Use of cancer-associated fibroblasts positive for transporter protein ABCA8 in the preparation of markers for diagnosing SETD2-deficient pancreatic cancer

Abstract

The present invention provides the use of cancer-associated fibroblasts positive for the transporter protein ABCA8 in the preparation of a marker for diagnosing pancreatic cancer with SETD2 deficiency. The present invention also provides the use of cancer-associated fibroblasts positive for the transporter protein ABCA8 as a marker for preparing a marker for diagnosing pancreatic cancer with high oxidative phosphorylation metabolism characteristics. The present invention also provides the use of a specific oxidative phosphorylation inhibitor in the preparation of a drug for treating pancreatic cancer with SETD2 deficiency, the structural formula of the specific oxidative phosphorylation inhibitor is shown below, and the present invention also provides the use of the transporter protein ABCA8 as a target in the preparation and screening of drugs for treating pancreatic cancer with SETD2 deficiency. The present invention identifies for the first time the existence of a new subpopulation of CAFs with ABCA8 markers and rich in lipids in SETD2-deficient pancreatic cancer, which provides lipids to tumor cells through the ABCA8 transporter, thereby enhancing tumor cell mitochondrial OXPHOS and maintaining tumor progression.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine and relates to a biomarker, specifically, the use of cancer-associated fibroblasts positive for the transporter protein ABCA8 in preparing a marker for diagnosing SETD2-deficient pancreatic cancer, and suggests the use of a specific oxidative phosphorylation inhibitor (S-Gboxin) in preparing a drug for treating SETD2-deficient pancreatic cancer. Background Art

[0002] Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer (hereinafter referred to as pancreatic cancer), characterized by high malignancy and poor prognosis. It is expected to become the second leading cause of cancer-related death in the world after lung cancer by 2030. So far, surgical resection is still the only radical cure for pancreatic cancer. However, PDAC has an insidious onset, and most patients have already developed local infiltration and distant metastasis when they seek medical treatment, lacking surgical indications. Therefore, radiotherapy and chemotherapy have become the main treatments for patients with advanced and distant metastases. However, pancreatic cancer is not sensitive to radiotherapy and chemotherapy so far. In addition, pancreatic cancer lacks effective targets and specific targeted drugs; immunotherapy has a very limited effect on pancreatic cancer. The above status of pancreatic cancer diagnosis and treatment suggests that strengthening basic research on the pathogenesis of pancreatic cancer is expected to optimize early diagnosis and develop new treatment strategies, which is of great clinical significance.

[0003] Pancreatic carcinogenesis is driven by both tumor cells and the tumor microenvironment (TME). Cancer-associated fibroblasts (CAFs) are the most abundant cells in the pancreatic cancer TME and play an important role in tumorigenesis and metastasis, metabolic reprogramming, immune evasion, and drug resistance. CAFs are highly heterogeneous in origin, phenotype, and function. Single-cell RNA sequencing (scRNA-seq) has identified the presence of myofibroblastic CAFs (myCAFs), inflammatory CAFs (iCAFs), and antigen-presenting CAFs (apCAFs) in mouse and human pancreatic tumors. Heterogeneous CAF subsets promote tumors or inhibit tumor progression by secreting cytokines and metabolites to support or inhibit tumor cells and surrounding immune cells. The heterogeneity of CAFs is regulated by both tumor cells and the TME; however, how tumor cell-intrinsic factors, especially epigenetic dysregulation, affect the phenotypic and functional heterogeneity of CAFs remains to be elucidated.

[0004] Epigenetic dysregulation is one of the important characteristics of tumor cells. In recent years, many studies have revealed that epigenetic dysregulation can regulate tumor occurrence and development from multiple perspectives by affecting stromal cells and immune cells to reshape TME. Histone methyltransferase SETD2 (SET domain containing protein 2) catalyzes the production of H3K36me3, and its mutation or abnormal expression is widely present in a variety of solid tumors including PDAC, becoming a common tumor suppressor mechanism. Therefore, the development of precision treatment plans for SETD2-deficient tumors has broad translational significance.

