能力与成克隆能力，同时具有与原位肿瘤组织相似的上皮细胞异质性，相比于传统肿瘤类器官，其表达或显著高水平表达多谱系细胞标志基因LGR5、OLFM4、CHGA、CHGB、MUC2、AQP8、LYPD8中的一种或多种，并能够捕获和长期维持中间可塑性状态，获得长期药物反应能力。
如本文所用的术语“具有增生性肠道隐窝组织特征的类器官”是指指分离人肠道的隐窝组织，并使用本发明的5C条件(例如，LDN214117，EGF，CHIR99021，WS6，SB202190)培养获得的类器官。其特点为中央囊腔显示出明显的收缩迹象，同时新生的隐窝样出芽结构显著增加，类似增生性息肉组织的表型。相比于传统条件下培养获得的类器官，其显著下调部分分化谱系基因，如CHGA、CHGB、MUC2、AQP8、LYPD8等；并且具有增生性肠道隐窝组织特征的类器官中IPS特征基因、肿瘤特异基因等有显著提升。具有增生性肠道隐窝组织特征的类器官为研究多种人体肠道疾病、以及结直肠肿瘤的起始发生提供了理想体外模型。
如本文所用的术语“中间可塑性状态”是指在结直肠病人肿瘤进展过程中出现，具有高度可塑性的状态。其特点是富集了多种不同程序，如营养外胚层细胞、胶质细胞和星形胶质细胞等，具有高度混合的转录程序，同时相比于在传统条件下培养获得的类器官或细胞，其也显著提高了先前报道的泛癌上皮-间充质可塑性标志基因(参见，David P.Cook 等，“Transcriptional census of epithelial-mesenchymal plasticity in cancer”，2022)，并显著高水平表达标志物DPP4，EMP1，ITGA2，ANXA1中的一种或多种。此外，在肿瘤的发生发展、恶性程度及抗放化疗治疗中均发挥重要作用。
如本文所用的术语“增生性肠道隐窝组织”是由于肠道粘膜长期受外界刺激，如炎症、感染等，出现的异常增生，其表现为黏膜的肥厚增生，隐窝结构的异常增多，增长，并且有可能进一步发展成腺瘤或者癌。
如本文所用的术语“基质胶”，也称为细胞外基质(ECM)，由多种多糖、水、弹性蛋白和糖蛋白组成。本发明方法中使用的ECM由MatrigelTM(BD Biosciences)提供，其包含层粘连蛋白、巢蛋白和胶原蛋白IV。
组合物
(1)ALK抑制剂
这类抑制剂与ALK受体结合，并阻止细胞因子与相对应受体的结合。
优选的ALK抑制剂包括LDN214117，其浓度可以使用常规浓度，例如为0.2μM-20μM，优选地1μM-4μM，最优选地为2μM。其结构式如下：
在本文公开的方法中也可使用其他的ALK抑制剂，它们均是本领域已知的并且是市售可获得的，包括但不限于：LDN193189，LDN214117，LDN212854，ML347，K02288。
(2)EGF激动剂
优选的EGF激动剂包括EGF，其浓度可以使用常规浓度，例如为10ng/ml-250ng/ml，优选地25ng/ml-100ng/ml，最优选地为50ng/ml。
在本文公开的方法中也可使用其他的EGF激动剂，它们均是本领域已知的并且是市售可获得的，包括但不限于：EGF，HB-EGF，AREG，EREG，NRG1，NRG2，NRG3，NRG4。
(3)WNT激动剂
WNT激动剂可用于激活细胞中TCF/LEF介导的转录。
优选地WNT激动剂包括CHIR-99021，其浓度可以使用常规浓度，例如为0.3μM-30μM，优选地1.5μM-6μM，最优选地为3μM。其结构式如下：
在本文公开的方法中也可使用其他的WNT激动剂，它们均是本领域已知的并且是市售可获得的，包括但不限于：CHIR-99021，CHIR-98014，CHIR-98023，CHIR-98024，GSK3β inhibitor XV，TD114-2，BIO-acetoxim
