Pharmaceutical composition for immunotherapy of MSS colorectal cancer and application of pharmaceutical composition

Abstract

The invention provides a pharmaceutical composition for treating MSS intestinal cancer, and belongs to the technical field of biological medicine, the pharmaceutical composition is used for deeply researching a treatment scheme for effectively inhibiting the MSS intestinal cancer, carrying out basic research from protein purification, proteomics, siRNA libraries and the like, and trying to develop a brand new treatment scheme and a treatment mode using nitixinone, sorafenib and PD1 antibodies; the three medicines are combined for use, so that a remarkable synergistic effect is achieved, and the tumor is successfully changed from cold to hot. The pharmaceutical composition provided by the invention has an important clinical value, breaks through the current difficulty in treatment of MSS intestinal cancer, and opens a brand new direction of application of immunotherapy in clinical advanced metastatic colorectal cancer.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and in particular relates to a drug combination for treating MSS colon cancer. Background Art

[0002] Colorectal cancer (CRC) is a common clinical malignant tumor, with the incidence and mortality ranking third and second among malignant tumors in the world, respectively. However, the early diagnosis and treatment rate of cancer patients is low, and the clinical diagnosis and treatment of advanced cases are not standardized. About 20% of CRC patients have metastasis at the time of initial diagnosis, making surgery more difficult. About 15% of colorectal cancers are microsatellite unstable (MSI) type, and these patients have a better survival rate and are more sensitive to immunotherapy. However, in clinically advanced metastatic colorectal cancer (mCRC), MSI-H patients account for only 5%, and the vast majority of patients are mismatch repair normal / microsatellite stable (pMMR / MSS), and about 90% of patients are insensitive to immunotherapy and cannot benefit from immune monotherapy. From the perspective of the immune microenvironment, MSS CRCs are mostly immune-exempt and immune-desert types. How to solve the treatment dilemma of immunotherapy in pMMR / MSS colorectal cancer has always been a hot exploration direction in colorectal cancer treatment.

[0003] It has been confirmed that dMMR / MSI-H type mCRC patients are the beneficiaries of immunotherapy. However, 95% of CRC patients are MSS / pMMR type patients, which are "cold tumors" and cannot benefit from immunotherapy alone. Previously, the REGONIVO study demonstrated for the first time the potential of regorafenib combined with nivolumab in MSS-type CRC, but subsequent studies failed to reproduce its excellent results, possibly due to the heterogeneity of the immune microenvironment (Fukuoka S, Hara H, Takahashi N, Kojima T, Kawazoe A, Asayama M, et al. Regorafenib Plus Nivolumab in Patients With Advanced Gastric or Colorectal Cancer: An Open-Label, Dose-Escalation, and Dose-Expansion Phase Ib Trial (REGONIVO, EPOC1603). J Clin Oncol. 2020 Jun 20; 38(18): 2053-2061.; Wang F, He MM, Yao YC, Zhao X, Wang ZQ, Jin Y, et al. Regorafenib plus toripalimab in patients with metastatic colorectal cancer: a phase Ib / II clinical trial and gut microbiome analysis. Cell Rep Med. 2021 Aug 27; 2(9): 100383.). In the third-line treatment of metastatic colorectal cancer (mCRC), the combination of cedabenb + bevacizumab + PD-1 antibody (CAPability-01) has been newly added to the CSCO 2024 version of the guidelines. Studies have shown that cedabenb can activate immune cells in the tumor immune microenvironment of MSS colorectal cancer, increase the infiltration of CD8+T cells, and change the tumor immune microenvironment from "cold to hot". This regimen significantly improves the efficacy and brings new treatment options to patients with pMMR / MSS colorectal cancer, exploring a new path in the "no man's land". However, more than 50% of pMMR / MSS colorectal cancer patients still have no obvious response and the cause and mechanism are unknown. It is urgent to break this dilemma and explore new clinical treatment options. Summary of the invention

[0004] In view of this, the purpose of the present invention is to provide a drug combination that effectively inhibits MSS colorectal cancer. The present invention has conducted in-depth research to explore effective treatment options for inhibiting MSS colorectal cancer. Starting from basic research on protein purification, proteomics, siRNA libraries, etc., an attempt has been made to develop a new treatment option, using a treatment model of nitisinone + sorafenib + PD1 antibody. The combined use of the three drugs achieved a significant synergistic effect, successfully turning the tumor "from cold to hot". This study has important clinical value, breaking the current dilemma and opening up a new direction for the application of immunotherapy in mCRC.

[0005] The invention provides a drug combination for treating MSS colorectal cancer, comprising sorafenib and a HDP-specific inhibitor.

[0006] Preferably, the HPD-specific inhibitor is nitisinone.

[0007] Preferably, the mass ratio of sorafenib to nitisinone is (5-7):(0.8-1.2).

[0008] Preferably, the dosage of sorafenib is 25-35 mg / kg, once a day; the dosage of nitisinone is 8-12 mg / kg, once every 2 days.

[0009] The present invention provides a drug combination for inducing apoptosis of MSS colorectal cancer cells, comprising sorafenib and nitisinone; the mass ratio of sorafenib to nitisinone is (8-12):(250-350).