[0005] SETD2 mutations are widely present in PDAC. The present invention identifies for the first time a new lipid-rich CAF subpopulation marked by ABCA8 in SETD2-deficient pancreatic tumors. This CAF subpopulation supplies lipid nutrients to tumor cells through the ABCA8 transporter, thereby increasing tumor cell mitochondrial oxidative phosphorylation (OXPHOS) and maintaining tumor progression. A specific OXPHOS high-activity inhibitor (S-Gboxin) can inhibit the growth of SETD2-deficient pancreatic tumors in mouse models and human tumor xenograft models (PDX). In general, the present invention emphasizes a new subtype of lipid-rich CAF marked by the transporter ABCA8, and OXPHOS inhibitors have therapeutic potential for SETD2-deficient pancreatic cancer with this lipid-rich subtype CAF, thereby providing a theoretical basis and potential strategy for the precise treatment of SETD2-deficient pancreatic cancer.

[0006] The transporter described in the present invention is named ABCA8a in mice and ABCA8 in humans.

[0007] S-Gboxin is a functional analog of Gboxin and an inhibitor of oxidative phosphorylation (OXPHOS). It can inhibit the growth of mouse and human glioblastoma (GBM) with an IC50 value of 470nM. The structural formula is shown below:

[0008] Summary of the invention

[0009] In view of the above technical problems in the prior art, the present invention provides the use of cancer-associated fibroblasts positive for the transporter protein ABCA8 in the preparation of a marker for diagnosing SETD2-deficient pancreatic cancer. The use of cancer-associated fibroblasts positive for the transporter protein ABCA8 in the preparation of a marker for diagnosing SETD2-deficient pancreatic cancer is intended to solve the technical problem of poor diagnostic effect for SETD2-deficient pancreatic cancer in the prior art.

[0010] The present invention provides the use of cancer-associated fibroblasts positive for transporter protein ABCA8 in preparing a marker for diagnosing SETD2-deficient pancreatic cancer.

[0011] The present invention also provides the use of transporter protein ABCA8 positive cancer-associated fibroblasts as a marker in preparing a kit for diagnosing SETD2-deficient pancreatic cancer.

[0012] The present invention also provides the use of transporter protein ABCA8-positive cancer-associated fibroblasts as markers for preparing a diagnosis of pancreatic cancer with high oxidative phosphorylation metabolic characteristics.

[0013] The present invention also provides the use of transporter protein ABCA8 positive cancer-associated fibroblasts as a marker in the preparation of a kit for diagnosing pancreatic cancer with high oxidative phosphorylation metabolic characteristics.

[0014] The present invention also provides a kit comprising a reagent for detecting cancer-associated fibroblasts positive for transporter protein ABCA8.

[0015] The present invention also provides a use of a specific oxidative phosphorylation inhibitor in the preparation of a drug for treating SETD2-deficient pancreatic cancer. The structural formula of the specific oxidative phosphorylation inhibitor is shown below:

[0016]

[0017] The present invention also provides the use of the transporter protein ABCA8 as a target in the preparation and screening of drugs for treating SETD2-deficient pancreatic cancer.

[0018] Furthermore, the SETD2 deficiency refers to SETD2 mutation or low expression.

[0019] Experiments have shown that SETD2 mutations are widespread in pancreatic cancer. A lipid-rich CAF subpopulation marked by ABCA8a was identified in SETD2-deficient pancreatic cancer in both mouse models and patient samples. This CAF population provides lipids to tumor cells through the ABCA8a transporter, thereby increasing tumor cell mitochondrial OXPHOS and maintaining tumor progression. In addition, given the metabolic characteristics dominated by oxidative phosphorylation (OXPHOS) in SETD2-deficient pancreatic cancer, the use of OXPHOS inhibitors (S-Gboxin) can inhibit the occurrence and development of SETD2-deficient pancreatic cancer in mouse models and human tumor xenograft models (PDX).

