Application of CXCL9 in the prediction of immunotherapy efficacy and prognosis of cholangiocarcinoma

Abstract

The present invention discloses the application of CXCL9 in the prediction of the efficacy of immunotherapy for cholangiocarcinoma and the prognosis judgment, which belongs to the field of biomedicine technology, and specifically relates to providing CXCL9 chemokine as a predictive biomarker for the efficacy of immunotherapy for cholangiocarcinoma. The discovery of this biomarker may improve the current efficacy prediction model of immunotherapy for cholangiocarcinoma, thereby more accurately screening out the patient population that may benefit from immunotherapy. The present invention verifies the role of CXCL9 in immunotherapy for cholangiocarcinoma through in vivo and in vitro experiments: overexpression of CXCL9 in mice enhances the efficacy of chemotherapy combined with immunotherapy for cholangiocarcinoma by promoting T cell infiltration activity; a cholangiocarcinoma cell-T lymphocyte co-culture model is constructed in vitro to clarify that the expression of CXCL9 in cholangiocarcinoma cells is closely related to the cytotoxicity of T lymphocytes.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to the application of CXCL9 in the prediction of immunotherapy efficacy and prognosis judgment of cholangiocarcinoma. Background Art

[0002] Biliary tract cancer (BTC) includes cholangiocarcinoma (cancer occurring in the liver, around the liver hilum, or at the distal end of the bile duct) and gallbladder cancer. For decades, the overall prognosis of biliary tract cancer has been almost stagnant. Due to the occult and atypical early clinical symptoms of biliary tract cancer, nearly two-thirds of biliary tract cancer patients are already in an unresectable or distant metastatic state when they are diagnosed, so systemic therapy is the main treatment option. However, the efficacy of chemotherapy alone is not significant. In the first-line treatment, the gemcitabine combined with cisplatin (GemCis) regimen can only extend the median overall survival (mOS) to 11.7 months, and it is urgent to find other treatment options.

[0003] In recent years, personalized precision treatment based on immunity and targeting has become a new treatment strategy that has changed clinical practice. Due to the low mutation frequency of targetable genes such as IDH1, FGFR and BRAF in biliary tract cancer (<5%), the population that benefits from second-line targeted therapy is still very limited. Immunotherapy has made breakthroughs in many solid tumors in recent years, but has made slow progress in the field of biliary tract cancer. It was not until the TOPAZ-1 (NCT03875235) study that a breakthrough in chemotherapy combined with immunotherapy was achieved in the first-line treatment of advanced biliary tract cancer. GemCis combined with durvalumab can extend mOS to 12.8 months, but the objective response rate (ORR) is only 26.7%, indicating that only a small number of people benefit from immunotherapy. Therefore, accurate screening of immune-dominant populations to protect non-responders from adverse reactions has important social benefits and is a research hotspot and difficulty in the current field of biliary tract cancer immunotherapy.

[0004] CXCL9 is a member of the CXC chemokine family, produced by macrophages, endothelial cells, hepatocytes and tumors under the induction of IFN-γ, and promotes tumor suppressive lymphocyte infiltration in solid tumors through its receptor CXCR3. CXCL9 has been shown to play a crucial role in enhancing the efficacy of immune checkpoint inhibition therapy in preclinical cancer models due to the recruitment and activation of T cells. Summary of the invention

[0005] At present, a variety of biomarkers, including programmed cell death ligand 1 (PD-L1) protein expression, tumor mutation burden (TMB), mismatch repair deficiency (dMMR) and microsatellite instability (MSI), have been confirmed to be associated with the benefit of immunotherapy for solid tumors, but the research on biliary malignancies is still in the exploratory stage. PD-L1 expression is a widely studied biomarker in biliary cancer, but it was not significantly associated with ICI treatment sensitivity in the TOPAZ-1 study.

[0006] The purpose of the present invention is to provide a biliary tract cancer biomarker CXCL9, which can be used to construct a prognostic evaluation model for biliary tract cancer patients receiving first-line chemotherapy combined with immunotherapy; and the chemokine CXCL9 can regulate the immune microenvironment of biliary tract cancer and is a potential immunotherapy target.

[0007] The technical solution adopted by the present invention to achieve the above-mentioned purpose is:

[0008] The application of CXCL9 as a marker in constructing models for assessing the prognosis of cancer immunotherapy includes:

[0009] Obtaining the expression level of the expression product of CXCL9 in the biological sample containing cancer cells of the subject, and evaluating the expression level of CXCL9; wherein any one of the results (1)-(2) is obtained:

[0010] Results (1): CXCL9 was highly expressed in cancer cells, indicating a good prognosis for the subject;

[0011] Result (2): CXCL9 was lowly expressed in cancer cells, indicating a poor prognosis for the subject.

[0012] Preferably, the subject receives first-line chemotherapy combined with immunotherapy; or, the cancer cells are biliary cancer cells; or, the subject is a human patient.

[0013] More preferably, the first-line chemotherapy combined with immunotherapy uses gemcitabine; and / or, the first-line chemotherapy combined with immunotherapy uses RMP1-14 antibody; and / or, the first-line chemotherapy combined with immunotherapy uses acetylcysteine ​​ester disulfide.

[0014] More preferably, acetylcysteine ​​ester disulfide is prepared by reacting acetylcysteine ​​and 2-propoxyethanol.

[0015] Preferably, the CXCL9 expression level is evaluated using the H-score system, where H-score>6 indicates high expression, and H-score≤6 indicates low expression.

[0016] More preferably, the method is a PCR method.

[0017] Preferably, the method comprises a reagent for detecting the expression level of CXCL9; or, comprises a method for detecting the expression level of CXCL9.

[0018] More preferably, a reagent for detecting CXCL9 RNA is included; or CXCL9 RNA is amplified by PCR.

[0019] The present invention discloses the use of a reagent and / or method for detecting the expression level of CXCL9 in preparing a model for evaluating the prognosis of cancer immunotherapy.

[0020] The present invention discloses the application of CXCL9 in preparing a product capable of regulating the immune microenvironment of biliary tract cancer and / or treating biliary tract cancer and / or promoting T cell infiltration activity and / or constructing a cholangiocarcinoma cell-T lymphocyte co-culture model.

[0021] Preferably, the above application includes a reagent for detecting the expression level of CXCL9.

[0022] The invention discloses a preparation method of acetylcysteine ​​ester disulfide. Acetylcysteine ​​is added into deionized water, stirred and mixed to dissolve, then sodium iodide and hydrogen peroxide are added, stirred and reacted for 0.5-3h at 20-40 DEG C, rotary evaporation is performed after the reaction is completed, then DMF and thionyl chloride are added, stirred and reacted for 1-3h, then 2-propoxyethanol is added, stirred and reacted for 2-5h, rotary evaporation is performed after the reaction is completed, and column chromatography purification is performed to obtain acetylcysteine ​​ester disulfide.

[0023] Preferably, acetylcysteine ​​is used in an amount of 4-12 wt % of deionized water.