(4)FGF激动剂
优选的FGF激动剂包括FGF2，其浓度可以使用常规浓度，例如为10ng/ml-1000ng/ml，优选地50ng/ml-200ng/ml，最优选地为100ng/ml。
在本文公开的方法中也可使用其他的FGF激动剂，它们均是本领域已知的并且是市售可获得的，包括但不限于：FGF1，FGF2，FGF3，FGF4，FGF5，FGF6，FGF7，FGF8，FGF9，FGF10，FGF11，FGF12，FGF13，FGF14，FGF15，FGF16，FGF17，FGF18，FGF19，FGF20，FGF21，FGF23。
(5)EBP1及IκB双重调节剂
优选的EBP1及IκB双重调节剂包括WS6，其浓度可以使用常规浓度，例如为0.05μM-5μM，优选地0.25μM-1μM，最优选地为0.5μM。其结构式如下：
在本文公开的方法中也可使用其他的EBP1及IκB双重调节剂，它们均是本领域已知的并且是市售可获得的，包括但不限于：WS3，(+)-Usnic acid，Pim-1/2 kinase inhibitor 1，RMC-5552，Sauchinone，PS-1145。
(6)p38抑制剂
优选的p38抑制剂包括SB202190，其浓度可以使用常规浓度，例如为1μM-100μM，优选地5μM-20μM，最优选地为10μM。其结构式如下：
在本文公开的方法中也可使用其他的p38抑制剂，它们均是本领域已知的并且是市售可获得的，包括但不限于：Pexmetinib，SB203580，BIRB796，LY2228820，VX-702，PH-797804，VX-745，TAK-715，BMS-582949。
具体实施方式
实施例1建立人肠道组织来源类器官
通过大规模筛选与组合，我们建立了一种新的人肠道组织来源类器官培养条件，其是在基础培养基中添加4个组分(4C；包括ALK抑制剂例如LDN214117、EGF激动剂例如EGF、WNT激动剂例如CHIR-99021、FGF激动剂例如FGF2)。相比传统条件培养的类器官，4C条件下培养的人类结肠类器官显示出显著更强的生长速率与类器官形成能力(图1，a-c)。于此同时，人肠道组织来源的类器官可以在4C条件下扩增超过20个代，并且维持了基因组稳定性(图1，d，e)。
接下来，我们对肠道组织来源类器官进行免疫荧光染色，鉴定其中各细胞谱系表达与分布特征。通过免疫荧光染色，我们观察到了多种谱系细胞的标志物，包括OLFM4代表的肠道干细胞，CHGA代表的肠内分泌细胞，MUC2代表的杯状细胞和APOA1代表的肠上皮细胞，均存在于肠道组织来源类器官中，而在传统肠道类器官中缺失，这与以往的文献报道相一致(图1，f)。
更进一步，我们通过单细胞转录谱(scRNA-seq)分析肠道组织来源类器官中的细胞谱系组成，并与传统肠道类器官和原位肠道隐窝组织进行比较。为了确认细胞异质性的长期维持情况，不同类型的类器官至少培养了2个月。我们使用已发表的人结直肠各谱系细胞的标记基因对类器官和原位肠道隐窝组织进行无监督聚类分析，得到不同细胞谱系的聚类图(图1，g，h)。值得注意的是，尽管存在不可避免的个体差异，我们发现肠道组织来源类器官与传统肠道类器官相比，分化细胞类型的丰富程度显著增加(图1，g，h)。重要的是，肠道组织来源类器官的谱系组成相比传统肠道类器官，更类似于原位肠道隐窝组织(图1，g，h)。每个细胞类群都表现出不同的基因表达模式，这些模式与人结直肠中相应的谱系一致(图1，i，j)。总的来说，这些结果表明肠道组织来源类器官可以捕获并维持与原位肠道隐窝组织相当的细胞谱系组成。
同时，我们测试了不同ALK抑制剂，均能成功构建肠道类器官，仅扩增速率略有差异，其中LDN-214117最优(图1，k，l)。