[0020] Compared with the prior art, the technical effect of the present invention is positive and obvious. The present invention identifies for the first time the existence of a new lipid-rich CAF subpopulation with ABCA8a marker in SETD2-deficient pancreatic cancer, which provides lipids to tumor cells through ABCA8a transporters, thereby enhancing tumor cell mitochondrial OXPHOS and maintaining tumor progression. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 The expression of SETD2 / H3K36me3 in human pancreatic cancer samples.

[0022] Figure 2 To identify a specific lipid-enriched CAF subpopulation with ABCA8a signature in SETD2-deficient pancreatic tumors.

[0023] Figure 3 Deletion of ABCA8a suppresses pancreatic tumor progression in SETD2-deficient mice.

[0024] Figure 4 Enhanced OXPHOS metabolic signature in SETD2-deficient pancreatic tumor cells.

[0025] Figure 5 OXPHOS inhibitors can suppress the progression of SETD2-deficient pancreatic cancer in mouse models.

[0026] Figure 6 Correlation of SETD2 / H3K36me3 with CAFs marked by ABCA8 in human pancreatic cancer samples.

[0027] Figure 7 The effects of OXPHOS inhibitors on human tumor xenograft (PDX) models. DETAILED DESCRIPTION

[0028] The present invention will be further described below in conjunction with specific embodiments with reference to the accompanying drawings for a better understanding of the present invention.

[0029] The following are specific embodiments of the present invention, which are used to describe the present invention rather than to limit the present invention.

[0030] The reagents and experimental methods used in the examples are as follows:

[0031] Molecular biochemical reagents: Citric acid tissue antigen retrieval solution (MVS-0101), DAB peroxidase substrate kit (DAB-2031) were purchased from Maixin Biotechnology; dimethyl sulfoxide (DMSO) and collagenase P were purchased from Sigma-Aldrich, USA; 30% H 2 O 2Solution, phosphate buffered saline (DPBS) was purchased from Shanghai Shenggong Bioengineering Co., Ltd.; type IV collagenase, bovine serum albumin (BSA) and DNase I were purchased from Shanghai Yishen Biotechnology Co., Ltd.; HBSS medium, newborn calf serum (NCS) and MEMα medium were Gibco brands; S-Gboxin was purchased from MCE (MedChemExpress); BODIPY 493 / 503 was purchased from Thermo Fisher Scientific; tissue cell triglyceride (TG) content enzymatic determination kit was purchased from Beijing Pulilai Gene Technology Co., Ltd.; free fatty acid (NEFA) test kit was purchased from Nanjing Jiancheng Bioengineering Institute; Seahorse XF cell mitochondrial stress test kit was purchased from Agilent Technologies Co., Ltd.

[0032] Example 1

[0033] Immunohistochemical staining of pancreatic ductal adenocarcinoma clinical tissue microarray was performed to determine the expression of SETD2 / H3K36me3 in human pancreatic cancer samples.

[0034] The pancreatic ductal adenocarcinoma clinical tissue microarray (C20150721, tissuemicroarray TMA) was performed in Renji Hospital (Shanghai, China) with approval from the local ethics committee and consent from the patients. Clinical parameters of pancreatic cancer patients, including age, sex, stage, pathological diagnosis, differentiation status, TNM status, and survival rate, were collected. None of the patients received preoperative chemotherapy or radiotherapy before surgery.

[0035] Immunohistochemical staining experiment: After dewaxing and rehydration, paraffin-embedded tissue sections were subjected to microwave antigen retrieval in citric acid tissue antigen retrieval solution for 15 minutes. Next, the sections were washed with PBS and then incubated in a 3% volume concentration of H 2 O 2 The cells were incubated in the solution for 10 minutes to eliminate endogenous peroxidase activity. After blocking with 5% goat serum at room temperature for 1 hour, target-specific antibodies were added: H3K36me3 (1:1000, Abcam) and SETD2 (1:300, CST); and incubated overnight at 4°C, followed by reaction with biotinylated goat anti-rabbit secondary antibody and detection using a commercial DAB peroxidase substrate kit (DAB-2031, Maixin Biotech).

[0036] Figure 1 A shows representative immunohistochemical images of SETD2 and H3K36me3 in Renji Hospital human pancreatic cancer tissue microarray (C20150721), with a scale bar of 100 μm.