[0024] Preferably, the amount of sodium iodide used is 0.5-2wt% of acetylcysteine.

[0025] Preferably, hydrogen peroxide is added in the form of a 30 wt % hydrogen peroxide aqueous solution, and the usage amount of the 30 wt % hydrogen peroxide aqueous solution is 80-100 wt % of the acetylcysteine.

[0026] Preferably, DMF is used in an amount of 1000-5000 wt % of acetylcysteine.

[0027] Preferably, the usage amount of thionyl chloride is 200-400wt% of acetylcysteine.

[0028] Preferably, the amount of 2-propoxyethanol used is 60-300 wt % of acetylcysteine.

[0029] Preferably, in the column chromatography, petroleum ether and ethyl acetate are used as eluents, and the volume ratio of petroleum ether to ethyl acetate in the eluent is 1:1-5.

[0030] The invention discloses a composite medicament comprising RMP1-14 antibody and gemcitabine.

[0031] Preferably, the RMP1-14 antibody and gemcitabine are mixed in a mass ratio of 1:2-10.

[0032] The invention discloses a composite medicament comprising RMP1-14 antibody, acetylcysteine ​​ester disulfide and gemcitabine.

[0033] Preferably, the RMP1-14 antibody, acetylcysteine ​​ester disulfide and gemcitabine are mixed in a mass ratio of 1:0.2-4:2-10.

[0034] The invention discloses a composite medicament comprising RMP1-14 antibody, acetylcysteine ​​ester disulfide, aminoacetamide and gemcitabine.

[0035] Preferably, the RMP1-14 antibody, acetylcysteine ​​ester disulfide, aminoacetamide and gemcitabine are mixed in a mass ratio of 1:0.2-4:0.2-2:2-10.

[0036] The invention discloses the use of acetylcysteine ​​ester disulfide and / or aminoacetamide in increasing the expression of CXCL9 in cells.

[0037] Preferably, acetylcysteine ​​ester disulfide, aminoacetamide and gemcitabine are mixed in a mass ratio of 1-20:1-10.

[0038] The present invention discloses a model for evaluating the prognosis of cancer immunotherapy, comprising:

[0039] Obtaining the expression level of the expression product of CXCL9 in the biological sample containing cancer cells of the subject, and evaluating the expression level of CXCL9; wherein any one of the results (1)-(2) is obtained:

[0040] Results (1): CXCL9 was highly expressed in cancer cells, indicating a good prognosis for the subject;

[0041] Result (2): CXCL9 was lowly expressed in cancer cells, indicating a poor prognosis for the subject.

[0042] Preferably, the CXCL9 expression level is evaluated using the H-score system, where H-score>6 indicates high expression, and H-score≤6 indicates low expression.

[0043] The purpose of the present invention is to provide a biliary tract cancer biomarker CXCL9, which can be used to construct a prognostic evaluation model for biliary tract cancer patients receiving first-line chemotherapy combined with immunotherapy; and the chemokine CXCL9 can regulate the immune microenvironment of biliary tract cancer and is a potential immunotherapy target.

[0044] The beneficial effects of the technical solution of the present invention are as follows: (1) The present invention provides CXCL9 chemokine as a predictive biomarker for the efficacy of immunotherapy for biliary tract cancer. The discovery of this biomarker may improve the current efficacy prediction model for immunotherapy for biliary tract cancer, thereby more accurately screening out the patient population that may benefit from immunotherapy. (2) The present invention aims to provide accurate immunotherapy strategies for biliary tract cancer patients by studying the role of CXCL9 molecular subtypes in immunotherapy for biliary tract cancer, guide clinicians to formulate personalized treatment plans for patients, improve the targetedness and effectiveness of treatment, and reduce unnecessary treatment and related economic burdens and adverse reactions. (3) In vitro and in vivo experiments verify the role of CXCL9 in immunotherapy for biliary tract cancer: overexpression of CXCL9 in mice enhances the efficacy of chemotherapy combined with immunotherapy for biliary tract cancer by promoting T cell infiltration activity; a cholangiocarcinoma cell-T lymphocyte co-culture model is constructed in vitro to clarify that the expression of CXCL9 in cholangiocarcinoma cells is closely related to the cytotoxicity of T lymphocytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 This is the result diagram of CXCL9 expression and patient prognosis;

[0046] Figure 2 This is the result diagram of the increase in CXCL9 expression level, CD274, PDCD1 expression level, and tumor microenvironment score;

[0047] Figure 3 This is the chemokine family expression diagram of patients in the response group;

[0048] Figure 4 Standardized enrichment scores, gene rankings, and P-value graphs for genes corresponding to different signaling pathways;

[0049] Figure 5 Figures of methods and results validated by in vivo and in vitro experiments;

[0050] Figure 6 Representative and statistical graphs of immunohistochemistry;

[0051] Figure 7 Representative flow cytometry images and statistical results;

[0052] Figure 8 Figure 1 shows the methods and results of the co-culture experiment;

[0053] Fig. 9 Results graphs validated in clinical settings;

[0054] Fig.10 is the liver weight graph;

[0055] Fig.11 This is a graph showing the expression of CXCL9. DETAILED DESCRIPTION

[0056] The technical solution of the present invention is further described in detail below in conjunction with specific implementations and drawings:

[0057] Embodiments of the present invention:

[0058] 1. Patient Enrollment and Data Collection

[0059] The study period of the method of the present invention is from November 2017 to August 2022. Patients are screened by inclusion and exclusion criteria, and eligible patients are registered and data are collected.

[0060] Screening criteria for patient enrollment were: patients aged 18 years or older, with histologically confirmed unresectable advanced or metastatic biliary adenocarcinoma (including intrahepatic or extrahepatic cholangiocarcinoma and gallbladder cancer) who had received first-line immune checkpoint inhibitors (ICIs) in combination with chemotherapy. Detailed inclusion and exclusion criteria are shown in the table below.

[0061]

[0062] Collection of patient data: All medical records of eligible patients were reviewed, and the genomic and transcriptomic landscapes of baseline tissues were analyzed. PFS was measured from the start of treatment to disease progression or death (whichever came first). OS was defined as the time from the start of treatment to death. Eligible patients who were receiving treatment or had no confirmed progression were reviewed at the time of the last follow-up. The above study was approved by the Ethics Committee of Zhejiang Cancer Hospital (approval number: IRB-2020-404) and complied with the Declaration of Helsinki. Informed consent was obtained from all participating patients.

[0063] 2. DNA Extraction, Library Preparation, and Targeted DNA Sequencing

[0064] Genomic DNA was extracted from formalin-fixed paraffin-embedded (FFPE) tumor samples using the QIAamp DNA FFPE Tissue Kit provided by Qiagen. Subsequently, the DNA quantity and quality were assessed using the Qubit 3.0 Fluorometer (Thermo Fisher Scientific) and Nanodrop 2000 (Thermo Fisher Scientific), respectively. The creation of sequencing libraries was completed using the KAPA Hyper Prep Kit from KAPA Biosystems. Hybridization-based target enrichment was performed using the Geneseeq Prime® Pan-Cancer Genome and the xGen Lockdown Hybridization and Washing Kit (Integrated DNA Technology). Library capture was performed using Life Technologies' Dynabeads M-270, followed by amplification using KAPA HiFi HotStart ReadyMix from KAPA Biosystems. Library quantification was performed using qPCR and the KAPA Library Quantification Kit from KAPA Biosystems. Finally, the library was sequenced on the Illumina Hiseq4000 platform.