[0010] Preferably, the concentration of sorafenib is 8-12 μM; the concentration of nitisinone is 250-350 μM.

[0011] The present invention provides an antibody-drug combination for treating MSS colorectal cancer, comprising sorafenib, an HDP-specific inhibitor and a PD1 antibody.

[0012] Preferably, the HDP-specific inhibitor is nitisinone; the mass ratio of sorafenib, nitisinone and PD1 antibody is (350-490): (56-84): (32-48).

[0013] Preferably, the dosage of sorafenib is 25-35 mg / kg, once a day; the dosage of nitisinone is 8-12 mg / kg, once every 2 days; the concentration of PD1 antibody is 8-12 mg / kg, twice a week.

[0014] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides a drug combination for treating MSS colorectal cancer, including sorafenib and an HPD inhibitor. The present invention has found that the specific HPD inhibitor nitisinone can enhance sorafenib-induced apoptosis of MSS colorectal cancer cells; enhance sorafenib to inhibit the growth of MSS colorectal cancer subcutaneous tumors in mice; sorafenib enhances nitisinone-induced cytotoxicity of MSS colorectal cancer glutamine-dependent cells by inhibiting glutamine metabolism; sorafenib combined with nitisinone can amplify oxidative stress-induced irreversible mitochondrial damage, release mtDNA, activate cGAS-STING pathway and type I interferon expression; the present invention uses organoids and immune-competent mouse MSS colorectal cancer subcutaneous tumor models to confirm that sorafenib combined with nitisinone promotes the immune response of MSS colorectal cancer and promotes the transformation of cold tumors to hot tumors.

[0015] Nitisinone combined with sorafenib induces irreversible mitochondrial damage by amplifying oxidative stress in MSS colorectal cancer cells, promoting the release of mtDNA into the cytoplasm and activating the downstream cGAS-STING pathway to promote the production of type I interferon, thereby recruiting CD8 + T cells and NK cells infiltrate and release cytokines, turning the tumor immune microenvironment from cold to hot. The present invention uses the immune-competent mouse MSS in situ colorectal cancer model to confirm that sorafenib combined with nitisinone and PD1 antibody induces MSS tumor regression without significant liver and kidney toxicity. This scheme significantly improves the anti-tumor effect, not only laying the foundation and providing a theoretical basis for MSS colorectal cancer immunotherapy, but also has important clinical value. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 Fasting combined with sorafenib significantly induced apoptosis of MSS tumor cells in a dose-time dependent manner;

[0017] Figure 2 The protein macromolecules in serum are important factors affecting the cytotoxicity of sorafenib;

[0018] Figure 3 Drugs targeting HDP (4-hydroxyphenylpyruvate dioxygenase) including the specific inhibitor nitisinone (orphan drug, FDA-approved for the treatment of tyrosinemia type 1 (HT-1)) can enhance sorafenib-induced apoptosis in MSS colorectal cancer cells;

[0019] Figure 4 To limit tyrosine metabolism, the specific inhibitor nitisinone was included to enhance the inhibition of sorafenib on the growth of MSS colorectal cancer subcutaneous tumors in mice;

[0020] Figure 5 Sorafenib enhances nitisinone-induced cytotoxicity in glutamine-dependent cells of MSS colorectal cancer line by inhibiting glutamine metabolism;

[0021] Figure 6 Sorafenib combined with nitisinone amplifies oxidative stress-induced irreversible mitochondrial damage, releases mtDNA, activates the cGAS-STING pathway and type I interferon expression;

[0022] Figure 7 The results of organoid and immune-competent mouse MSS colorectal cancer subcutaneous tumor models confirmed that sorafenib combined with nitisinone promoted the immune response of MSS colorectal cancer and promoted the transformation of cold tumors to hot tumors.

[0023] Figure 8 The MSS in situ colorectal cancer model in immune-competent mice confirmed that sorafenib combined with nitisinone synergistically with PD1 antibody induced MSS tumor regression without significant liver and kidney toxicity. DETAILED DESCRIPTION

[0024] The invention provides a drug combination for treating MSS colorectal cancer, comprising sorafenib and a HDP-specific inhibitor.

[0025] In the present invention, the HPD specific inhibitor is nitisinone; the mass ratio of sorafenib to nitisinone is preferably (5-7):(0.8-1.2), more preferably (5.5-6.5):1, and most preferably 6:1; the concentration of sorafenib is 25-35 mg / kg, preferably 28-33 mg / kg, more preferably 30 mg / kg; the concentration of nitisinone is 8-12 mg / kg, preferably 9-11 mg / kg, more preferably 10 mg / kg. In the specific implementation of the present invention, the preferred scheme is that the dosage of sorafenib is 30 mg / kg, once a day; the dosage of nitisinone is 10 mg / kg, once every 2 days.