[0037] Figure 1 B. The expression of SETD2 and H3K36me3 in the human pancreatic cancer tissue microarray of Renji Hospital (excluding samples that fell off the slide during the experiment). The results showed that the level of SETD2 was positively correlated with the level of H3K36me3 in human pancreatic cancer samples (Chi-square test, P<0.0001).

[0038] Example 2

[0039] Mouse pancreatic orthotopic tumor model: Trypsin digestion of Setd2-deficient and wild-type controls (Setd2 KO and Setd2 WT ) pancreatic tumor cells, remove the supernatant after centrifugation, wash with DPBS 2-3 times, and count the cells respectively. KO and Setd2 WT The cells were resuspended in DPBS containing 10% Matrigel at a density of 1x10 6 Cells / 25μl can be used for inoculation of one mouse. Tumor cells were injected into the pancreas of 8-week-old, healthy, female C57BL / 6J mice. The abdomen was closed and the endothelium and outer skin were sutured. If there were no special instructions, the mice were killed 2 weeks after tumor cell inoculation and the corresponding tumor tissues were collected for subsequent experiments.

[0040] Collagenase method to separate mouse pancreatic in situ tumor cells: Take the pancreatic tissue of tumor-bearing mice and temporarily place it in FACS buffer (HBSS + 2% NCS) (on ice), and remove the clean mucosa and lymph node tissue. Put the pancreatic tissue into a 1.5ml EP tube, add 200μl FACS-prepared enzyme digestion solution (composed of collagenase IV with a final concentration of 2mg / ml), cut it with scissors, and then transfer the tissue to a 20ml glass bottle, add 5ml of enzyme digestion solution, place it in a 37℃ water bath, and incubate it for 30 minutes. Then, filter it into a 15ml centrifuge tube with a 200-mesh gauze, centrifuge it at 50g for 1 minute, take the supernatant (the purpose is to remove the acinar cells); then centrifuge it at 350g for 5 minutes, and discard the supernatant. Suspend the precipitated cells in red blood cell lysis buffer to lyse the red blood cells, resuspend them in 100μl FACS buffer, and then proceed with subsequent processing.

[0041] Isolation of human pancreatic cancer primary cells by collagenase method: Primary pancreatic tumor cells were obtained from patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) in Renji Hospital. The method was similar to that of isolating mouse pancreatic tumor primary cells, and they were obtained by collagenase digestion (collagenase IV, 2 mg / ml, digestion for 30-60 minutes).

[0042] Flow staining analysis: The primary mouse pancreatic tumor cells separated by collagenase method were resuspended in 100 μl FACS buffer, and flow cytometry antibodies were added in proportion, and incubated in a 4°C refrigerator away from light for 30 minutes. Then 1 ml FACS buffer was added to each tube to wash away the antibodies, centrifuged at 350g for 5 minutes, the supernatant was discarded, and the cells were resuspended in 200-300 μl FACS buffer for flow analysis; or resuspended in 500-1000 μl FACS buffer for flow sorting.

[0043] Detection of neutral lipid BODIPY in cells: CAFs (DAPI - CD45.2 - PDPN + ). BODIPY 493 / 503 flow cytometry analysis: Prepare staining solution (DPBS + 0.5% BSA) in a volume of 1 ml for each sample to maintain a final concentration of BODIPY 493 / 503 of 1 μM, and incubate in a 37°C oven in the dark for 30 minutes. Add 1 ml of DPBS buffer to each tube to wash away the antibody, centrifuge at 350g for 5 minutes, discard the supernatant, resuspend in 200-300 μl DPBS buffer and immediately perform flow cytometry analysis. Immunofluorescence staining with BODIPY 493 / 503: Incubate fixed cells with BODIPY 493 / 503 solution (1 μM in 1 ml DPBS + 0.5% BSA staining solution) in a 37°C oven in the dark for 30 minutes. Wash the cells and counterstain with DAPI to visualize the cell nucleus. Then, use an Olympus A1Si laser scanning confocal microscope to visualize the stained cells.