[0065] 3. Bioinformatics Analysis of DNA Sequencing Data

[0066] Trimmomatic was used to perform quality control on FASTQ files, trim leading or trailing low-quality bases (bases with a quality score lower than 20) and remove N bases. Subsequently, the sequencing data were aligned to the reference human genome (hg19) using Burrows-Wheeler Aligner (BWA-mem, v0.7.12), and the alignment results were deduplicated using Sambaba. Base quality realignment and indel realignment were performed using the Genome Analysis Toolkit (GATK 3.4.0). Variant calling, including single nucleotide variants (SNVs) and insertions / deletions (INDELs), was performed using VarScan2. Genomic fusions were identified using FACTERA with default parameters, and copy number variations (CNVs) were detected using CNVkit with default parameters, applying a threshold of 0.6 for copy number loss and 2.0 for copy number gain. Tumor mutation burden (TMB) was defined as the number of somatic mutations, coding mutations, base substitution mutations, and indel mutations per megabase of the genome examined. Microsatellite instability (MSI) was defined as samples showing instability in more than 40% of the 52 indel sites covered by the panel.

[0067] 4. RNA Sequencing and Bioinformatics Analysis

[0068] Total RNA was extracted from formalin-fixed paraffin-embedded (FFPE) tumor samples using the RNeasy FFPE kit (QIAGEN), and RNA quantity was assessed using the Bioanalyzer 2100 (Agilent Technologies). To eliminate ribosomal RNA and any remaining genomic DNA, enzymatic digestion was performed using RNase H and DNase. Subsequently, sequencing libraries were constructed using the KAPA Stranded RNA-Seq Library Preparation Kit with RiboErase (HMR) (KAPA Biosystems). Library quality was verified using the Agilent High Sensitivity DNA Kit on the Bioanalyzer 2100 (Agilent Technologies) and then sequenced on the Illumina HiSeq NGS platform (Illumina).

[0069] Illumina bcl2Fastq software (v2.19.0.316) was used for base calling to generate sequence reads in FASTQ format. Trimmomatic (v0.36) was used to remove adapters and low-quality reads. Transcriptome mapping was performed by STAR software (v2.7.3a) and the data were aligned to the reference human genome (hg19). Gene-level quantification was performed using RSEM (version 1.2.31), and gene expression was quantified as transcripts per million (TPM). Gene set enrichment analysis (GSEA) was performed using fgsea (v1.20.0), and statistical significance was defined as |NES| ≥ 1.5 and P-adjusted < 0.1.

[0070] 5. Immune Infiltration Analysis

[0071] The proportion of tumor-infiltrating immune cells was estimated using xCell. The TME score was estimated using the R package TMEscore (https: / / github.com / DongqiangZeng0808 / TMEscore). T cell dysfunction and exclusion scores were assessed using Tumor Immune Dysfunction and Exclusion (TIDE, http: / / tide.dfci.harvard.edu).

[0072] 6. Statistical Analysis

[0073] In genomic and transcriptomic comparisons, Fisher’s exact test was used to assess differences in categorical variables between the groups, whereas Wilcoxon rank-sum test was used to determine differences in continuous variables between these groups. In animal and cell line experiments, Student’s unpaired t-test and one-way ANOVA (Tukey’s and Bonferroni’s multiple comparison tests) were used, and Wilcoxon rank-sum test was used to assess differences between IHC staining of patient resection specimens. The Cochran–Armitage test was used to assess trends (either increasing or decreasing) in proportions of specific binary categorical variables, whereas Jonckheere’s trend test was used to assess trends regarding ordinal variables. Spearman’s Rho correlation coefficient was used to analyze associations between continuous features. Survival analysis involved the generation of PFS and OS curves with their respective 95% confidence intervals (CIs) using the Kaplan–Meier method and comparisons using the log-rank test. Hazard ratios (HRs) were calculated and compared using univariate Cox proportional hazards regression models. To account for multiple comparisons, false discovery rate (FDR) correction was implemented.

[0074] Unless otherwise stated, two-tailed P values ​​< 0.05 were considered statistically significant. All statistical analyses were performed using R software (v4.1.2).

[0075] 7. Clinical Validation Cohort

[0076] The validation cohort was an independent multicenter retrospective cohort of 40 patients with biliary tract cancer. The inclusion criteria were as follows: (1) aged 18 years or older; (2) histologically confirmed unresectable advanced or metastatic biliary tract adenocarcinoma; (3) receiving first-line immune checkpoint inhibitor combined with chemotherapy; (4) with complete clinical data, survival follow-up data, and efficacy evaluation data; (5) no active malignant tumors other than biliary tract cancer within 5 years.

[0077] Each patient had an available baseline paraffin tumor tissue sample, which was subsequently used for sectioning and immunohistochemistry (IHC) staining. This study was approved by the Ethics Committee of Zhejiang Cancer Hospital (approval number: IRB-2023-851) and complied with the Declaration of Helsinki. Informed consent was obtained from all participating patients.

[0078] 8. Cell Lines and Mice

[0079] Human cholangiocarcinoma (CCA) cell line RBE cells were purchased from Procell (CL-0191). RBE cells were cultured in RPMI 1640 supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin. Cell cultures were maintained at 37°C and 5% CO2. C57BL / 6 mice were purchased from Shanghai Model Organism Center. All animal experiments were approved by the Animal Experiment Ethics Committee of Zhejiang Cancer Hospital (2023-06-014) and performed in accordance with the Guide for the Care and Use of Laboratory Animals.

[0080] 9. Plasmid Construction and Lentivirus Infection

[0081] The plasmids used in the present invention were constructed by Genechem, including pT3-myr-AKT-HA, pT3-EF1a-YapS127A and pCMV(CAT)T7-SB100. pCMV-CXCL9-3×FLAG-Neo and pCMV-3×FLAG-Neo plasmids were obtained from Miaoling Biology, China. All plasmids used in vivo were purified by GoldHi EndoFree Plasmid Maxi Kit (Cwbio). Control lentivirus CON313 and shRNA lentivirus LV-CXCL9-RNAi (Hu6-MCS-CMV-puro) were constructed by Genechem. The sequences of the two CXCL9 shRNAs are as follows: 5'-GATGTGAAGGAACTGATTAAA-3' (sh1-CXCL9); 5'-GCTACACTGAAGAATGGAGTT-3' (sh2-CXCL9). Purified shRNA-encoding lentivirus was transduced into 50% confluent CCA cells using HiTransG (Genechem). Transduced cells were selected with puromycin and stably expressing cell lines were eventually established.