[0026] The present invention also provides a drug combination for inducing apoptosis of MSS colorectal cancer cells, comprising sorafenib and nitisinone; the mass ratio of sorafenib to nitisinone is (8-12):(250-350), preferably 10:(280-330), and more preferably 1:30. In the present invention, the concentration of sorafenib is 8-12 μM, preferably 9-11 μM, and more preferably 10 μM; the concentration of nitisinone is 250-350 μM, preferably 280-330 μM, and more preferably 300 μM. In the present invention, the MSS colorectal cancer cells are preferably SW480 and WiDr cell lines and primary MSS colorectal cancer cell lines derived from patients.

[0027] The present invention provides an antibody-drug combination for treating MSS colorectal cancer, comprising sorafenib, an HPD-specific inhibitor and a PD1 antibody. In the present invention, the HPD-specific inhibitor is nitisinone; the mass ratio of sorafenib, nitisinone and PD1 antibody is (350-490): (56-84): (32-48), preferably (400-440): (65-75): (38-42), and more preferably 42:7:4. In the specific implementation of the present invention, the concentration of sorafenib is 30 mg / kg, and the dosage is 1 time / day; the concentration of nitisinone is 10 mg / kg, and the dosage is 1 time / 2 days; the concentration of PD1 antibody is 10 mg / kg, and the dosage is 2 times / week.

[0028] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0029] Example 1

[0030] 1. Fasting combined with sorafenib significantly induced apoptosis of MSS tumor cells in a dose-time dependent manner

[0031] Experimental methods:

[0032] Cells: MSS colorectal cancer cell lines: SW480, WiDr, HT29 were purchased from ATCC, and primary colorectal cancer cells were isolated, cultured, and identified from tumor tissues removed from untreated MSS colorectal cancer patients in the Sixth Affiliated Hospital of Sun Yat-sen University (ethics number 2024ZSLYEC-403, and the patient signed the informed consent form);

[0033] Culture method of MSS colorectal cancer cell line: DMEM culture medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin. The culture environment is 37°C and 5% CO2. When the cell confluence is about 90%, add 4 mL PBS (without Ca 2+ / Mg 2+ ), shake gently and then discard by suction to remove residual serum and dead cells. Add 2 mL of trypsin-EDTA (0.25%) and incubate at 37°C for 1-3 minutes (observe cells become round and fall off under a microscope). Add 3 mL of complete medium to terminate digestion, pipette evenly and transfer to a new culture flask or plate for subsequent experiments.

[0034] Experimental groups: 1 to 12 are: 1: DMSO, 2: Cisplatin (CDDP) 20μM, 3: Irinotecan 10μg / mL, 4: Docetaxel 2μM, 5: ADM (Doxorubicin) 1μM, 6: Auranofin 1μM, 7: Sorafenib 10μM, 8: Regorafenib 10μM, 9: 2-DG 2.5mM, 10: Erlotinib 10μM, 11: Sunitinib 10μM, 12: Linsitinib 10μM.

[0035] Treat in a 37°C, 5% CO2 incubator for about 48 hours, and perform crystal violet staining. Aspirate the culture medium in the well plate, avoid touching the cell layer, and wash twice with PBS; add 200μL of pre-cooled methanol to each well, let stand at room temperature for 10 minutes → aspirate methanol. Add staining solution: add 100μL of 0.5% crystal violet solution to each well (cover the cell layer); stain at room temperature and avoid light for 20-30 minutes; remove the staining solution: gently aspirate the staining solution to avoid cell shedding; gently rinse each well with ultrapure water 3-4 times until the background is colorless; invert the well plate on absorbent paper and dry at room temperature for 30 minutes; take pictures of the entire plate.

[0036] CCK8 detection was performed according to the instructions in the manual.

[0037] Experimental results:

[0038] The results are as follows Figure 1Fasting combined with sorafenib significantly induced apoptosis of MSS tumor cells in a dose-time dependent manner. A. Crystal violet staining was used to detect the cytotoxic effects of potential / commonly used anti-tumor drugs on MSS colorectal cancer cell lines cultured in normal control medium (Ctrl M) and Fasting culture conditions (FM). Among them: 1: DMSO, 2: CDDP 20μM, 3: Irinotecan 10μg / mL, 4: Docetaxel 2μM, 5: ADM 1μM, 6: Auranofin 1μM, 7: Sorafenib 10μM, 8: Regorafenib 10μM, 9: 2-DG 2.5mM, 10: Erlotinib 10μM, 11: Sunitinib10μM, 12: Linsitinib 10μM; B. CCK8 was used to detect the cytotoxic effect of sorafenib at different concentrations on MSS colorectal cancer cell lines and primary colorectal cancer cells under normal culture and Fasting culture conditions; C. CCK8 was used to detect the growth inhibitory effect of sorafenib on MSS colorectal cancer cells under normal control and Fasting culture conditions; D. High-content microscopy was used to detect the induction of MSS colorectal cancer tumor apoptosis by sorafenib under normal culture and Fasting culture conditions over time, and PI positive staining indicated apoptotic cells.

[0039] The results showed that sorafenib significantly inhibited cell growth and enhanced its apoptosis-inducing ability under fasting conditions, and it was dose- and time-dependent.