[0044] Detection of triglycerides and free fatty acids (NEFA): According to the above method, CAFs (DAPI - CD45.2 - PDPN + ), and then the subsequent metabolic indexes were detected. According to the manufacturer's instructions, triglycerides and free fatty acids (NEFA) were measured on the corresponding cells using the corresponding detection kits (such as Figure 2 shown).

[0045] Figure 2 A shows the expression of Setd2 by flow cytometry KO and Setd2 WT ABCA8a in tumors + and ABCA8a - Representative plots and statistical analysis of CAFs suggesting ABCA8a + CAFs in Setd2 KO The proportion in tumors was significantly higher.

[0046] Figure 2 B shows Setd2 KO ABCA8a in pancreatic tumors + and ABCA8a - Representative immunofluorescence staining images and flow cytometry statistical analysis of BODIPY 493 / 503.

[0047] Figure 2 C shows the result from Setd2 KO ABCA8a sorted in tumors + and ABCA8a - Levels of cellular triglycerides (TG) and free fatty acids (NEFA) in CAFs. - Compared with CAF cells, cells from Setd2 KO ABCA8a sorted in tumors + CAF cells contained more neutral lipids (BODIPY), as well as more abundant intracellular triglycerides (TGs) and free fatty acids (NEFAs), confirming their lipid-rich phenotype.

[0048] Figure 2 D Flow cytometry showed that ABCA8 is also present in pancreatic cancer patients + CAF.

[0049] Example 3

[0050] Primary pancreatic stellate cells (PSC) isolation and iPSC generation: Five pancreata from C57BL / 6J mice (total weight of approximately 1 g) were minced and digested in a dissociation buffer containing 1.3 mg / ml collagenase P and 10 mg / ml DNase I in FACS buffer (HBSS+2% NCS) at 37°C for 12 minutes. The digested pancreas was filtered through a 100 μm filter and washed in FACS buffer. Centrifuged at 50 g for 1 minute to remove acinar cells, the cell suspension was transferred to another tube, and centrifuged at 350 g for 5 minutes. After removing the supernatant, the precipitated cells were suspended in erythrocyte lysis buffer to lyse the erythrocytes. The cells were then inoculated in MEMα medium containing 10% fetal bovine serum and 1% penicillin-streptomycin by volume at 37°C and 5% CO 2 We then used SV40 TAg to immortalize PSCs and generate iPSCs.

[0051] Mouse subcutaneous tumor formation experiment: 5×10 5 Mouse pancreatic cancer cell line (Setd2 KO and Setd2 WT) alone or separately and 5×10 5 Fibroblasts (iPSCs) were mixed and resuspended in DPBS containing 10% Matrigel, and then injected into the groin of 8-week-old female C57BL / 6J mice on both sides. After 2 weeks, the size of the formed tumor was observed and measured.

[0052] Abca8a knockout transgenic mice - / - From Jicui Pharmaceutical Biotechnology Co., Ltd., and control Abca8a + / + Mouse, Setd2 KO In the pancreatic in situ tumor formation experiment, mice were killed 2 weeks after tumor cell inoculation, and the corresponding tumor tissues were collected for subsequent experiments.

[0053] Figure 3 A shows the tumors of mice in different groups, and the scale bar is 1 cm.

[0054] Figure 3 B is the statistical graph of tumor weights of mice in different groups.

[0055] Figure 3 C shows the pancreatic tumors and pancreas / body weight ratios of mice in different groups, with the scale bar being 1 cm.

[0056] Figure 3 D is the H&E staining of tumors in different groups of mice and the statistical diagram of tumor area.

[0057] Figure 3 E shows the expression of Abca8a by flow cytometry + / + and Abca8a - / - Statistical analysis of ABCA8a levels in mouse CAFs suggests that Abca8a - / - The level of ABCA8a on CAFs was significantly decreased.