实施例2建立人结直肠肿瘤组织来源类器官
我们将4C条件用于培养人结直肠肿瘤组织，有意思的是，尽管存在不可避免的个体差异，但与传统条件下培养的人结直肠肿瘤类器官(传统肿瘤类器官)相比，在该条件下培养的人结直肠肿瘤类器官(结直肠肿瘤组织来源类器官)始终显示出更大的尺寸与更高的形成效率，以及更强大的长期扩张能力(图2，a-c)。于此同时，人结直肠肿瘤组织来源类器官可以在该条件下扩增超过20个代，并维持典型形态及扩增能力(图2，d)。
接下来，我们以原位肿瘤组织作为对照，鉴定结直肠肿瘤组织来源类器官的特征。在免疫缺陷小鼠肾囊下异种移植结直肠肿瘤组织来源类器官后，我们在体内重建了人结直肠肿瘤组织。组织病理学特征分析显示，结直肠肿瘤组织来源类器官来源的异种移植物与相对应患者原位肿瘤组织具有相似的结构及肿瘤标记物染色分布，包括Ki-67、CK20、OLMF4和β-catenin(图2，e)。为了进一步研究结直肠肿瘤组织来源类器官的分子特征，我们进行了整体转录谱测序(RNA-seq)分析，并与肠道组织来源类器官进行比较。基因集富集分析进一步证实了结直肠肿瘤组织来源类器官富集了肿瘤相关特征基因群(图2，f)。我们采用最近报道的人结直肠肿瘤特征基因集，发现其在结直肠肿瘤组织来源类器官中也高度富集(图2，g)。此外，整体转录谱的降维分析显示，来自结直肠肿瘤组织的结直肠肿瘤组织来源类器官与来自肠道组织的肠道组织来源类器官具有截然不同的性质(图2，h，i)。总的来说，这些数据表明结直肠肿瘤组织来源类器官的基因表达与结直肠肿瘤更加相似。
为了确定结直肠肿瘤组织来源类器官的基因组特征，我们进行了全外显子组测序(WES)，并比较了结直肠肿瘤组织来源类器官和相对应原位肿瘤组织的基因突变。我们发现，结直肠肿瘤组织来源类器官表现出与配对原位肿瘤非常相似的点突变模式(图2，j)。基因突变变异的热图显示，每个患者特征性的突变基因也在结直肠肿瘤组织来源类器官中得到重现(图2，k)。总的来说，这些结果表明结直肠肿瘤组织来源类器官可以忠实地反应原发肿瘤的组织病理学和基因组特征。
接下来，我们进行了单细胞转录谱(scRNA-seq)分析，以进一步研究结直肠肿瘤组织来源类器官中的细胞谱系组成，并与传统肿瘤类器官和原位肿瘤组织进行比较。为了确认细胞异质性的长期维持情况，不同类型的肿瘤类器官至少培养了2个月。我们使用已发表的人结直肠各谱系细胞的标记基因对肿瘤类器官和原位肿瘤组织进行无监督聚类分析，得到不同细胞谱系的聚类图(图3，a)。值得注意的是，尽管存在不可避免的个体差异，我们发现结直肠肿瘤组织来源类器官与传统肿瘤类器官相比，分化细胞类型的丰富程度显著增加(图3，b)。例如，以OLFM4为标记基因的干细胞类群，以CHGA、CHGB为标记基因的肠内分泌细胞、以MUC2为标记基因的杯状细胞，以及以AQP8、LYPD8为标记基因的肠上皮细胞等，在结直肠肿瘤组织来源类器官中有显著提升。重要的是，结直肠肿瘤组织来源类器官的谱系组成相比传统肿瘤类器官，更类似于原位肿瘤上皮细胞(图3，b)。每个细胞类群都表现出不同的基因表达模式，这些模式与人体内组织相应的谱系一致(图3，c，d)。总的来说，这些结果表明结直肠肿瘤组织来源类器官可以捕获并维持与原位肿瘤相当的上皮异质性。
考虑到4C条件可以同时支持正常类器官与肿瘤类器官的培养和扩张，我们对培养的肿瘤类器官中肿瘤纯度进行了估计，以排除结直肠肿瘤组织来源类器官中正常组织污染的可能性。基于单细胞转录谱测序数据，我们通过inferCNV分析体细胞拷贝数改变(CNAs)来识别肿瘤细胞。该分析可以有效地将具有CNAs的恶性细胞与具有正常核型的非恶性细胞分开。我们发现结直肠肿瘤组织来源类器官大量保留了相应原位肿瘤的DNA拷贝数的损失和增加(图3，e)。根据恶性细胞比例计算肿瘤纯度，我们发现原位肿瘤、传统肿瘤类器官和结直肠肿瘤组织来源类器官中恶性细胞的比例分别为87％、90％和94.2％(图3，e)，这表明结直肠肿瘤组织来源类器官具有高纯度的肿瘤细胞。总的来说，这些结果表明结直肠肿瘤组织来源类器官在上皮异质性、肿瘤纯度等方面与原位肿瘤相似。