[0040] 2. Effect of protein macromolecules in serum on sorafenib cytotoxicity

[0041] Experimental methods: Fetal bovine serum was divided into two parts: ultrafilter FBS (ufFBS) and small molecular inorganic matter (smFBS) using 10kD protein ultrafiltration centrifuge tubes; cells were treated with DMEM medium containing low glucose (1g / L glucose + 10% FBS), low serum (4.5g / L glucose + 1% FBS), 10% heated serum (56℃, 30min), 4.5g / L glucose + 10% ufFBS and 4.5g / L glucose + 10% smFBS combined with 10μM sorafenib for 48h. Cell apoptosis was detected by flow cytometry and apoptosis detection kits. The fetal bovine serum was divided into two parts: large molecular protein (ultrafilter FBS, ufFBS) and small molecular inorganic matter (small molecular fraction of FBS, smFBS) using 30kD and 50kD protein ultrafiltration concentration centrifuge tubes, and then combined with 10μM sorafenib for 48 hours to detect cell apoptosis.

[0042] The apoptosis detection kit was operated according to the kit instructions.

[0043] Experimental results:

[0044] The results are as follows Figure 2 As shown: Protein macromolecules in serum are important factors affecting the cytotoxicity of sorafenib. A. Protein ultrafiltration tubes separate FBS into small molecular fractions (small molecular fraction of FBS, smFBS) and large molecular proteins (ultrafilter FBS, ufFBS); B. Schematic diagram for screening factors that may affect the anti-tumor effect of sorafenib. C. Flow cytometry and apoptosis detection kits were used to detect the effects of different components of serum (small molecular fraction of FBS, smFBS and large molecular protein ufFBS) separated by ultrafiltration tubes on sorafenib-induced apoptosis under different conditions, including Fasting (1g / L glucose + 1% FBS), low glucose (1g / L glucose), low serum (1% FBS), heated serum (heated FBS) and ultrafiltration tubes; D. Flow cytometry was used to detect the effects of different components of serum (small molecular fraction of smFBS and large molecular protein ufFBS) separated by 50kD ultrafiltration tubes on sorafenib-induced apoptosis.

[0045] The results showed that the components that inhibited the apoptosis of MSS colorectal cancer cells by sorafenib were mainly large molecular proteins existing in serum and could be retained by 50kD protein ultrafiltration tubes.

[0046] 3. The specific inhibitor nitisinone enhances sorafenib-induced apoptosis of MSS colorectal cancer cells

[0047] Experimental method: After the serum was subjected to 50kD protein ultrafiltration tube to intercept the macromolecular protein ufFBS, it was further desalted with PBS. The collected ufFBS was separated into 5 parts (labeled as F1-F5) according to the molecular weight using a protein purifier, and then the effects of these 5 parts of ufFBS on the apoptosis-inducing ability of sorafenib were measured respectively. It was found that F4 significantly inhibited the ability of sorafenib to induce apoptosis of MSS colorectal cancer. Proteomics was used to determine the differential proteins between F4 and F1. Subsequently, the siRNA library was used to inhibit the expression of the target protein and verify its combined effect of sorafenib in inducing cell apoptosis and inhibiting growth. The siRNA transfection was performed according to the instructions, and the proteomics was commissioned to a well-known domestic protein detection company.

[0048] Experimental results:

[0049] The results are as follows Figure 3 Results: Targeting HDP (4-hydroxyphenylpyruvate dioxygenase), including the specific inhibitor nitisinone (orphan drug, FDA-approved for the treatment of tyrosinemia type 1 (HT-1)), can enhance sorafenib-induced apoptosis in MSS colorectal cancer cells. A. Flow chart of serum desalting, replacement and separation; B. The protein purifier divides the macromolecular proteins in the serum after desalting, replacement and separation into 5 parts according to molecular weight, namely Fraction 1-5 (F1, F2, F3, F4, F5); C. Flow cytometry and AnnexinV FITC-PI double staining were used to detect the effects of macromolecular proteins in F1, F2, F3, F4 and F5 parts of the serum separated by the protein purifier on sorafenib-induced tumor apoptosis; D. Proteomics detection of protein expression profiles of F1 and F4 parts, and the volcano map showed that the top9 differentially expressed proteins were significantly highly expressed; E. RT-PCR detection of the target gene silencing efficiency of the candidate siRNA library; F. CCK-8 detection of the toxic effect of silencing the target gene in the candidate siRNA library combined with sorafenib on MSS colorectal cancer cells; G. CCK-8 and AnnexinV FITC-PI double staining apoptosis detection of the toxic effect of nitisinone, a drug that inhibits the action of HPD target gene, combined with sorafenib on MSS colorectal cancer cells.

[0050] The results show that the macromolecular protein in serum significantly inhibits the effect of sorafenib in MSS colorectal cancer, further confirming that HPD protein from serum may be a sensitizing target of sorafenib. The combination of sorafenib and siRNA-specific molecules and the small molecule inhibitor nitisinone confirmed that the apoptosis of MSS colorectal cancer cells was significantly induced by sorafenib. This suggests that this combined strategy may bring new options for the treatment of MSS colorectal cancer.