[0058] The above suggests that different levels of Abca8a in iPSCs can only affect Setd2 KO Tumor progression, Setd2 WT There was no significant effect on tumor progression. + / + Compared with mice, Abca8a - / - Mouse Setd2 KO The tumors shrank significantly. The above results prove that ABCA8a plays a role in promoting Setd2 KO Role in tumor progression.

[0059] Example 4

[0060] RNA sequencing (RNA-seq) and analysis: RNA from Setd2 was obtained by sorting according to Example 2. WT and Setd2KO Tumor cells of pancreatic orthotopic tumor (DAPI - CD45.2 - EpCAM + ) for RNA extraction. The RNA-seq transcriptome library was prepared using the TruSeqTM RNA Sample Preparation Kit (Illumina) according to the instructions. After quality control of the raw data, it was aligned to the reference mouse genome mm10 for subsequent analysis.

[0061] Seahorse XF Cell Mitochondrial Stress Assay (OCR):

[0062] Preparations the day before the experiment: (1) Turn on the Agilent Seahorse XFe / XF analyzer and computer and allow to warm up overnight (at least 5 hours). (2) Hydrate the sensor cartridge and place at 37°C in a CO-free environment. 2 Incubate overnight in an incubator. (3) SeahorseXF cell culture microplates were pre-coated with a mixture of 50 μl NaOH (1 M), 2850 μl NaHCO3 (0.1 M) and 100 μl Cell-Tak.

[0063] On the day of the experiment: (1) Prepare the probe plate: Replace the probe plate that was hydrated the day before with Seahorse hydration solution and place it at 37°C in a CO-free environment. 2 Place in an incubator for at least 1 hour to rehydrate the probe plate. After hydration, use a pipette to add three inhibitor working solutions of oligomycin, FCCP and rotenone / antimycin A to each well. (2) Prepare the detection solution: Add glucose, glutamine and sodium pyruvate to Seahorse XFDMEM culture medium. Make the final concentration of added glucose 10mmol / L, glutamine 2mmol / L and sodium pyruvate 1mmol / L. (3) Sorting the Setd2 cells according to Example 2 WT and Setd2 KO Tumor cells of pancreatic orthotopic tumor (DAPI - CD45.2 - EpCAM + ), centrifuge the cell plate at 200 g for 1 minute (zero brake-off) at 5000 cells / 50 μl of test solution to allow the cells to fall to the bottom of the plate. Then place the cell plate at 37°C without CO 2 Place the plate in an incubator and allow the cells to adhere for 30 minutes. Gently add 130 μl of the test solution to each well along the side of the cell plate to avoid disturbing the cells. At this point, place the cell plate back at 37°C with no CO. 2 Equilibrate in the incubator for 20 minutes. Set up the test machine and start the machine within 1 hour.

[0064] For human pancreatic cancer-specific analysis: Gene Set Enrichment Analysis (GSEA) bioinformatics analysis was performed using TCGA-PAAD (Pan Cancer Atlas).

[0065] Figure 4 A Using an orthotopic mouse pancreatic tumor model, transcriptome analysis revealed Setd2 KO OXPHOS is elevated in pancreatic tumor cells.

[0066] Figure 4 B and Figure 4 C detected Setd2 KO and Setd2 WT The oxidative respiration capacity of pancreatic orthotopic tumor cells further confirmed that Setd2 KO Increased mitochondrial respiration in pancreatic tumor cells.

[0067] Figure 4 D compared the KRAS mut TP53 WT SETD2 WT Patients (hKC) and KRAS mut TP53 mut SETD2 WT Patients (hKPC) and KRAS mut TP53 WT SETD2 mut Transcriptome differences between human KSCs and SETD2-deficient pancreatic cancer patients further confirmed that OXPHOS was elevated in tumor cells.

[0068] Example 5

[0069] Drug administration treatment of mice: The establishment of the mouse orthotopic tumor model was carried out according to Example 2. The mice were intraperitoneally injected with the OXPHOS inhibitor S-Gboxin (10 mg / kg) as the S-Gboxin experimental group. The mice were intraperitoneally injected with the same volume of DMSO as the control group. After 7 days of the establishment of the mouse orthotopic tumor model, the drug treatment was performed every other day. After a total of 8 doses, the pancreatic tissue containing the tumor was collected on the 21st day for subsequent related detection and analysis. HE staining of all mouse tissue samples was completed by Shanghai Ruiyu Biotechnology Co., Ltd.

