Application of serum exosome-derived CXCL4 in colorectal cancer diagnosis, treatment selection and prognosis evaluation

Abstract

The invention relates to application of CXCL4 derived from serum exosome in colorectal cancer diagnosis, treatment selection and prognosis evaluation, and belongs to the technical field of tumor treatment. According to the invention, the expression level of the CXCL4 gene derived from the serum exosome for immunotherapy of patients suffering from colorectal cancer is obviously higher than that of the CXCL4 gene derived from the serum exosome for immunotherapy of the patients suffering from colorectal cancer, and the CXCL4 gene can be used as a marker for distinguishing the non-sensitive type and the sensitive type for immunotherapy of the patients suffering from colorectal cancer and is used for guiding a subsequent treatment scheme.

Description

Technical Field

[0001] The present invention relates to the technical field of tumor treatment, and in particular to the application of serum exosome-derived CXCL4 in colorectal cancer diagnosis, treatment selection and prognosis evaluation. Background Art

[0002] Currently, the more mature and clinically applicable predictive markers for immunotherapy are: tumor mutational burden (TMB), PD-L1, and microsatellite stability (MSS) / microsatellite instability (MSI) classification. TMB is defined as the number of non-synonymous sequence gene mutations in the coding region of the cancer genome, usually calculated as the number of mutations per megabyte of genomic sequence. Anti-tumor immune responses can be driven by TMB-related new peptides, which are recognized as non-self by T cells, leading to sustained clinical responses to immunotherapy. Multiple studies conducted in patients with non-small cell lung cancer and melanoma have clearly identified TMB as a marker for the evaluation of PD-L1 response in patients with these two types of tumors. Currently, the most widely used FDA-approved marker for immunotherapy of colorectal cancer (CRC) is MSS / MSI classification. MSI-type tumors refer to those with a highly unstable microsatellite phenotype, which will produce a large number of frameshift mutations and then generate new peptide chains. These frameshift peptides, as new antigens, are highly immunogenic and enhance the immune response of tumor-infiltrating lymphocytes. MSS-type tumors refer to tumors with a relatively stable microsatellite phenotype. Due to endogenous factors of tumor cells, insufficient T lymphocyte infiltration in the tumor microenvironment and excessive infiltration of tumor-associated macrophages jointly mediate an immune-rejecting tumor microenvironment. In 2017, the National Comprehensive Cancer Network guidelines of the United States were updated to recommend PD-1 / PD-L1 treatment as the last-line treatment for patients with advanced MSI-type CRC, which brought a breakthrough in the treatment of advanced CRC that was in trouble. In the 2023 National Comprehensive Cancer Network guidelines of the United States, MSS / MSI classification officially became the basis for selecting PD-1 / PD-L1 treatment strategies for all stages of CRC patients.

[0003] However, tumor heterogeneity means that TMB is only part of the anti-tumor immune response characteristics, and the predictive value of TMB in CRC immunotherapy is controversial. Whole exome sequencing is the gold standard method for detecting TMB, but it is expensive. In addition, mutation types such as reading frame shifts and rearrangements generated in the non-coding regions of the genome cannot be detected by whole exome sequencing, and more expensive whole genome sequencing is required. TMB was originally a marker determined based on clinical studies in European populations. Recent studies have found that TMB is not suitable for Asian populations, and there are significant differences in the positive thresholds between the two populations. In short, the above shortcomings limit the widespread application of TMB as a predictive marker for CRC immunotherapy.

[0004] Among the markers predicting the sensitivity of PD-1 / PD-L1 therapy, the detection of PD-L1 expression is relatively widely used, but its objective response rate is still less than 50%. Usually, the tumor tissue that needs to be removed for PD-L1 detection is mainly detected by immunohistochemistry, and the quantitative indicators are the tumor cell positive proportion score, the immune cell positive proportion score and the comprehensive positive score. However, due to intratumor heterogeneity, the same tumor tissue may be partially positive and partially negative, and may also undergo dynamic changes. The expression of tissue PD-L1 cannot perfectly predict the effect of immunotherapy. Recent studies have found that the PD-L1 protein in tumor cells is subject to a variety of post-translational modifications, such as palmitoylation, methylation and glycosylation, which can also interfere with the results of immunohistochemistry.