实施例3结直肠肿瘤组织来源类器官维持“中间可塑性状态”及其长期药物反应能力的评估
为了探索结直肠肿瘤组织来源类器官能否捕获原位肿瘤组织的特征，我们首先在单细胞水平上分析了人类结直肠肿瘤的转录谱特征。我们重新分析了最近公开的单细胞转录谱数据集，该数据集由来自63名结直肠肿瘤患者的49155个上皮细胞组成，无监督聚类显示了10个不同的细胞分群(图4，a)。通过对不同细胞分群和程序的比较，我们发现细胞分群7富集了先前报道的泛癌上皮-间充质可塑性标志基因(图4b)，表明细胞分群7是一种中间塑性状态，这种状态积极参与并可能促进显着的表型转变，包括细胞谱系转换、上皮-间质转变和癌胚重编程过程。因此，我们将这些细胞的状态定义为中间可塑性状态(IPS)。
接下来，我们研究结直肠肿瘤组织来源类器官能否捕获具有IPS特征的细胞群。我们使用至少培养了2个月的结直肠肿瘤组织来源类器官和传统肿瘤类器官，进行单细胞转录谱分析。并使用标签转移来注释肿瘤类器官中的细胞分群情况，我们观察到细胞分群7特征基因仅在结直肠肿瘤组织来源类器官中富集(图4c，d)。与这一结果一致，我们发现结直肠肿瘤组织来源类器官中IPS细胞的占比显著增加，泛癌上皮-间充质可塑性标志基因显著提升，而这群细胞在传统肿瘤类器官中并不存在(图4e，f)。为了比较结直肠肿瘤组织来源类器官和原位肿瘤组织中捕获的IPS的特征，我们整合了不同类型的测序数据。值得注意的是，结直肠肿瘤组织来源类器官中IPS细胞的转录组谱与原位肿瘤组织中高度相似(图4g)。我们进一步验证了IPS代表性基因DPP4、ITGA2、ANXA1和EMP3在结直肠肿瘤组织来源类器官中显著上调(图4h)。总的来说，这些数据表明结直肠肿瘤组织来源类器官可以通过维持具有IPS特征的细胞群，并进一步反映人结直肠肿瘤的表型可塑性。
为了进一步探索结直肠肿瘤组织来源类器官的功能，我们检测了结直肠肿瘤组织来源类器官对放化疗药物的反应，并与传统肿瘤类器官进行比较。这些肿瘤来源于一名未接受任何辅助治疗的AJCC III期中分化腺癌患者，并在不同条件下培养。我们采用一线化疗药物FOLFOXIRI 检测肿瘤类器官的药物反应。细胞活力用细胞计数试剂盒-8(CCK-8)测定，并绘制肿瘤类器官对治疗的剂量-反应曲线。我们分别检测了肿瘤类器官在早期传代(从肿瘤组织培养)和晚期传代(培养至少2个月)时的治疗响应。我们发现，在早期传代时期，结直肠肿瘤组织来源类器官和传统肿瘤类器官，具有相似的对辐照和FOLFOXIRI的响应，结直肠肿瘤组织来源类器官细胞活力略有增加(图4，i)。值得注意的是，与结直肠肿瘤组织来源类器官相比，传统肿瘤类器官在长期传代后，对辐照和化疗药物的抵抗能力显著下降(图4，i)。而结直肠肿瘤组织来源类器官在传代晚期对辐照和FOLFOXIRI的抗性得到进一步增强(图4，i)。这些结果表明，结直肠肿瘤组织来源类器官作为一种稳定的表型，可以长期维持肿瘤的耐药性，而传统肿瘤类器官缺乏耐药肿瘤细胞的维持和扩增。
实施例4发现通过增强“中间可塑性状态”使结直肠肿瘤组织来源类器官获得上皮-间充质可塑性并增加转移潜力的小分子