[0051] 4. Restriction of tyrosine metabolism (including the specific inhibitor nitisinone) enhances the inhibition of sorafenib on the growth of MSS colorectal cancer subcutaneous tumors in mice

[0052] Test method: According to 5×10 5 Dosage of SW480 cells / mouse SW480 cells were inoculated into 4-6 week old female BALB / C nude mice and administered after tumor formation for about 7-8 days. Sorafenib was used at a concentration of 30 mg / kg, intragastrically, once a day; and nitisinone was used at a concentration of 10 mg / kg, intragastrically, once every 2 days. Tumor size was measured twice a week with a vernier caliper, and mice were weighed and the data were recorded. H&E staining, TUNEL, and Ki67 were performed according to the common methods / instructions. Tyrosine-phenylalanine-deficient mouse feed was commissioned to be customized by a well-known domestic company.

[0053] Experimental results:

[0054] The results are as follows Figure 4 Figure 1 shows: Restriction of tyrosine metabolism, including the specific inhibitor nitisinone, enhances the inhibition of sorafenib on the growth of MSS colorectal cancer subcutaneous tumors in mice. AC. Tyr-D diets and HPD-specific small molecule inhibitors combined with sorafenib inhibit the growth of SW480 mouse subcutaneous tumors. A: Mouse SW480 subcutaneous tumor imaging, B: Mouse SW480 subcutaneous tumor weight, C: Mouse SW480 subcutaneous tumor growth curve; D. H&E staining of the whole tumor (largest cross-section); E: H&E staining and TUNEL staining of each treatment group of SW40 subcutaneous tumor; FH. Ki67 staining (G) and Ki67+ cell number statistics (H) of each treatment group of SW40 subcutaneous tumor.

[0055] Results: The mouse MSS colorectal cancer subcutaneous tumor model confirmed that targeting tyrosine metabolism through the HPD inhibitor nitisinone or tyrosine restricted diet combined with sorafenib significantly inhibited the growth of MSS colorectal cancer.

[0056] 5. Sorafenib enhances the cytotoxicity of nitisinone-induced glutamine-dependent cells in the MSS colorectal cancer line by inhibiting glutamine metabolism.

[0057] Experimental method: LC-MS metabolite detection method: Collect 5×10 6cells, add 1ml 0.9% saline to wash the cells twice until the cell culture medium is colorless; add 1ml pre-cooled 80% methanol (pre-cooled in a -20℃ refrigerator overnight, water and methanol need to be mass spectrometry grade) to the cells, oscillate for 10s to mix them evenly; scrape the cell metabolites with a cell scraper, and absorb the cell metabolites into a 1.5ml centrifuge tube, vortex for 5min; centrifuge at 15000rcf at 4℃ for 15min; take the supernatant and centrifuge and concentrate it in a new 1.5ml centrifuge tube, the dry sample can be stored in a -20℃ refrigerator or tested on a machine.

[0058] Based on GC-MS detection of U- 13 C-labeled glutamine-tracing assay: Prepare special culture medium: Use glutamine-free culture medium, add stable isotope labeled glutamine [U-13C5]glutamine according to the original formula concentration, filter with 0.22μm filter membrane to sterilize, and then add 10% FBS. After 24 hours of plating, aspirate the culture medium, wash twice with PBS, and replace the above special culture medium after aspiration, and treat with corresponding drugs for about 20 hours. Metabolites were extracted by washing twice with 0.9% saline and transferring to ice; 500 μl of pre-cooled methanol was used to terminate metabolism, and then 200 ul of ice-cold water (containing 1 μg of norvaline as an internal standard) was added for 1 min; cells were evenly scraped off with a pipette tip, and the mixture was transferred to a 1.5 ml EP tube; 500 μl of pre-cooled chloroform was added; after vortexing at room temperature for 15 min, centrifuged at 4°C, 14000×g for 10 min; about 400 μl of the upper layer (polar metabolites) was collected and transferred to a new EP tube, vacuum-dried at 4°C, and the sample was stored at -20°C;

[0059] Polar metabolite derivatization step: polar metabolites were taken out from -20°C, the sample cover was opened, and vacuum dried at room temperature; during the drying process, a 2% (w / v) methoxyamine hydrochloride pyridine solution was prepared: 20 mg methoxyamine hydrochloride was weighed, poured into a 1.5 ml EP tube, 1 ml pyridine was added, and vortexed to mix; after the polar metabolites were dried, 20 μl of 2% (w / v) methoxyamine hydrochloride prepared in step b was added to each sample, vortexed for about 1 min, and then placed in a 37°C dry bath for 60 min; 30 μl N-methyl-N-(tert-butyl-dimethylsilyl) trifluoroacetamide + 1% tert-butyldimethylchlorosilane (TBDMS) was added, and the sample was dried at 45°C for 30 min; centrifuged at 12000 rpm at room temperature for 3 min; an inner liner was placed in the injection vial, 20 μl pyridine was added first, and then 40 μl supernatant was added, and the cover was closed for loading.