[0005] Although the response rate of MSI CRC to immunotherapy is significantly higher than that of MSS CRC, the proportion of patients who are sensitive to immunotherapy in MSI CRC is only 50%-70%, and some patients with advanced MSS CRC also benefit from immunotherapy. MSS / MSI classification has a certain suggestive effect on the immunotherapy efficacy of CRC, but it cannot make an accurate prediction. TMB, PD-L1 and MSS / MSI detection all require tumor tissue. Due to the invasiveness and accessibility of tissue biopsy, many tumor patients are unable to predict the immunotherapy efficacy.

[0006] Therefore, there is an urgent need to find non-invasive immunotherapy predictive markers that can obtain tumor molecular information in real time to guide clinical precision treatment. Summary of the invention

[0007] The purpose of the present invention is to overcome the shortcomings of the prior art and provide an XX material having XX advantages and a preparation method and application thereof.

[0008] To achieve the above object, the technical solution adopted by the present invention is:

[0009] In a first aspect, the present invention provides a use of a reagent for detecting CXCL4 in the preparation of a product for performing immunophenotyping diagnosis, treatment regimen selection and / or prognosis evaluation of colorectal cancer by detecting the level of CXCL4 in a serum exosome sample obtained from a subject, wherein the CXCL4 is used as a biomarker; the colorectal cancer phenotyping diagnosis is to classify the colorectal cancer as immunotherapy-sensitive or immunotherapy-insensitive according to the level of CXCL4 in the serum exosome sample of the subject; the treatment regimen selection is to select whether to administer immunotherapy to the subject suffering from colorectal cancer or adjust the existing treatment regimen according to the level of CXCL4 in the serum exosome sample of the subject; the prognosis evaluation is to evaluate the subject suffering from colorectal cancer as having a good prognosis or a poor prognosis according to the level of CXCL4 in the serum exosome sample of the subject.

[0010] As a preferred embodiment of the first aspect, the typing diagnosis includes: typing colorectal cancer as immunotherapy-sensitive or immunotherapy-insensitive according to the CXCL4 level in the subject's serum exosome sample, wherein if the CXCL4 level is higher than a statistically determined population standard level, the colorectal cancer is typed and diagnosed as immunotherapy-insensitive colorectal cancer, and if the CXCL4 level is lower than the standard level, the colorectal cancer is typed and diagnosed as immunotherapy-sensitive colorectal cancer; and / or

[0011] The treatment regimen selection includes: determining whether to administer immunotherapy or a combination of immunotherapy and an immunopotentiator to the subject suffering from colorectal cancer according to the CXCL4 level in the subject's serum exosome sample, wherein if the CXCL4 level is lower than the control level, immunotherapy is selected for the subject, and if the CXCL4 level is higher than the control level, immunotherapy is not selected for the subject or immunotherapy and an immunopotentiator are selected for the subject;

[0012] The prognosis assessment includes: performing a prognosis assessment on the subject suffering from colorectal cancer according to the CXCL4 level in the subject's serum exosome sample, wherein if the CXCL4 level is lower than the control level, the subject is assessed to have a good prognosis, and if the CXCL4 level is higher than the control level, the subject is assessed to have a poor prognosis.

[0013] As a preferred embodiment of the first aspect, the control level is: the CXCL4 molecule level measured in a non-tumor normal biological sample, a tumor sample collected from the same subject at different time points, a population standard level determined by statistics, or a standardized level.

[0014] As a preferred embodiment of the first aspect, the control level is the CXCL4 level detected by qRT-PCR and calculated using T-test.

[0015] As a preferred embodiment of the first aspect, the immunotherapy is one or more therapies selected from the following group: immune checkpoint inhibition therapy, cellular immunotherapy, antigen chimeric receptor cell therapy, T cell receptor T cell therapy, bispecific antibody therapy, tumor vaccine therapy, and oncolytic virus therapy.

[0016] As a preferred embodiment of the first aspect, the immune checkpoint inhibition therapy includes PD-1 inhibitor therapy, PD-L1 inhibitor therapy, and CTLA-4 inhibitor therapy.

[0017] As a preferred embodiment of the first aspect, the cellular immunotherapy includes adoptive cell therapy.

[0018] As a preferred embodiment of the first aspect, the antigen chimeric receptor cell therapy includes CAR-T therapy and / or CAR-NK therapy.

[0019] As a preferred embodiment of the first aspect, the CXCL4 is a substance selected from the following group: CXCL4 gene, CXCL4 mRNA, cDNA, CXCL4 protein, or active or characteristic fragments of any of the foregoing.