[0082] 10. Orthotopic Cholangiocarcinoma Animal Model

[0083] Experimental groups were set up, and orthotopic cholangiocarcinoma was induced in 6-week-old C57BL / 6 mice by tail vein hydrodynamic injection (HTVi). 30 μg of pT3-EF1a-YapS127A, 20 μg of pT3-myr-AKT-HA, and 2 μg of pCMV(CAT)T7-SB100 transposase plasmids were diluted in 2 ml of 0.9% NaCl, filtered through a 0.22 μm filter, and injected into the tail vein of mice within 6 seconds. To induce CXCL9 overexpression, 20 μg of pCMV-CXCL9-3×FLAG-Neo plasmid was diluted in 2 ml of 0.9% NaCl, filtered through a 0.22 μm filter, and injected into the tail vein of mice treated as above. A control group was set up. The control group also induced orthotopic cholangiocarcinoma in 6-week-old C57BL / 6 mice by tail vein hydrodynamic injection (HTVi). The difference was that the pCMV-CXCL9-3×FLAG-Neo plasmid injected in the above step was replaced by pCMV-3×FLAG-Neo plasmid.

[0084] The experimental group adopted a chemoimmunotherapy treatment regimen, and mice were intraperitoneally injected with RMP1-14 antibody (5 mg / kg) and gemcitabine (25 mg / kg) on ​​days 10 and 17 after treatment. The control group mice were intraperitoneally injected with an equal volume of 0.9% saline. The mice were euthanized on day 24. The experimental and control groups each consisted of five mice, and the livers were weighed and compared between the groups. Subsequently, sections of each liver sample were stained with immunohistochemistry (IHC), and three mice from each group were selected for flow cytometry analysis. RMP1-14 was obtained from Bixcell (BE0146). Gemcitabine was purchased from Zhejiang Cancer Hospital.

[0085] 11. Flow Cytometry of Immune Cells in Mouse Cholangiocarcinoma

[0086] Mouse CCA tissue was cut into 1 mm-sized fragments using ophthalmic scissors and passed through a 70 μm filter. The obtained cell suspension was collected in DMEM medium supplemented with 10% FBS. Next, immune cells were separated by Percoll gradient method (GE Healthcare). After treatment with red blood cell lysis buffer (Biosharp, BL503B), the separated immune cells were washed with PBS and counted for further flow cytometry. Cytokine-producing immune cells were activated by leukocyte activation cocktail (BD Biosciences, 550583) for 7 hours for subsequent flow cytometry antibody staining. Flow cytometry antibodies are as follows: anti-mouse CD45 (BD Pharmingen, 557659), anti-mouse CD8a (BD Pharmingen, 557959), anti-mouse TNF (BD Pharmingen, 566510), anti-mouse GZMB (BD Pharmingen, 566510). 11-8898-82), anti-mouse perforin (BD Pharmingen, 12-9392-82), and anti-mouse IFN-γ (BD Pharmingen, 566097). After staining, immune cells were analyzed using an LSRFortessa flow cytometer (BD Biosciences). The expression level of each molecule was characterized by mean fluorescence intensity and analyzed using FlowJo v10.8 software.

[0087] 12. PMBC Isolation and T Cell Activation

[0088] Peripheral blood mononuclear cells (PBMCs) were obtained from heparinized peripheral blood by density gradient centrifugation using lymphocyte separation medium (Solarbio, P8610). Blood was donated from normal young adults aged 20-25 years. To prepare fresh complete ImmunoCult™-XF medium (STEMCELL, 100-0956), human recombinant IL-2 (STEMCELL, 78036.1) was added to ImmunoCult™-XF T Cell Expansion Medium at the time of use at a final concentration of 10 ng / mL. PBMCs were then cultured at 1×10 6 Cells were plated at a density of 10 cells / mL in fresh complete ImmunoCult™-XF T Cell Expansion Medium. CD3 / CD28 T Cell Activator (STEMCELL, 10971) was added to the cell suspension at a volume of 25 µL / mL. Cells were incubated at 37°C and 5% CO2 for 3 days.

[0089] 13. T cell activation assay in co-culture model

[0090] RBE cells and activated T cells were seeded on 6-well plates at a ratio of 1:2. 5 RBE cells and 1.0×10 6 T cells were added to the plate together for co-culture. After 24 hours of co-culture, the activation status of T cells was detected by flow cytometry. The flow cytometry antibodies were as follows: anti-human CD45 (BD Pharmingen, 557833), anti-human CD3 (BD ​​Pharmingen, 566684), anti-human granzyme B (BD Pharmingen, 561142), anti-human TNF (BD Pharmingen, 560679), and anti-human perforin (BD Pharmingen, 563576).

[0091] 14. Fluorescence quantitative reverse transcription PCR

[0092] For relative gene expression analysis, RNA was extracted from cell pellets or tumor tissues using Trizol reagent (Invitrogen). 1 µg of total RNA was used to synthesize cDNA using the Hifair® III 1st Strand cDNA Synthesis System (Yeasen). cDNA (0.5-1 μg) was amplified using Hieff UNICON® Universal Blue qPCR SYBR Master Mix (Yeasen) and corresponding primers (hCXCL9 forward: 5'-CTTTTCCTCTTGGGCATCATCT-3'; hCXCX9 reverse: 5'-AGGAACAGCGACCCTTTTCCA-3'; mCXCL9 forward: 5'-GGAGTTCGAGGAACCCTAGTG-3'; mCXCX9 reverse: 5'-GGGATTTGTAGTGGATCGTGC-3'). Cycling parameters were as follows: 3 min: 95°C; 40X (15 sec: 95°C; 1 min: 60°C). Gene expression levels were normalized to the Actin housekeeping gene and quantified using the CT calculation method.

[0093] 15. Immunohistochemical Staining

[0094] Mouse and human cholangiocarcinoma tissues were collected after formalin fixation, paraffin embedding, and sectioning. The sections were dewaxed, rinsed with distilled water, and then subjected to antigen retrieval and washed with PBS for 5 minutes × 3 times. The primary antibodies used for IHC of mouse cholangiocarcinoma tissues included CD3 (Abcam, ab16669, 1:500), CD4 (Abcam, ab183685, 1:200) and CD8 (Abcam, ab217344, 1:500). For immunohistochemical staining of human cholangiocarcinoma tissues, the primary antibodies included CD3 (ZSGB-BIO, ZM-0417, 1:100), CD4 (Biolynx, I1030, 1:100), CD8 (Biolynx, I1036, 1:100), and CXCL9 (Abcam, ab202961, 1:400). Then, goat anti-rabbit IgG H&L (Biotin) (dilution ratio 1:1000) was added to the tissue sections, incubated for 30 minutes, and washed with PBS for 5 minutes × 3 times; then DAB color development and hematoxylin counterstaining of cell nuclei were performed using a DAB color development kit; finally, the tissue sections were dehydrated and blocked with neutral gel.