通过小分子筛选，我们发现WS6，一种EBP1及IκB双重调节剂，能够显著诱导结直肠肿瘤组织来源类器官提升上皮-间充质样转变，表现为肿瘤细胞大量呈现间充质样的梭形形态(图5a，b)。此外，免疫荧光分析显示WS6处理的结直肠肿瘤组织来源类器官失去了顶端-基底极性，并表现出细胞-细胞粘附性降低(图5a，b)。qPCR分析进一步显示，经过WS6处理的结直肠肿瘤组织来源类器官，多种上皮-间充质样转变相关的标记基因表达显著升高，包括VIM、LGALS1、TNC和HTRA1(图5c)。基因集变异分析(GSVA)显示，11个已被报道的上皮-间充质样转变相关基因集中有10个在WS6处理后显著提升(图5d)。这些结果表明，WS6的加入可显著诱导结直肠肿瘤组织来源类器官发生上皮-间充质样转变。
通过注释标签转移来分析加入WS6处理与否的结直肠肿瘤组织来源类器官的单细胞转录谱数据，我们发现WS6的添加将“中间可塑性状态”细胞的比例大约从6％增加到14％(图5e，f)。
我们从一名没有转移的AJCC II期结直肠肿瘤患者中分离其肿瘤组织并培养成类器官，分别给与或不给与WS6处理，并使用小鼠体内异 种移植体系验证WS6是否能够提升结直肠肿瘤组织来源类器官的转移潜力。我们将经过或不经过WS6处理的结直肠肿瘤组织来源类器官植入免疫缺陷小鼠的脾脏，发现只有经过WS6处理的结直肠肿瘤组织来源类器官在腹腔和盆腔中引起转移(图5，g，h)。此外，经过WS6处理的结直肠肿瘤组织来源类器官在小鼠腹腔中形成的转移瘤数量和转移瘤的平均亮度显著增加(图5，g，h)。总的来说，这些数据表明WS6处理增加了结直肠肿瘤组织来源类器官的转移潜力。
实施例5发现启动“中间可塑性状态”的小分子组合
IPS在结直肠肿瘤组织来源类器官中的捕获促使我们研究在肠道上皮细胞中诱导IPS程序能否触发相似的表型转变。有趣的是，我们没有在正常结肠隐窝来源的肠道组织来源类器官中观察到IPS细胞的富集。通过大规模小分子筛选与优化，我们建立了一种新的小分子组合，其是在基础培养基中添加5个组分(5C；包括ALK抑制剂例如LDN214117、EGF激动剂例如EGF、WNT激动剂例如CHIR-99021、EBP1及IκB双重调节剂例如WS6和p38抑制剂例如SB202190)，能够显著启动“中间可塑性状态”的表达。通过将分离的人肠道隐窝组合或者将实施例1中获得的人肠道组织来源类器官或增生性肠道隐窝组织在新的5C条件下培养，IPS特征基因相比肠道组织来源类器官及传统肠道类器官明显更加富集(图6a)，同时多个上皮-间充质可塑性标志基因显著提升(图6b)。与此结果一致，我们进行了单细胞转录谱分析，发现与肠道组织来源类器官和传统条件培养的类器官相比，具有增生性肠道隐窝组织特征的类器官中IPS细胞的比例显著增加(图6c，d)。总之，我们建立了新的小分子组合，能够在肠道上皮细胞中显著诱导IPS程序的表达。
实施例6建立具有增生性肠道隐窝组织特征的人肠道类器官
有趣的是，与肠道组织来源类器官相比，在5C条件下培养的人肠道组织类器官，中央囊腔显示出明显的收缩迹象(图6e)。相对应，其新生的隐窝样出芽结构的显著增加(图6f)。同时，多种分化功能谱系基因 (例如CHGA、CHGB、MUC2、AQP8、LYPD8)有显著降低(图6h)，这与在增生性息肉组织中观察到的表型相似，我们将之定义为具有增生性肠道隐窝组织特征的人肠道类器官。重要的是，具有增生性肠道隐窝组织特征的类器官可以作为一种稳定表型长期扩展(图6e，f，h)。这表明5C条件诱导得到的增生性肠道隐窝特征并非类器官的短暂应激反应，而是一种稳定的状态转变，模拟了体内肿瘤发生过程早期，正常肠道隐窝组织经历的增生阶段。因此，使用5C条件直接培养增生性人肠道隐窝组织，也能很好维持起增殖性肠道隐窝组织的特征。并且我们发现，与肠道组织来源类器官和传统条件下培养的类器官相比，近期报道的结直肠肿瘤特征基因在具有增生性肠道隐窝组织特征的类器官中表达显著增强，尽管其程度仍低于在结肠炎相关癌症组织(图6g)。总的来说，我们建立了一个高效稳定的化学重编程系统，在肠道上皮细胞中诱导IPS程序，并进一步培养出具有增生性肠道隐窝组织特征的类器官，其主要用于研究多种疾病模型，包括健康肠道-＞增生肠道-＞肿瘤的转变过程，为治疗肿瘤提供新的理论指导或潜在靶点。