[0070] Figure 5 A and Figure 5 B: In Setd2 KO and Setd2 WT In an orthotopic pancreatic tumor model, the OXPHOS inhibitor S-Gboxin significantly inhibited Setd2KO Tumor growth, but Setd2 WT The tumor had less impact.

[0071] Example 6

[0072] Human pancreatic cancer samples were obtained from patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) at Renji Hospital affiliated to Shanghai Jiao Tong University School of Medicine. The correlation between H3K36me3 expression in tumor cells and the proportion of CAFs with dual expression of ABCA8 and the known marker FAP was analyzed.

[0073] Figure 6 A is obtained by flow cytometry in accordance with Example 2 from tumor cells (DAPI - CD45 - EpCAM + ) and ABCA8 + FAP + CAFs ratio (the special lipid-rich CAF subpopulation identified in Example 2 also expresses FAP, hereinafter referred to as ABCA8 + FAP + CAFs refers to this special CAF subpopulation).

[0074] Figure 6 B shows ABCA8 from human pancreatic cancer samples + FAP + There was a negative correlation between the proportion of CAFs and the H3K36me3 level in tumor cells.

[0075] Figure 6 C shows representative HE and corresponding H3K36me3 immunohistochemical staining in human pancreatic cancer tissues. Scale bars = 2 mm and 100 μm. Green arrows indicate fat-like cells. This suggests that fat-like cells and less differentiated tumor cells exist in human pancreatic cancer tissues with low H3K36me3 expression.

[0076] Example 7

[0077] Establishment of human xenograft model (PDX): Human pancreatic cancer samples were obtained from patients diagnosed with pancreatic ductal adenocarcinoma (PDAC) in Renji Hospital affiliated to Shanghai Jiao Tong University School of Medicine, with numbers 15031901, 2021011, 2021031, 15052004, 2022003, 2022002 and 2021023. Fresh pancreatic cancer tissues obtained from patients were cut into 2-3 mm 3 The fragments were implanted subcutaneously on the back of 5-week-old female BALB / c nude mice (F1 mice). The tumor size reached 1-2 cm 3When the xenografts reach 1-2 cm, it means the transplantation is successful. Then these xenografts from F1 mice are cut into small pieces and implanted subcutaneously into other mice (F2 mice). 3 When the size of the cells reached 1.37 mm / s, they were cut into small pieces and then transplanted into F3 mice for subsequent drug administration. All 5-week-old female BALB / c nude mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd.

[0078] Drug administration of human xenograft model (PDX): Mice were intraperitoneally injected with OXPHOS inhibitor S-Gboxin (10 mg / kg) as the S-Gboxin experimental group. Mice were intraperitoneally injected with the same volume of DMSO as the control group. Closely observe the vital signs of mice, measure the weight of mice and tumor size every week for subsequent experiments. After 16 days of PDX model establishment, drug administration was performed every other day. After a total of 5 doses, tumor tissue was collected on the 24th day for subsequent related detection and analysis.

[0079] Figure 7 A shows representative HE and corresponding immunohistochemical staining of H3K36me3 in seven PDX tumors.

[0080] Figure 7 B shows the H3K36me3 levels and ABCA8 in tumor cells of these seven PDX models + FAP + The proportion of CAFs was negatively correlated.

[0081] Figure 7 C: Tumors with lower H3K36me3 levels (PDX15031901) showed higher sensitivity to the OXPHOS inhibitor S-Gboxin (***, P ≤ 0.001), while tumors with higher H3K36me3 levels (PDX2021023) showed a modest response to the OXPHOS inhibitor S-Gboxin (*, 0.01

Claims

1. Use of a specific oxidative phosphorylation inhibitor in the preparation of a drug for treating SETD2-deficient pancreatic cancer, wherein the structural formula of the specific oxidative phosphorylation inhibitor is as follows: 。

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