[0095] Tumor sections were screened at low magnification (100×) and immunoreactivity was assessed at high magnification (400×). All IHC-stained sections were examined and evaluated by two independent pathologists in a blinded manner. The proportion of CD3, CD4, and CD8-positive immune cells in mouse tumor tissues was defined as the proportion of positive immune cells to all nucleated cells in a certain field of view. The H-scoring system was used to evaluate the intensity of CXCL9 expression in the external cohort (N=40). The formula of the H-scoring system is as follows: Hscore=(∑IS×AP), where IS represents the staining intensity and AP represents the percentage of positively stained cells. IS depends on the staining of the cells: no staining is 0 points; weak staining is 1 point; moderate staining is 2 points; strong staining is 3 points, and the percentage of AP-stained cells: 0% is 0 points; 1%-25% is 1 point; 26%-50% is 2 points; 51%-75% is 3 points; 76%-100% is 4 points. The median of H-score was set as the cutoff value, and the expression of CXCL9 in the external cohort was divided into a high expression group (H-score>6) and a low expression group (H-score≤6).

[0096] The results of the above research implementation methods of the present invention are as follows:

[0097] 1. Patient characteristics

[0098] The study prospectively enrolled 125 patients with advanced BTC who received first-line immune checkpoint inhibitors combined with chemotherapy between November 2017 and August 2022, including 54 patients with gallbladder cancer (GC), 57 patients with intrahepatic cholangiocarcinoma (ICC), and 14 patients with extrahepatic cholangiocarcinoma (ECCA). The median age was 63 years (range, 34 to 82 years), and 52.8% (66 / 125) were male. The median follow-up time of the entire cohort was 14.8 months, and the median PFS and OS were 6.9 months (95% CI: 6.2-7.9) and 11.8 months (95% CI: 10.3-14.8), respectively. The median PFS of GC, ICC, and ECCA patients were 6.83, 6.93, and 6.77 months, and the median OS were 11.10, 12.75, and 8.61 months, respectively, and no significant differences were observed (PFS: P = 0.720, OS: P = 0.714).

[0099] All 125 patients underwent targeted DNA sequencing, and an additional 62 patients underwent RNA sequencing. The most mutated genes were TP53 (64 / 125, 51.2%), KRAS (34 / 125, 27.2%), ERBB2 (18 / 125, 14.4%), and ARID1A (17 / 125, 13.6%).

[0100] 2. Relationship between CXCL9 expression and patient prognosis

[0101] The relationship between CXCL9 expression and patient prognosis Figure 1 As shown in Figure 2, patients with elevated CXCL9 expression at the RNA expression level showed a higher disease remission rate. Figure 1 As shown in A, P = 0.014; prolonged PFS as Figure 1 As shown in B, median values, CXCL9 high vs. low: 7.66 vs. 6.67 months, P = 0.018; and longer OS as Figure 1 As shown in C, the median, CXCL9 high expression and low expression: 16.92 months and 10.58 months, P = 0.010.

[0102] 3. Analysis of the immune microenvironment and downstream pathways in patients with CXCL9 gene mutation

[0103] Given the key role of CXCL9 in immune regulation, the present invention then used RNA sequencing data to study the tumor immune microenvironment associated with its expression level.

[0104] Increased CXCL9 expression levels were associated with increased expression levels of immune checkpoint-related genes. Figure 2As shown in Figures AC, for example, CD274 (PD-L1, P < 0.001) and PDCD1 (PD-1, P < 0.001), as well as significantly improved tumor microenvironment (TME) score (P < 0.001), indicated immune activation. These findings highlight the enhanced immune activity in this subgroup of patients.

[0105] The expression levels of three genes of the chemokine family CXCL9 (P=0.014), CXCL10 (P=0.011), and CXCL11 (P=0.008) in the response group were significantly higher, and the expression level of the CXCL14 gene was lower (P=0.039). Figure 3 shown.

[0106] In patients with high CXCL9 gene expression, the expression of immune-related A (interferon-γ), B (interferon-α), C (E2F transcription factor), D (IL6-JAK-STAT3 signaling pathway), E (inflammatory response), F (G2M immune checkpoint), G (complement), H (IL2-STAT5 signaling pathway), and I (tumor necrosis factor-α / NF-kB pathway) was upregulated; while in patients with high CXCL9 gene expression, the expression of immune-related J (transforming growth factor-β pathway) and K (epithelial-mesenchymal transition) was downregulated. The gene standardized enrichment scores (NES), gene ranks, and P values ​​corresponding to different signaling pathways are shown in Figure 3. Figure 4 As shown, A stands for INTERFERONˍGAMMAˍRESPONSE, B stands for INTERFERONˍALPHAˍRESPONSE, C stands for E2FˍTARGETS, D stands for IL6ˍJAKˍSTAT3ˍSIGNALING, E stands for INFLAMMATORYˍRESPONSE, F stands for G2MˍCHECKPOINT, G stands for COMPLEMENT, H stands for IL2ˍSTAT5ˍSIGNALING, I stands for TNFAˍSIGNALINGˍVIAˍNFKB, J stands for TGFˍBETAˍSIGNALING, and K stands for EPITHELIALˍMESENCHYMALˍTRANSITION.

[0107] 4. In vitro and in vivo experiments verified that high expression of CXCL9 enhanced the efficacy of immunotherapy combined with chemotherapy and affected the infiltration and activation of T cells in CCA

[0108] The present invention established a mouse cholangiocarcinoma model by hydrodynamic transfection of AKT / YAP (S127A) plasmids. At the same time, the present invention co-injected the constructed CXCL9 overexpression plasmid into the mouse by hydrodynamic co-injection to achieve overexpression of CXCL9 in cholangiocarcinoma tissues. Quantitative PCR analysis and immunohistochemical staining of CXCL9 mRNA expression confirmed the successful overexpression of CXCL9 in the CCA model. RMP1-14 antibody and gemcitabine were intraperitoneally administered to mice on days 10 and 17. The mice were killed on day 24 and the livers were collected. There were 6 mice in each group. The results are as follows Figure 5 As shown in Figure 2, the process of establishing a cholangiocarcinoma mouse model is as follows: Figure 5 As shown in Figure A. The results showed that compared with the control group, immunotherapy combined with chemotherapy delayed the progression of cholangiocarcinoma in mice. It is worth noting that the overexpression of CXCL9 further enhanced the efficacy of chemotherapy combined with immunotherapy and significantly reduced the tumor burden in mice. The tumor size of each group is shown in Figure Figure 5 As shown in B, the liver weight of each group is Figure 5 As shown in C. Quantitative PCR analysis of CXCL9 mRNA expression confirmed the successful overexpression of CXCL9 in the cholangiocarcinoma model. The difference in CXCL9 mRNA expression in the overexpression group compared with the control group was >2, and P < 0.05 was considered successful overexpression. The difference in CXCL9 mRNA expression in the tumor tissue of the overexpression group mice compared with the control group was 9.787, P < 0.001. The qPCR statistical results of the control group and the overexpression group are shown in Figure 5 As shown in D.