Claims (25)

  1. 一种组合物，其特征在于，包括(1)ALK抑制剂，(2)WNT激动剂，(3)EGF激动剂和(4)FGF激动剂，优选地所述组合物用于获得和/或培养人肠道组织来源类器官或人结直肠肿瘤组织来源类器官。
  2. 根据权利要求1所述的组合物，其中，所述(1)ALK抑制剂选自由以下各项组成的组中的一种或多种：LDN193189，LDN214117，LDN212854，ML347，K02288；优选为LDN214117。
  3. 根据权利要求1所述的组合物，其中，所述(2)WNT激动剂选自由以下各项组成的组中的一种或多种：CHIR-99021，CHIR-98014，CHIR-98023，CHIR-98024，GSK3βinhibitor XV，TD114-2，BIO-acetoxim；优选为CHIR-99021。
  4. 根据权利要求1所述的组合物，其中，所述(3)EGF激动剂选自由以下各项组成的组中的一种或多种：EGF，HB-EGF，AREG，EREG，NRG1，NRG2，NRG3，NRG4；优选为EGF。
  5. 根据权利要求1所述的组合物，其中，所述(4)FGF激动剂选自由以下各项组成的组中的一种或多种：FGF1，FGF2，FGF3，FGF4，FGF5，FGF6，FGF7，FGF8，FGF9，FGF10，FGF11，FGF12，FGF13，FGF14，FGF15，FGF16，FGF17，FGF18，FGF19，FGF20，FGF21，FGF23；优选为FGF2。
  6. 根据权利要求1所述的组合物，其还包含(5)EBP1及IκB双重调节剂；
    任选地，所述(5)EBP1及IκB双重调节剂选自由以下各项组成的组中的一种或多种：WS3，WS6，(+)-Usnic acid，Pim-1/2激酶抑制剂1，RMC-5552，Sauchinone，PS-1145；优选为WS6。
  7. 获得和/或培养人肠道组织来源类器官或人结直肠肿瘤组织来源类器官的方法，其特征在于，在培养基中进行培养，优选地在培养基中将正常人肠道隐窝组织或人结直肠肿瘤组织进行培养，其中所述培养基包含权利要求1-6任一项所述的组合物。
  8. 根据权利要求7所述的方法，其中，使用DMEM/F12作为基础培养基；
    优选地，所述基础培养基中还添加有HEPES、GlutaMAX、B27、1mM N-Acetylcysteine。
  9. 权利要求7所述的方法获得的人肠道组织来源类器官或人结直肠肿瘤组织来源类器官。
  10. 权利要求9所述的人肠道组织来源类器官或人结直肠肿瘤组织来源类器官，其特征在于，其表达标志基因LGR5、OLFM4、CHGA、CHGB、MUC2、AQP8、LYPD8中的一种或多种。
  11. 权利要求9所述的人结直肠肿瘤组织来源类器官，其包括具有“中间可塑性状态”细胞，所述“中间可塑性状态”细胞提高泛癌上皮-间充质可塑性标志基因，并显著提高标志物DPP4，EMP1，ITGA2，ANXA1中的一种或多种的表达。
  12. 权利要求9所述的人结直肠肿瘤组织来源类器官，其能够长期维持“中间可塑性状态”细胞。
  13. 权利要求9所述的人结直肠肿瘤组织来源类器官，其用于体外研究肿瘤抗治疗能力、转移侵袭等能力，用于筛选高效抑制或杀伤肿瘤细胞的治疗化合物，或用作重建肿瘤发展过程的体外模型。
  14. 组合物，其包括(1)ALK抑制剂，(2)WNT激动剂，(3)EGF激动剂，(4)EBP1及IκB双重调节剂和(5)p38抑制剂，优选地所述组合物用于获得和/或培养具有增生性肠道隐窝组织特征的类器官和/或“中间可塑性状态”细胞。