[0060] The Seahorse XF24 metabolic analyzer was used to analyze mitochondrial respiratory function and glycolytic capacity: tumor cells with good growth status were inoculated in XF24-well cell culture plates overnight; MSS colorectal cancer cell lines were treated with corresponding drugs for about 20 hours, and the Seahorse XF24 metabolic analyzer combined with Mito Fuel Flex Test Kit was used to analyze mitochondrial respiratory function;

[0061] Transmission electron microscopy was used to detect changes in mitochondrial morphology: different cells were treated for about 12 hours, and cells were collected: cells were digested with trypsin, and cells were collected by centrifugation until the cell sediment was visible to the naked eye, ranging from sesame to mung bean size (1000 rpm, 5 min). After discarding the fixative, new electron microscopy fixative was added and fixed at room temperature for 2 h, and then transferred to 4°C for storage; specimens fixed in electron microscopy fixative were trimmed, post-ES fixated, dehydrated, and resin infiltrated, and then imported epoxy resin 812 was used as an embedding agent to embed tissue specimens in the resin; the resin-embedded samples were sliced, generally with a thickness of about 1.5 μm, and the slices were fished out with anti-stripping glass slides; samples were negatively stained: high-density heavy metal salts were used to form an electron-impermeable surrounding layer on the periphery of the specimens; transmission electron microscopy was used to take pictures: the stained copper grid was observed on the machine (magnification 0.2K-600K), and the appropriate magnification and field of view were selected according to the actual situation to take pictures.

[0062] Routine experiments such as Western blot, Incucyte-ZOOM live cell real-time monitoring imager, JC-1 membrane potential detection, GSSG / GSH ratio, ADP / ATP ratio, mitochondrial membrane permeability detection and ROS determination were performed according to the experimental instructions or reagent instructions.

[0063] Experimental results:

[0064] The results are as follows Figure 5 Sorafenib enhances the cytotoxicity of nitisinone-induced glutamine-dependent cells in MSS colorectal cancer line by inhibiting glutamine metabolism. A. Schematic diagram of tyrosine metabolism; B. LC / MS detection of changes in metabolites in cells of different treatment groups; C. Glutamine uptake by cells in different drug treatment groups; D. 13Schematic diagram of glutamine metabolism in C5-glutamine labeled tracer cells; E. GC / MS detection of glutamine metabolites in cells of different treatment groups; F. Western blot detection of protein changes in different treatment groups; G. Statistics of protein changes in Figure F; H. Incucyte Zoom detection of cell growth under different drug treatments; I. Changes in GSSG / GSH ratio in cells of different drug treatment groups; J. Flow cytometry combined with DCFH-DA probe detection of ROS levels in different drug treatment groups; K. Flow cytometry combined with Mito-SOX probe detection of mitochondrial ROS levels in different drug treatment groups; L. Seahorse energy metabolism analyzer detection of mitochondrial function in different drug treatment groups; L. Changes in ADP / ATP levels in cells of different drug groups; N. Changes in mitochondrial membrane potential of cells without drug treatment using flow cytometry combined with JC-1 probe; O. Fluorescence microscopy combined with CalceinAM probe detection of mitochondrial membrane permeability transition pore opening; P. Transmission electron microscopy detection of mitochondrial morphological changes in different drug treatment groups.

[0065] The results showed that Nitisinone enhanced sorafenib sensitivity mainly by interfering with mitochondrial homeostasis: inducing increased glutamine dependence, increased oxidative stress, and aggravated mitochondrial dysfunction.

[0066] 6. Sorafenib combined with nitisinone amplifies oxidative stress-induced irreversible mitochondrial damage, releases mtDNA, activates the cGAS-STING pathway and type I interferon expression.

[0067] Experimental method: Immunofluorescence detection of DNA released from mitochondria of tumor cells into the cytoplasm: MSS colorectal cancer cell lines with good growth status were inoculated in confocal dishes overnight; drugs were added for different durations (see figure captions for details); 4% paraformaldehyde was fixed, and PBS was washed three times; 0.5% Triton X-100, PBS was washed three times / 3min, and normal goat serum was used for blocking at room temperature for 30min; primary antibodies of mitochondria-specific TOM20 antibody and DNA antibody diluted in goat serum were used, incubated at 4℃ overnight, and washed 2-3 times with PBS. Fluorescent secondary antibodies (rabbit anti-594, mouse anti-488) were added, incubated in a wet box at 20-37℃ for 1h, and washed three times with PBST, each time for 3min; DAPI was added and incubated in the dark for 5min, the specimens were stained for nuclei, and excess DAPI was washed off with PBST for 5min×4 times; and then images were collected and observed under a confocal microscope.

[0068] Ethidium Bromide (EtBr) was used to construct mitochondrial DNA deletion (ρ 0) Cell model: Medium preparation: Add sodium pyruvate (final concentration 100μg / mL), uridine (final concentration 50μg / mL), and EtBr (final concentration 100ng / mL) to complete medium (DMEM+10%FBS+1%P / S). Inoculate cells at a density of 30-50%, place in pre-treated medium containing EtBr and continue to culture for 4-8 weeks, and replace fresh medium containing EtBr every 2-3 days. Passage regularly (when the cell density reaches 80%) and keep the density low (to avoid excessive growth leading to energy stress). ρ 0 Cells were verified by MitoTracker staining.