[0109] Next, the present invention analyzed the immune microenvironment of mouse cholangiocarcinoma tissue. As expected, immunotherapy combined with chemotherapy induced a strong anti-tumor immune response. Immunohistochemical staining showed that immunotherapy combined with chemotherapy increased the infiltration of CD3+, CD4+, and CD8+ T cells in mouse tumor tissue. Figure 6 and Figure 7 shown.

[0110] Strikingly, overexpression of CXCL9 further increased the number of tumor-infiltrating T cells in the treatment group, as shown in the representative immunohistochemical graphs and statistical graphs of each group. Figure 6 As shown in A and B. The present invention further used flow cytometry to analyze the activity of T cells. The results showed that compared with the control group, immunotherapy combined with chemotherapy treatment of mice overexpressing CXCL9 significantly enhanced the production of tumor killing factor-α, γ-interferon and perforin in the CD8+T cell subsets, while the overall increase trend of GZMB was not significant. The representative flow cytometry graphs and statistical results of each group are shown in Figure 7 As shown in A and B.

[0111] In order to confirm the effect of CXCL9 on T cell function, the present invention established a CCA cell-T cell co-culture model. Activated T cells isolated from human peripheral blood mononuclear cells (PBMCs) were co-cultured with control or CXCL9 knockdown RBE cholangiocarcinoma cells, and then their cytotoxicity was detected by flow cytometry. The results are as follows: Figure 8 The co-culture experiment flow chart is shown in Figure 8 As shown in A. Consistent with the observation results of the present invention in mouse cholangiocarcinoma tissue, CXCL9 knockdown in RBE cells significantly reduced the production of granzyme B (P<0.05) and perforin (P<0.05) by T cells in the co-culture model, and the production level of tumor killing factor-α showed a downward trend. Representative flow cytometry graphs and statistical results of each group are shown in Figure 8 As shown in B.

[0112] 5. Clinical validation that high expression of CXCL9 enhances the efficacy of combined immunotherapy and chemotherapy and affects the infiltration and activation of T cells in CCA

[0113] To validate the above results in a clinical setting, we initiated an independent multicenter CCA cohort, which included 40 patients who received first-line chemoimmunotherapy. Immunohistochemical staining of CXCL9, CD3, CD4, and CD8 was performed on tumor resection specimens from these patients. Fig. 9 The results showed that patients with increased CXCL9 expression showed a higher response rate ( Fig. 9 A, P = 0.014) and prolonged PFS ( Fig. 9 In addition, the distribution of CD3+T, CD4+T, and CD8+T cells in patients with high CXCL9 expression was significantly higher than that in patients with low CXCL9 expression. Representative immunohistochemistry diagrams are shown in Figure 2. Fig. 9 As shown in C, the corresponding statistical results are as follows Fig. 9 As shown in D. In summary, CXCL9 plays a key role in the efficacy of chemotherapy combined with immunotherapy and T cell activation in cholangiocarcinoma.

[0114] Example 1: Method for constructing a CXCL9-based efficacy prediction model for biliary tract cancer immunotherapy

[0115] Obtain bile duct cancer cells from a patient with bile duct cancer, detect the expression level of CXCL9 in the bile duct cancer cells, and evaluate the expression level of CXCL9; wherein any one of the results (1)-(2) is obtained:

[0116] Results (1): CXCL9 was highly expressed in biliary cancer cells, indicating a good prognosis for biliary cancer patients;

[0117] Result (2): CXCL9 was lowly expressed in biliary tract cancer cells, suggesting a poor prognosis for biliary tract cancer patients.

[0118] The expression of CXCL9 was evaluated using the H-score system, and the formula of the H-score system is as follows: Hscore = (∑IS×AP), where IS represents the staining intensity and AP represents the percentage of cells with positive staining. IS depends on the staining of the cells: no staining is 0 points; weak staining is 1 point; moderate staining is 2 points; strong staining is 3 points, and the percentage of AP-stained cells is: 0% is 0 points; 1%-25% is 1 point; 26%-50% is 2 points; 51%-75% is 3 points; 76%-100% is 4 points. The median of the H-score was set as the cutoff value, H-score>6 is high expression, and H-score≤6 is low expression.

[0119] Example 2: Method for constructing an in situ cholangiocarcinoma animal model

[0120] A mouse CCA model was established by hydrodynamic transfection of pT3-EF1a-YapS127A, pT3-myr-AKT-HA, and pCMV(CAT)T7-SB100 plasmids, and a CXCL9 overexpression plasmid was constructed and co-injected into mice by hydrodynamics.

[0121] Orthotopic cholangiocarcinoma was induced in 6-week-old C57BL / 6 mice by tail vein hydrodynamic injection (HTVi). 30 μg of pT3-EF1a-YapS127A, 20 μg of pT3-myr-AKT-HA, and 2 μg of pCMV(CAT)T7-SB100 transposase plasmids were diluted in 2 ml of 0.9% NaCl, filtered through a 0.22 μm filter, and injected into the tail vein of mice within 6 seconds. To induce CXCL9 overexpression, 20 μg of pCMV-CXCL9-3×FLAG-Neo plasmid was diluted in 2 ml of 0.9% NaCl, filtered through a 0.22 μm filter, and injected into the tail vein of the above-treated mice.

[0122] Example 3: Method for constructing a cholangiocarcinoma cell-T cell co-culture model

[0123] Activated T cells isolated from PBMCs were co-cultured with RBE cells containing sh-Con or sh-CXCL9. RBE cells and activated T cells were seeded on 6-well plates at a ratio of 1:2. 5×10 5 RBE cells and 1.0×10 6 T cells were added to the plate for co-culture.

[0124] Example 4: A method for enhancing the therapeutic efficacy of immunotherapy combined with chemotherapy

[0125] Compound drug: RMP1-14 antibody and gemcitabine. RMP1-14 antibody and gemcitabine are mixed at a mass ratio of 1:5.

[0126] Enhanced immune combined with chemotherapy: mice with in situ cholangiocarcinoma and CXCL9 overexpression were obtained according to the method of Example 2. After the mice with in situ cholangiocarcinoma and CXCL9 overexpression were cultured for 14 days, the composite agent of Example 4 was diluted in 0.9% NaCl, intraperitoneally injected, and cultured for 24 days. The amount of the composite agent used for mice was measured by RMP1-14 antibody, and the amount of RMP1-14 antibody used was 5 mg / kg.