  15. 根据权利要求14所述的组合物，其中，所述(1)ALK抑制剂选自由以下各项组成的组中的一种或多种：LDN193189，LDN214117，LDN212854，ML347，K02288；优选为LDN214117。
  16. 根据权利要求14所述的组合物，其中，所述(2)WNT激动剂选自由以下各项组成的组中的一种或多种：CHIR-99021，CHIR-98014，CHIR-98023，CHIR-98024，GSK3βinhibitor XV，TD114-2，BIO-acetoxim；优选为CHIR-99021。
  17. 权利要求14所述的组合物，其中，所述(3)EGF激动剂选自由以下各项组成的组中的一种或多种：EGF，HB-EGF，AREG，EREG，NRG1，NRG2，NRG3，NRG4；优选为EGF。
  18. 权利要求14所述的组合物，其中，所述(4)EBP1及IκB双重调节剂选自由以下各项组成的组中的一种或多种：WS3，WS6，(+)-Usnic acid，Pim-1/2 kinase inhibitor 1，RMC-5552，Sauchinone，PS-1145；优选为WS6。
  19. 权利要求14所述的组合物，其中，所述(5)p38抑制剂选自由以下各项组成的组中的一种或多种：SB202190，Pexmetinib，SB203580，BIRB796，LY2228820，VX-702，PH-797804，VX-745，TAK-715，BMS-582949；优选为SB202190。
  20. 获得和/或培养人具有增生性肠道隐窝组织特征的肠道类器官和/或“中间可塑性状态”细胞的方法，其特征在于，在培养基中进行培养，优选地在培养基中将人正常肠道隐窝组织或增生性肠道隐窝组织或权利要求9所述的人肠道组织来源类器官进行培养，其中所述培养基包含权利要求14-19中任一项所述的组合物。
  21. 权利要求20所述的方法，其中，使用DMEM/F12作为基础培养基；
    优选地，所述基础培养基中还添加有HEPES、GlutaMAX、B27、1mM N-Acetylcysteine。
  22. 权利要求21所述的方法获得的人具有增生性肠道隐窝组织特征的肠道类器官和/或“中间可塑性状态”细胞。
  23. 权利要求22所述的人具有增生性肠道隐窝组织特征的肠道类器官，其特征在于，显著下调部分分化谱系基因，如CHGA、CHGB、MUC2、AQP8、LYPD8中的一种或多种。
  24. 权利要求22所述的人具有增生性肠道隐窝组织特征的肠道类器官，其特征在于可以长期维持“中间可塑性状态”细胞。
  25. 权利要求22所述的人具有增生性肠道隐窝组织特征的肠道类器官，其用于体外研究肿瘤起始发生，用于筛选高效抑制或杀伤肿瘤细胞的治疗化合物，或用作研究肿瘤发生的体外模型。
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Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190367882A1 (en) * 2016-12-05 2019-12-05 Children's Hospital Medical Center Colonic organoids and methods of making and using same
CN114085803A (zh) * 2020-08-24 2022-02-25 北京大学 建立具有再生特性的类器官培养体系
CN114181903A (zh) * 2021-12-06 2022-03-15 浙江弘瑞医疗科技有限公司 结直肠癌类器官培养基及无支架3d培养方法