[0069] Experimental results:

[0070] The results are as follows Figure 6As shown: Sorafenib combined with nitisinone amplifies oxidative stress to induce irreversible mitochondrial damage, releases mtDNA, activates cGAS-STING pathway and type I interferon expression. A. Confocal microscopy detected mtDNA release in cells treated with different drugs for about 20 hours; B. Statistical graph of mtDNA release in cells treated with different drugs (result graph in A); C. Western Blot detected cGAS-STING pathway protein expression in cells treated with different drugs for about 24 hours; D. RT-qPCR detected type I interferon mRNA expression in cells treated with different drugs for about 20 hours; E, mitotracker detection of mitochondrial WT and ρ0 cell lines constructed with ethidium bromide EB; F. Western Blot analysis of cGAS-STING pathway protein expression in WT and ρ0 cells lacking mitochondria treated with different drugs for about 20 hours; G. Fluorescence microscopy combined with mitochondrial membrane potential probes JC-1 and CalceinAM probes to detect mitochondrial membrane potential and permeability transition pore opening after about 20 hours of drug treatment; H. Confocal microscopy to detect cell mtDNA release after about 20 hours of drug treatment and mitochondrial ROS scavenger MitoTEMPO; IH corresponding to the statistical chart of mtDNA release in each group of cells; J. Fluorescence microscopy combined with mitochondrial membrane potential probes JC-1 and CalceinAM probes to detect mitochondrial membrane potential and permeability transition pore opening after about 20 hours of drug treatment; AM probe was used to detect mitochondrial membrane potential and permeability transition pore opening after drug treatment for about 44 hours; K. Confocal microscopy was used to detect the release of mitochondrial mtDNA in cells after different drug treatments for about 44 hours and the action of the antioxidant NAC; LK was a statistical chart of mtDNA release in each group of cells; M. The effect of the antioxidant NAC on the growth of the corresponding treated cells; N. The effect of different cell death inhibitors on the growth of the corresponding treated cells, Z-VAD-FMK (apoptosis), ferrostatin-1 (Fer-1; ferroptosis), disulfiram (pyroptosis) and necrosulfonamide (NSA; necroptosis); O. Morphological changes of mitochondrial WT and ρ0 cells lacking ρ0 under different drug treatment conditions.

[0071] The results showed that sorafenib combined with nitisinone amplified oxidative stress to induce irreversible mitochondrial damage, released mtDNA, activated the cGAS-STING pathway and type I interferon expression. The activation of the cGAS-STING pathway by mtDNA release may induce the transformation of MSS cold tumors to hot tumors and improve immune response.

[0072] 7. Organoid and immune-competent mouse MSS colorectal cancer subcutaneous tumor models confirmed that sorafenib combined with nitisinone promoted the immune response of MSS colorectal cancer and promoted the transformation of cold tumors to hot tumors.

[0073] Experimental methods: Organoid culture:

[0074] 1) Tissue collection and pretreatment: Fresh colon cancer tissues were collected after clinical surgery, washed with PBS containing penicillin / streptomycin (5%), and cut into small pieces; digested with collagenase IV, filtered, and the separated colon cancer cells were collected.

[0075] 2) Cultivation of colorectal cancer organoid cultures:

[0076] ① After pretreatment of colorectal cancer tissue, the mixture of separated colorectal cancer cells and matrix gel was applied to a 24-well plate in a hemispherical shape at 50 μL / well. The culture plate was placed in a 5% CO2 37°C incubator for 30 minutes until the matrix gel was completely solidified;

[0077] ② Slowly add 500 μL of organoid culture medium pre-restored to room temperature to each well along the wall of the well, and culture in a 5% CO2 37°C incubator;

[0078] ③After 3 days, observe the formation of organoids under a microscope. If the particle size is larger than 30-50 μm, it is considered that the organoids have been formed. Replace the culture medium every 3 days for cultivation, and continue to observe the size of the organoids. If most of the organoids are larger than 30-50 μm under a microscope and do not increase in size, the organoids can be collected for subsequent experiments.

[0079] 3) Sensitivity of colorectal cancer organoid cultures to corresponding metabolic target inhibitors:

[0080] ① Subculture the successfully cultured organoids;

[0081] ②Different concentrations of nitisinone + sorafenib were treated for 72h ( Figure 7 or nitisinone + sorafenib treatment for 24 hours followed by co-culture with autologous PBMCs for 72 hours ( Figure 7 C in );

[0082] ③ Take photos to observe the growth of organoids, and use propidium iodide (PI) to detect the death of organoid cells;

[0083] ④Evaluate the sensitivity of nitisinone combined with sorafenib on colorectal cancer organoids.

[0084] H&E, Ki67, TUNEL and multiple immunohistochemical staining were performed according to routine experimental procedures or kit instructions.