[0020] As a preferred embodiment of the first aspect, the product is a kit or a combination of a reagent and a detection device.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] Compared with traditional tissue biopsies, liquid biopsies represented by exosomes have the characteristics of non-invasiveness and dynamism, and have become the hottest research direction in the field of tumor markers. Exosomes are relatively stable because they are wrapped in vesicle structures. Detection of proteins carried by exosomes can directly reflect the characteristics of the source cells. At the same time, tumor cells, immune cells, and various stromal cells can secrete exosomes and release them into the blood. Detection of exosomes in the blood can accurately reflect the state of the tumor microenvironment in real time. At the same time, it was found that the expression level of CXCL4 gene derived from serum exosomes in patients with colorectal cancer who are not sensitive to immunotherapy is significantly higher than that in sensitive patients. Therefore, it can be used as a marker to distinguish between non-sensitive and sensitive types of colorectal cancer patients for immunotherapy, and to guide subsequent treatment plans. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1Schematic diagram of high expression of CXCL4 in serum exosomes of MSS-type CRC patients (Figure A is a schematic diagram of the morphology of exosomes separated from serum by transmission electron microscopy; Figure B is a schematic diagram of the purity of exosomes separated from serum by protein immunoblotting; Figure C is a volcano map showing the difference in serum EV proteins between MSS-type and MSI-type CRC patients; Figure D is a heat map showing the protein difference multiple, with 26 proteins greater than 1.5 times and p<0.05, of which 16 were upregulated in the MSS group and 10 were downregulated in the MSS group; Figure E is a schematic diagram of the difference in CXCL4 from serum EVs of CRC cohort samples collected in the early stage of ELISA detection between the MSS group and the MSI group; Figure F is a schematic diagram of the difference in CXCL4 between MSS-type and MSI-type CRC patients by immunohistochemistry);

[0024] Figure 2 Schematic diagram of CXCL4 as a predictive marker for CRC immunotherapy (Figure A is a schematic diagram showing that the expression of CXCL4 in CRC tumor tissues of patients with immunotherapy-resistant CRC is significantly higher than that of patients with immunotherapy-sensitive CRC in the public database; Figure BD is a schematic diagram showing that the expression of CXCL4 in tumor tissues of the public database in Gao cohort (Figure B), Cho cohort (Figure C) and Amato cohort (Figure D) can significantly distinguish between patients with immunotherapy-resistant and sensitive CRC; Figure EI is a schematic diagram showing that the public database shows that CRC patients with high expression of CXCL4 in tumor tissues in Lauss cohort (Figure E), VanAllen cohort (Figure F), Kim cohort (Figure G), Cho cohort (Figure H) and Nathanson cohort (Figure I) have a poor response to immunotherapy);

[0025] Figure 3 Schematic diagram of exosome-derived CXCL4 being a new predictive marker for immunotherapy of CRC (Figure A is a schematic diagram of protein immunoblotting to detect the expression levels of exosome-derived CXCL4 secreted by various types of cells; Figure B is a schematic diagram of ELISA to detect the difference in CXCL4 expression levels, where R is the immunotherapy-sensitive group and NR is the immunotherapy-non-sensitive group). DETAILED DESCRIPTION

[0026] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments.

[0027] Example 1 Screening of markers for predicting sensitivity of colorectal cancer to immunotherapy

[0028] 4D-DIA deep proteomics analysis was performed on the serum exosomes collected from 40 CRC patients. The patients were divided into MSS group and MSI group according to the MSS / MSI typing results of pathological tissues. The differences in serum exosome-derived proteins between the two groups were compared as follows:

[0029] 1. Extraction of serum exosomes:

[0030] Fresh blood collected from 40 CRC patients was centrifuged at 4000 g for 20 minutes in a vacuum tube, and the supernatant was taken to obtain serum, which was then stored at -80°C.

[0031] The collected frozen serum was thawed evenly at 4°C and then centrifuged at 10,000 g for 20 minutes. The resulting suspension was diluted with 4 mL of cold PBS and transferred to a 0.22 μm filter. After centrifugation at 120,000 g for 90 minutes, the supernatant was removed, the precipitate at the bottom of the tube was collected, the precipitate was resuspended in 4 mL of PBS, and centrifuged again at 120,000 g for 90 minutes. After removing the supernatant, the resulting precipitate was a highly pure exosome. The resulting exosome sample was resuspended in 100 μL of PBS for further use.

[0032] Exosome identification: The exosomes obtained above were identified, and the results of projection electron microscopy showed that the exosomes with complete morphology were successfully isolated (see Appendix Figure 1 A); Western blotting results showed that the isolated exosomes contained exosome-specific markers CD63 and TSG101, but did not contain endoplasmic reticulum markers GRP94 and Calnexin, proving that high-purity exosomes (EVs) were successfully isolated (see Appendix Figure 1 B).