[0127] Example 5: A method for enhancing the therapeutic efficacy of immunotherapy combined with chemotherapy

[0128] Preparation of acetylcysteine ​​ester disulfide: Add acetylcysteine ​​to deionized water, stir and mix to dissolve, then add sodium iodide and hydrogen peroxide, stir and react at 30°C for 2h, and then add DMF and thionyl chloride to stir and react for 2h, and then add 2-propoxyethanol to stir and react for 3h, and then stir and evaporate after the reaction is completed. Purify by column chromatography to obtain acetylcysteine ​​ester disulfide. The amount of acetylcysteine ​​used is 8wt% of deionized water, the amount of sodium iodide used is 1wt% of acetylcysteine, hydrogen peroxide is added in the form of 30wt% hydrogen peroxide aqueous solution, the amount of 30wt% hydrogen peroxide aqueous solution used is 100wt% of acetylcysteine, the amount of DMF used is 3000wt% of acetylcysteine, the amount of thionyl chloride used is 300wt% of acetylcysteine, and the amount of 2-propoxyethanol used is 100wt% of acetylcysteine. In the column chromatography, petroleum ether and ethyl acetate were used as eluents, and the volume ratio of petroleum ether to ethyl acetate in the eluent was 1:3.

[0129] Compound drug: RMP1-14 antibody, acetylcysteine ​​ester disulfide and gemcitabine. RMP1-14 antibody, acetylcysteine ​​ester disulfide and gemcitabine are mixed in a mass ratio of 1:0.5:5.

[0130] Enhanced immune combined with chemotherapy treatment: mice with in situ cholangiocarcinoma and CXCL9 overexpression were obtained according to the method of Example 2. After the mice with in situ cholangiocarcinoma and CXCL9 overexpression were cultured for 14 days, the composite agent of Example 5 was diluted in 0.9% NaCl, intraperitoneally injected, and cultured for 24 days. The amount of the composite agent used for mice was measured by RMP1-14 antibody, and the amount of RMP1-14 antibody used was 5 mg / kg.

[0131] Example 6: Composite agent under high expression of CXCL9 and its use

[0132] Compared with Example 5, this embodiment is different in that it is a composite medicine.

[0133] Compound drug: RMP1-14 antibody, acetylcysteine ​​ester disulfide and gemcitabine. RMP1-14 antibody, acetylcysteine ​​ester disulfide and gemcitabine are mixed in a mass ratio of 1:2:5.

[0134] Comparative Example 1: A method for enhancing the therapeutic efficacy of immunotherapy combined with chemotherapy

[0135] The difference between this comparative example and Example 4 is that the immunotherapy is enhanced and combined with chemotherapy.

[0136] Enhanced immune combined with chemotherapy treatment: mice with orthotopic cholangiocarcinoma were obtained according to the method of Example 2, without constructing CXCL9 overexpression, and after culturing the mice with orthotopic cholangiocarcinoma for 14 days, the composite agent of Example 4 was diluted in 0.9% NaCl, intraperitoneally injected, and cultured for 24 days. The amount of the composite agent used for mice was measured by RMP1-14 antibody, and the amount of RMP1-14 antibody used was 5 mg / kg.

[0137] Comparative Example 2: A method for enhancing the therapeutic efficacy of immunotherapy combined with chemotherapy

[0138] The difference between this comparative example and Example 5 is that the immunotherapy is enhanced and combined with chemotherapy.

[0139] Enhanced immune combined with chemotherapy treatment: mice with orthotopic cholangiocarcinoma were obtained according to the method of Example 2, without constructing CXCL9 overexpression, and after culturing the mice with orthotopic cholangiocarcinoma for 14 days, the composite agent of Example 5 was diluted in 0.9% NaCl, intraperitoneally injected, and cultured for 24 days. The amount of the composite agent used for mice was measured by RMP1-14 antibody, and the amount of RMP1-14 antibody used was 5 mg / kg.

[0140] Comparative Example 3: A method for enhancing the therapeutic efficacy of immunotherapy combined with chemotherapy

[0141] The difference between this comparative example and Example 4 is that the immunotherapy is enhanced and combined with chemotherapy.

[0142] Enhanced immunotherapy combined with chemotherapy: Mice with in situ cholangiocarcinoma and CXCL9 overexpression were obtained according to the method of Example 2. After culturing the mice with in situ cholangiocarcinoma and CXCL9 overexpression for 14 days, 0.9% NaCl, the same amount as that used in Example 4, was intraperitoneally injected and cultured for 24 days.

[0143] Example 7: A method for culturing bile duct cancer cells

[0144] Culture to improve CXCL9 expression in cholangiocarcinoma cells: RBE cells were inoculated on 6-well plates and cultured in culture medium at 37°C and 5% CO2. The culture medium consisted of a culture medium and a supplement, the culture medium was RPMI 1640, and the supplements were fetal bovine serum, penicillin / streptomycin, and acetylcysteine ​​ester disulfide. The amount of fetal bovine serum added to the culture medium was 10wt%, the amount of penicillin / streptomycin added was 1wt%, and the amount of acetylcysteine ​​ester disulfide added was 5mg / L.

[0145] Example 8: A method for culturing bile duct cancer cells

[0146] Compared with Example 7, the difference between this example and Example 7 lies in the culture medium of RBE cells.

[0147] The culture medium is composed of a culture medium and a supplement, the culture medium is RPMI 1640, and the supplement is 10% fetal bovine serum, 1% penicillin / streptomycin, acetylcysteine ​​ester disulfide and aminoacetamide. The amount of fetal bovine serum added to the culture medium is 10wt%, the amount of penicillin / streptomycin added is 1wt%, the amount of acetylcysteine ​​ester disulfide added is 5mg / L, and the amount of aminoacetamide added is 2mg / L.

[0148] Example 9: A method for culturing bile duct cancer cells

[0149] Compared with Example 7, the difference between this example and Example 7 lies in the culture medium of RBE cells.

[0150] The culture medium is composed of a culture medium and a supplement, the culture medium is RPMI 1640, and the supplement is 10% fetal bovine serum, 1% penicillin / streptomycin, acetylcysteine ​​ester disulfide and aminoacetamide. The amount of fetal bovine serum added to the culture medium is 10wt%, the amount of penicillin / streptomycin added is 1wt%, the amount of acetylcysteine ​​ester disulfide added is 5mg / L, and the amount of aminoacetamide added is 5mg / L.

[0151] Comparative Example 4: A method for culturing bile duct cancer cells

[0152] The difference between this comparative example and Example 7 lies in the culture medium of RBE cells.

[0153] Culture to improve CXCL9 expression in cholangiocarcinoma cells: RBE cells were inoculated on 6-well plates and cultured in culture medium at 37°C and 5% CO2. The culture medium consisted of a culture medium and a supplement, the culture medium was RPMI 1640, and the supplements were fetal bovine serum, penicillin / streptomycin, and acetylcysteine ​​ester disulfide. The amount of fetal bovine serum added to the culture medium was 10wt%, the amount of penicillin / streptomycin added was 1wt%, and the amount of acetylcysteine ​​ester disulfide added was 0.05mg / L.

[0154] Comparative Example 5: A method for culturing bile duct cancer cells

[0155] The difference between this comparative example and Example 7 lies in the culture medium of RBE cells.

[0156] Culture to improve CXCL9 expression in cholangiocarcinoma cells: RBE cells were seeded on 6-well plates and cultured in culture medium at 37°C and 5% CO2. The culture medium consisted of a culture medium and a supplement, the culture medium was RPMI 1640, and the supplements were fetal bovine serum and penicillin / streptomycin. The amount of fetal bovine serum added to the culture medium was 10wt%, and the amount of penicillin / streptomycin added was 1wt%.