[0085] Experimental results:

[0086] The results are as follows Figure 7Shown: Organoids and immune-competent mouse MSS colorectal cancer subcutaneous tumor models confirm that sorafenib combined with nitisinone promotes the immune response of MSS colorectal cancer and promotes the transformation of cold tumors to hot tumors. A. Schematic diagram of the separation and activation of autologous PBMCs from CRC patient tumor tissue for organoid culture medium; B. Survival of patient colorectal cancer organoids under different drug treatments, and propidium bromide PI-positive cells are dead tumor cells. C. Survival of patients’ colorectal cancer organoids and autologous PBMCs after co-culture; D. Schematic diagram of construction of subcutaneous tumor mouse model with CT26 mouse colorectal cancer cells and drug administration method; E. Tumor imaging of mice after treatment with different drugs; NS: Nitisinone+Sorafenib, DS: Tyr-Ddiets+Sorafenib; F. Tumor growth curves of mice in different drug treatment groups; G. H&E staining results of whole mouse tumor tissue and local H&E staining; H. Ki67 staining results of mouse tumor tissue; I. TUNEL staining results of mouse tumor tissue; J. CD8+ / IFNγ / GZMB staining results of whole mouse tumor tissue and local.

[0087] Results show that the synergistic effect of nitisinone and sorafenib enhances the anti-PD-1 efficacy by activating the tumor immune microenvironment, suggesting potential clinical application value.

[0088] 8. The MSS in situ colorectal cancer model in immunocompetent mice confirmed that sorafenib combined with nitisinone synergistically induced MSS tumor regression with PD1 antibodies without significant liver and kidney toxicity.

[0089] ① Experimental methods: Construction and grouping of in situ mouse model of colorectal cancer: 1×10 6 CT26 cells expressing luciferase were inoculated into female 4-6 week old BALB / C nude mice to form tumors subcutaneously. 3 When the tumor tissue was removed, the mice were killed, washed with PBS, cut into pieces of about 0.5-1.0 mm in size, and stored in DMEM without FBS. Female BALB / C mice aged 4-6 weeks with healthy immunity were anesthetized, and the tumor tissue was inoculated into the cecum. The incision was sutured, and the implanted tumor was observed regularly. In the first week of tumor inoculation, tumor growth was observed using a small animal in vivo imaging device. Different combinations of nitisinone + sorafenib + PD1 antibody were given to the successfully transplanted tumor model, and tumor growth, metastasis and survival of mice were observed.

[0090] ②Evaluate the anti-tumor efficacy of nitisinone in combination with sorafenib.

[0091] The results are as follows Figure 8Shown: The MSS orthotopic colorectal cancer model in immune-competent mice confirmed that sorafenib combined with nitisinone and PD1 antibody induced MSS tumor regression without significant liver and kidney toxicity. A. Schematic diagram of the construction of the orthotopic mouse colorectal cancer model; B. Small animal in vivo imaging device monitoring the growth of orthotopic tumors after different drug treatments; C. H&E staining and KI67 staining of tumor tissues after different drug treatments; D. H&E staining of the liver and kidneys of mice after different drug treatments. E. Schematic diagram of the mechanism of the three-drug combination of nitisinone + sorafenib + PD1 antibody inducing the transformation of MSS colorectal cancer cold tumors to hot tumors.

[0092] The results show that the excellent efficacy and safety of the triple therapy (nitisinone + sorafenib + PD1 antibody combination) in the in situ colorectal cancer model provides a new strategy and lays a theoretical foundation for the treatment of MSS CRC, an "immunotherapy desert" tumor.

[0093] As can be seen from the above examples, the present invention uses an immunocompetent mouse MSS in situ colorectal cancer model to demonstrate that sorafenib combined with nitisinone synergizes with PD1 antibodies to induce MSS tumor regression without significant liver and kidney toxicity. This scheme significantly improves the anti-tumor effect, not only laying the foundation and providing a theoretical basis for MSS colorectal cancer immunotherapy, but also has important clinical value.

[0094] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A drug combination for treating MSS colorectal cancer, characterized in that: Including sorafenib and HDP-specific inhibitors.

2. The drug combination according to claim 1, characterized in that The HDP specific inhibitor is nitisinone.

3. The drug combination according to claim 2, characterized in that The mass ratio of sorafenib to nitisinone is (5-7):(0.8-1.2).

4. The pharmaceutical combination according to claim 3, characterized in that The dosage of sorafenib is 25-35 mg / kg, once a day; the dosage of nitisinone is 8-12 mg / kg, once every 2 days.

5. A drug combination for inducing apoptosis of MSS colorectal cancer cells, characterized in that: The invention comprises sorafenib and nitisinone; the mass ratio of sorafenib to nitisinone is (8-12):(250-350).

6. The pharmaceutical combination according to claim 5, characterized in that The concentration of sorafenib is 8-12 μM; the concentration of nitisinone is 250-350 μM.

7. An antibody-drug combination for treating MSS colorectal cancer, characterized in that: Including sorafenib, HDP-specific inhibitors and PD1 antibodies.

8. The antibody-drug combination according to claim 6, characterized in that The HDP-specific inhibitor is nitisinone; the mass ratio of sorafenib, nitisinone and PD1 antibody is (350-490): (56-84): (32-48).

9. The antibody-drug combination according to claim 7, characterized in that The dosage of sorafenib is 25-35 mg / kg, once a day; the dosage of nitisinone is 8-12 mg / kg, once every 2 days; the concentration of PD1 antibody is 8-12 mg / kg, twice a week.

Images