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP3842527A4 (en) * 2018-08-24 2022-05-18 Sumitomo Chemical Company Limited CELL AGGREGATION WITH OLFACTORY NEURON OR PRODUCER CELL THEREOF AND METHODS FOR PRODUCTION THEREOF
EP4043553A4 (en) * 2019-10-01 2023-11-22 Kyoto University METHOD FOR ISOLATING URETERAL NODLE CELLS
US20230340419A1 (en) * 2020-04-27 2023-10-26 University Of Southern California Methods for generation of mouse and human ureteric bud organoids and collecting duct organoids
CN112626023B (zh) * 2021-01-12 2023-08-04 中国人民解放军海军军医大学 脊髓olig2+神经祖细胞诱导及自我更新培养体系、诱导方法及应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20190367882A1 (en) * 2016-12-05 2019-12-05 Children's Hospital Medical Center Colonic organoids and methods of making and using same
CN114085803A (zh) * 2020-08-24 2022-02-25 北京大学 建立具有再生特性的类器官培养体系
CN114181903A (zh) * 2021-12-06 2022-03-15 浙江弘瑞医疗科技有限公司 结直肠癌类器官培养基及无支架3d培养方法

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
SHOICHI DATE, TOSHIRO SATO: "Mini-Gut Organoids: Reconstitution of the Stem Cell Niche", ANNUAL REVIEW OF CELL AND DEVELOPMENTAL BIOLOGY., ANNUAL REVIEWS, US, vol. 31, no. 1, 13 November 2015 (2015-11-13), US , pages 269 - 289, XP055471729, ISSN: 1081-0706, DOI: 10.1146/annurev-cellbio-100814-125218 *
XIA SUN, LIXING ZHAN: "Establishment and application of gut organoids", JOURNAL OF COLORECTAL & ANAL SURGERY, vol. 26, no. 3, 28 June 2020 (2020-06-28), pages 251 - 256, XP093212839, ISSN: 1674-0491, DOI: 10.19668/j.cnki.issn1674-0491.2020.03.001 *

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