[0157] Test Example 1:

[0158] 1. Cytotoxicity

[0159] Intrahepatic bile duct cells of normal mice were cultured in RPMI1640 containing 10wt% fetal bovine serum and 1wt% penicillin / streptomycin at 37°C and 5% CO2. Cytotoxicity experiments were performed when the cell density reached 70-80%. The toxicity of acetylcysteine ​​ester disulfide to intrahepatic bile duct cells was tested by MTT method. The relationship between the amount of acetylcysteine ​​ester disulfide added and RPMI 1640 was 100mg / L. A blank group and a control group were set up. No acetylcysteine ​​ester disulfide was added to the blank group and the control group, no MTT solution was added to the blank group, and MTT solution was added to the control group.

[0160] Cell survival rate = (A 实验 -A 空白 ) / (A 对照 -A 空白 ).

[0161] In the present invention, multiple experiments were conducted using the above concentration of acetylcysteine ​​ester disulfide, and the average cell survival rate obtained was 99.89%, indicating that the acetylcysteine ​​ester disulfide prepared in the present invention has substantially no cytotoxicity.

[0162] 2. Liver weight

[0163] The present invention cultured mice according to Examples 4-6 and Comparative Examples 1-3, and then tested their liver weights. The test results are as follows: Fig.10 As shown, Example 4, compared with Comparative Example 1, shows that when CXCL9 is overexpressed, RMP1-14 antibody and gemcitabine are used in the same amount, and have a better effect; Example 4, compared with Comparative Example 3, shows that the co-use of RMP1-14 antibody and gemcitabine can inhibit tumor biology and reduce liver weight; Examples 5-6, compared with Example 4, show that the use of acetylcysteine ​​ester disulfide can reduce liver weight and inhibit the growth of tumor cells; Example 5, compared with Comparative Examples 1-2, shows that when CXCL9 is overexpressed, the co-use of RMP1-14 antibody, acetylcysteine ​​ester disulfide and gemcitabine has a better effect.

[0164] 3. CXCL9 expression

[0165] The present invention cultured cholangiocarcinoma cells according to Examples 7-9 and Comparative Examples 4-5, and then tested the expression of CXCL9 in the cholangiocarcinoma cells. The expression of Comparative Example 5 was taken as unit 1. The test results are as follows: Fig.11 As shown, Example 7, compared with Comparative Examples 4-5, shows that the use of acetylcysteine ​​ester disulfide can increase the expression of CXCL9 in cholangiocarcinoma cells, and if the use of acetylcysteine ​​ester disulfide is too little, the use of acetylcysteine ​​ester disulfide cannot increase the expression of CXCL9 in cholangiocarcinoma cells; Examples 8-9, compared with Example 7, show that when using acetylcysteine ​​ester disulfide, the use of aminoacetamide can increase the expression of CXCL9 in cholangiocarcinoma cells.

[0166] The embodiments and / or implementation methods described above are only used to illustrate the preferred embodiments and / or implementation methods for realizing the technology of the present invention, and are not intended to limit the implementation methods of the technology of the present invention in any form. Any technical personnel in this field may make slight changes or modifications to other equivalent embodiments without departing from the scope of the technical means disclosed in the content of the present invention, but they should still be regarded as technologies or embodiments that are essentially the same as the present invention.

[0167] This article uses specific examples to illustrate the principles and implementation methods of this application. The description of the above embodiments is only used to help understand the method and its core ideas of this application. The above is only the preferred implementation method of this application. It should be pointed out that due to the limitations of textual expression and the objective existence of infinite specific structures, ordinary technicians in this technical field can make several improvements, modifications or changes without departing from the principles of this application, and can also combine the above technical features in an appropriate manner; these improvements, modifications, changes or combinations, or the direct application of the concept and technical solution of the invention to other occasions without improvement, should be regarded as the scope of protection of this application.

Claims

1. Use of gemcitabine, RMP1-14 antibody, acetylcysteine ​​disulfide and CXCL9 as a marker in preparing a model for evaluating the prognosis of cancer immunotherapy, include: Obtain a biological sample containing cancer cells from a subject, detect the expression level of the expression product of CXCL9, and evaluate the expression level of CXCL9; wherein any one of the results (1)-(2) is obtained: Results (1): CXCL9 was highly expressed in cancer cells, indicating a good prognosis for the subject; Result (2): CXCL9 in cancer cells was lowly expressed, indicating a poor prognosis for the subject; The subject receives first-line chemotherapy combined with immunotherapy; the cancer cells are bile duct cancer cells; the subject is a patient with bile duct cancer; the first-line chemotherapy combined with immunotherapy uses gemcitabine, RMP1-14 antibody and acetylcysteine ​​ester disulfide; In the preparation of acetylcysteine ​​ester disulfide, acetylcysteine ​​is added to deionized water, stirred and mixed to dissolve, and then sodium iodide and hydrogen peroxide are added, stirred and reacted at 20-40° C. for 0.5-3 hours, and after the reaction is completed, DMF and thionyl chloride are added and stirred and reacted for 1-3 hours, and then 2-propoxyethanol is added and stirred and reacted for 2-5 hours. After the reaction is completed, the reaction is carried out by rotary evaporation, and column chromatography is purified to obtain acetylcysteine ​​ester disulfide; the amount of acetylcysteine ​​used is 4:1 of deionized water. -12wt%; the amount of sodium iodide used is 0.5-2wt% of acetylcysteine; hydrogen peroxide is added in the form of a 30wt% hydrogen peroxide aqueous solution, and the amount of the 30wt% hydrogen peroxide aqueous solution used is 80-100wt% of acetylcysteine; the amount of DMF used is 1000-5000wt% of acetylcysteine; the amount of thionyl chloride used is 200-400wt% of acetylcysteine; the amount of 2-propoxyethanol used is 60-300wt% of acetylcysteine; The CXCL9 expression level was evaluated using the H-score system, with H-score>6 indicating high expression and H-score≤6 indicating low expression.

2. The use of gemcitabine, RMP1-14 antibody, acetylcysteine ​​ester disulfide and CXCL9 as a marker according to claim 1 in preparing a model for evaluating the prognosis of cancer immunotherapy, characterized in that: A reagent for detecting the expression level of CXCL9; or a method for detecting the expression level of CXCL9.

3. Use of gemcitabine, RMP1-14 antibody, acetylcysteine ​​ester disulfide and CXCL9 as a marker in the preparation of a model for evaluating the prognosis of cancer immunotherapy according to claim 1, characterized in that: A reagent for detecting CXCL9 RNA is included; or, CXCL9 RNA is amplified by PCR.

4. The use of gemcitabine, RMP1-14 antibody, acetylcysteine ​​ester disulfide and CXCL9 as a marker according to claim 2 in preparing a model for evaluating the prognosis of cancer immunotherapy, characterized in that: The method is a PCR method.

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