[0033] 2. Use ELISA kit (CXCL4: Neobioscience, EHC135.96) to detect chemokine 4 (CXCL4, chemokine (CXC motif) ligand 4) in serum-derived exosomes:

[0034] A total of 10 μL of exosome samples were mixed with 90 μL of RIPA protein lysis buffer on ice for 60 min and then diluted with 200 μL of PBS. Next, 100 μL of the diluted samples were added to a 96-well plate for ELISA assay, following the manufacturer's instructions. Finally, the absorbance at 450 nm was measured using a synergyH1 multimode reader (BioTek, Vermont, USA).

[0035] Test results: The volcano plot shows the difference in serum EVs proteins between MSS and MSI CRC patients ( Figure 1 C); The heat map shows that there are 26 proteins with a difference of more than 1.5 times and p < 0.05, of which 16 are up-regulated in the MSS group and 10 are down-regulated in the MSS group ( Figure 1 D).

[0036] The results of ELISA showed that the expression of CXCL4 from serum EVs was significantly different between the MSS group and the MSI group ( Figure 1 E)

[0037] 3. Quantitative proteomics analysis of serum-derived exosomes using 4D-DIA:

[0038] Add SDT lysis buffer to the exosome sample and boil it in a water bath for 15 minutes. Centrifuge at 14000g for 15 minutes, take the supernatant and filter it with a 0.22μm centrifuge tube, and collect the filtrate. Then perform Filter-Aided Sample Preparation (FASP) enzymatic hydrolysis on the sample, take the peptide mixture of all samples, and grade it through the Agilent 1260infinity II HPLC system. The sample is separated by the nanoliter flow rate Evosep One system, which is connected to the mass spectrometer timsTOFPro equipped with a CaptiveSpray ion source. Dilute each sample peptide to 10ng / ul, add iRT peptides to mix, take 200ng peptides from each sample, desalt with Evotips, and load the sample to complete DIA mass spectrometry analysis.

[0039] Test results: Immunohistochemistry results showed that the tumor tissues of MSS CRC patients expressed CXCL4 more than those of MSI CRC patients (see Appendix Figure 1 F).

[0040] The above results indicate that serum exosome-derived CXCL4 can be used as a marker to predict the sensitivity of colorectal cancer immunotherapy.

[0041] In this embodiment, the MSS / MSI typing is specifically as follows: microsatellite (MS) refers to a DNA sequence in the genome that is repeated in tandem in units of a few nucleotides (mostly 1-6). The expression of MMR proteins (MLH1, MSH2, MSH6, PMS2) is detected by immunohistochemistry. If all four proteins are positively expressed, the mismatch repair function is normal, which is defined as the MSS (microsatellite stability) type; if any one of the proteins is missing, the mismatch repair function is defective, which is defined as the MSI (microsatellite instability) type.

[0042] Example 2 Validation of CXCL4 as a marker for predicting sensitivity of colorectal cancer to immunotherapy using a public database

[0043] To verify the feasibility of CXCL4 as a marker for predicting the sensitivity of colorectal cancer to immunotherapy, we analyzed the information of the public database (Gene Expression Omnibus (GEO)) and found that the expression of CXCL4 in CRC tumor tissues was significantly increased in patients with immunotherapy-resistant CRC compared with patients with sensitive CRC (see Appendix). Figure 2 A). The expression of CXCL4 in tumor tissue can significantly distinguish ICIs (immune checkpoint inhibitors, ICIs)-resistant and sensitive CRC patients (Appendix Figure 2 BD), and CRC patients with high expression of CXCL4 have a poor response to ICIs treatment (Appendix Figure 2 EI).

[0044] Example 3 Cell experiment validates CXCL4 as a marker for predicting sensitivity of colorectal cancer to immunotherapy

[0045] To further verify the feasibility of CXCL4 as a marker for predicting the sensitivity of colorectal cancer to immunotherapy, we extracted exosomes from the culture medium of cell lines (CRC cells and normal intestinal epithelial cells) and found by western blotting that the expression of exosome-derived CXCL4 secreted by CRC cells was significantly higher than that of normal intestinal epithelial cells (CCD841, HIEC-6 and NCM460). Among them, the CXCL4 levels of immunotherapy-resistant CRC cell lines (SW620 and SW480) were higher than those of immunotherapy-sensitive CRC cell lines (LoVo, HCT116 and SW1116) (Supplementary Figure 2). Figure 3 A).

[0046] We also collected serum exosomes from immunotherapy-sensitive CRC patients and immunotherapy-resistant CRC patients for testing. The ELISA results also showed that the expression of CXCL4 in serum exosomes from immunotherapy-resistant CRC patients was significantly higher (Appendix Figure 3 B).

[0047] Therefore, through the above verification, the results show that serum exosome-derived CXCL4 can be used as a marker to predict the sensitivity of colorectal cancer immunotherapy, and the expression level of CXCL4 can be used to determine whether CRC patients are resistant or sensitive to immunotherapy. The specific judgment is: when the expression level of serum exosome-derived CXCL4 is greater than 3423.61pg / mL, CRC patients can be judged as resistant to immunotherapy, and when the expression level of CXCL4 is less than 3423.61pg / mL, CRC patients can be judged as sensitive to immunotherapy.

[0048] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. Use of a reagent for detecting CXCL4 in the preparation of a product for colorectal cancer immunophenotyping diagnosis, treatment regimen selection and / or prognosis evaluation by detecting the level of CXCL4 in a serum exosome sample obtained from a subject, wherein the CXCL4 is used as a biomarker; characterized in that, The colorectal cancer typing diagnosis is to type the colorectal cancer into immunotherapy-sensitive type or immunotherapy-insensitive type according to the CXCL4 level in the subject's serum exosome sample; The treatment regimen selection is to select whether to administer immunotherapy to the subject suffering from colorectal cancer or adjust the existing treatment regimen according to the CXCL4 level in the subject's serum exosome sample; The prognostic assessment is to assess the subject suffering from colorectal cancer as having a good prognosis or a poor prognosis according to the level of CXCL4 in the subject's serum exosome sample.

2. The use according to claim 1, characterized in that The typing diagnosis includes: typing the colorectal cancer into immunotherapy-sensitive or immunotherapy-insensitive types according to the CXCL4 level in the subject's serum exosome sample, wherein if the CXCL4 level is higher than a statistically determined population standard level, the colorectal cancer is typed and diagnosed as immunotherapy-insensitive colorectal cancer, and if the CXCL4 level is lower than the standard level, the colorectal cancer is typed and diagnosed as immunotherapy-sensitive colorectal cancer; and / or The treatment regimen selection includes: determining whether to administer immunotherapy or a combination of immunotherapy and an immunopotentiator to the subject suffering from colorectal cancer according to the CXCL4 level in the subject's serum exosome sample, wherein if the CXCL4 level is lower than the control level, immunotherapy is selected for the subject, and if the CXCL4 level is higher than the control level, immunotherapy is not selected for the subject or immunotherapy and an immunopotentiator are selected for the subject; The prognosis assessment includes: performing a prognosis assessment on the subject suffering from colorectal cancer according to the CXCL4 level in the subject's serum exosome sample, wherein if the CXCL4 level is lower than the control level, the subject is assessed to have a good prognosis, and if the CXCL4 level is higher than the control level, the subject is assessed to have a poor prognosis.

3. The use according to claim 2, characterized in that The control level is: the CXCL4 molecule level measured in a non-tumor normal biological sample, a tumor sample collected from the same subject at different time points, a population standard level determined by statistics, or a standardized level.

4. The use according to claim 3, characterized in that The control level is the CXCL4 level detected by qRT-PCR and calculated using T-test.

5. The use according to claim 1, characterized in that The immunotherapy is one or more therapies selected from the following groups: immune checkpoint inhibition therapy, cellular immunotherapy, antigen chimeric receptor cell therapy, T cell receptor T cell therapy, bispecific antibody therapy, tumor vaccine therapy, and oncolytic virus therapy.

6. The use according to claim 5, characterized in that The immune checkpoint inhibition therapy includes PD-1 inhibitor therapy, PD-L1 inhibitor therapy, and CTLA-4 inhibitor therapy.

7. The use according to claim 5, characterized in that The cellular immunotherapy includes adoptive cell therapy.

8. The use according to claim 5, characterized in that The antigen chimeric receptor cell therapy includes CAR-T therapy and / or CAR-NK therapy.

9. The use according to claim 1, characterized in that The CXCL4 is a substance selected from the following group: CXCL4 gene, CXCL4 mRNA, cDNA, CXCL4 protein or active or marked fragments of any of the foregoing.

10. The use according to claim 1, characterized in that The product is a kit or a combination of a reagent and a detection device.

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