Method for assisting in identifying PD-L1 expression of colorectal cancer patient by utilizing patient ascites source PTC (Positive Temperature Coefficient)

Abstract

The invention provides a method for auxiliary identification of PD-L1 expression of a colorectal cancer patient by using a patient ascites-derived tumor-like cell cluster (PTC), and a kit for identifying the PD-L1 expression of the colorectal cancer patient by using the PD-L1 expression of the colorectal cancer patient. PTC is a result of self-assembly and proliferation of tumor cells, primary epithelial cells, interstitial cells, fibroblasts and immune cells, an ascites sample can be utilized to be cultured into tumor microspheres within a short time, and the obtained tumor microspheres have high consistency with original tumors. PD-L1 detection can be provided for patients who cannot obtain tissue slices by utilizing the PTC derived from malignant effusion of colorectal cancer patients, and the feasibility of receiving immunotherapy is provided for the patients.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a method for assisting in identifying PD-L1 expression in ascites samples of colorectal cancer patients. Background Art

[0002] With the continuous advancement and development of technology, the methods of tumor treatment are also constantly being optimized and innovated. Immunotherapy is currently a relatively new drug treatment method, which mainly uses immune checkpoint inhibitors to activate the body's immune system to treat cancer. Commonly used immune checkpoint inhibitors in colorectal cancer include pembrolizumab, etc., but not all patients can benefit from immune checkpoint inhibitors, so it is necessary to predict the patient's sensitivity to the drug by testing relevant indicators. The detection indicators commonly used to predict the sensitivity of immune checkpoint inhibitors in colorectal cancer mainly include microsatellite high instability or mismatch repair gene deficiency (MSI-H / dMMR). Some literature also points out that the expression level of PD-L1 can also predict the sensitivity of colorectal cancer patients to immune checkpoint inhibitor treatment. Tumor tissues with high expression of PD-L1 are more sensitive to treatment with immune checkpoint inhibitors. Therefore, before immunotherapy, PD-L1 testing is required for patient tissue specimens. Although immunohistochemistry (IHC) can be used as a predictive marker for immunotherapy to screen potential beneficiaries and predict efficacy, there are still many problems with the practice and results of PD-L1 testing. For some patients who cannot obtain surgical tissue, the accuracy of PD-L1 testing in malignant effusion is different from that of tissue specimens. Therefore, the use of PTC technology to construct PTC tumor microspheres derived from ascites of colorectal cancer patients can provide a new solution for PD-L1 detection.

[0003] PTC is a new in vitro culture method for primary tumor cells derived from patients (Patient-Derived Tumor-like Cell Clusters, PTC). PTC is the result of self-assembly and proliferation of tumor cells, primary epithelial cells, stromal cells, fibroblasts and immune cells. By obtaining patient tissue samples, they self-assemble to form tumor microspheres after a series of in vitro cultures. The self-assembly cycle of PTC is short, and the self-aggregation process between cells can be completed in 3-6 days. It simulates the tumor microenvironment to a certain extent, truly reflects the situation of the tumor in the body, reflects the heterogeneity of the tumor, and has a high degree of consistency with the corresponding tumor tissue in pathology, genetics, and function. The present invention is based on PTC technology, and uses PTC to construct PTC tumor microspheres based on the ascites of colorectal cancer patients, detect the PD-L1 expression level of PTC tumor microspheres, and provide these colorectal cancer patients with the feasibility and effect prediction of receiving immunotherapy. Summary of the invention

[0004] The present invention provides a method for using PTC derived from ascites of patients to assist in identifying PD-L1 expression in patients with colorectal cancer. The method provides PD-L1 detection for colorectal cancer patients who can only provide ascites.

[0005] The technical solution of the present invention:

[0006] Step 1: Construction of a PTC model derived from ascites of colorectal cancer patients

[0007] Step 2: Based on the culture results of the PTC model, collect tumor microspheres and make paraffin sections

[0008] Step 3: Based on PTC paraffin sections, PD-L1 expression was identified by immunohistochemistry.

[0009] Furthermore, the specific method for constructing the PTC model derived from ascites of colorectal cancer patients in step 1 includes the following steps:

[0010] 1.1 Fresh ascites from colorectal cancer patients was centrifuged, washed with PBS and centrifuged again;

[0011] 1.2 Resuspend the precipitate in 1.1 with PBS, place it horizontally and slowly add it to the cell separation solution at a ratio of 1:1, and centrifuge at 2500r for 20min;

[0012] 1.3 Absorb the middle layer liquid of the centrifuge tube in 1.2, centrifuge at 2000r for 5 minutes, discard the supernatant, resuspend the precipitate with colorectal cancer-specific PTC culture medium, and inoculate it into a low-adsorption well plate to obtain a PTC model derived from colorectal cancer patients.

[0013] Furthermore, in step 2, based on the culture results of the PTC model, the specific method of collecting tumor microspheres and making paraffin sections includes the following steps:

[0014] 1.1 Collect the cultured microtumor PTC by centrifugation, discard the supernatant and fix with 4% paraformaldehyde;

[0015] 1.2 Dehydrate the microtumor PTC fixed in step 1.1 step by step with gradient alcohol, then permeabilize with xylene, and finally add paraffin for infiltration embedding;

[0016] 1.3 Slice the paraffin block embedded in 1.2 and bake the slices to obtain paraffin sections of the PTC model.

[0017] Furthermore, the specific method for identifying the expression of PD-L1 in the paraffin sections of the PTC model in step 3 includes the following steps:

[0018] 1.1 Place the slices in a 60°C oven and bake for 2-3 hours, then dewax the paraffin slices to water by passing through xylene and gradient alcohol step by step;

[0019] 1.2 Incubate the dewaxed sections in step 1.1 with 3% H2O2 at room temperature for 10 min to block endogenous catalase, and then rinse with PBS three times;

[0020] 1.3 Add the slices prepared in step 1.2 to 0.01 M citric acid repair solution (pH 6.0), heat in a microwave oven at medium heat for 5 min, repeat twice, cool to room temperature, and rinse three times with PBS;

[0021] 1.4 Block the sections in step 1.3 with 10% normal goat serum for 1 h, remove the serum and add PD-L1 primary antibody (22C3, Dako), incubate at 4°C overnight, and wash twice with PBS;

[0022] 1.5 Add secondary antibody to the slices prepared in step 1.4 and incubate for 30 min, then wash twice with PBS;

[0023] 1.6 Add DAB to the slices prepared in step 1.5 for staining, control the staining time under the microscope, and wash with PBS three times;

[0024] 1.7 Add hematoxylin to the slices prepared in step 1.6 for counterstaining, rinse with tap water after 1 minute, differentiate with 1% hydrochloric acid for 3 seconds, and rinse with tap water;

[0025] 1.8 Place the sections stained in step 1.7 into gradient alcohol and xylene in sequence, with each level lasting 5 minutes;

[0026] 1.9 Add neutral resin to the slices prepared in step 1.7 and examine under a microscope. DETAILED DESCRIPTION

[0027] The technical solution of the present invention will be further described below, but the present invention is not limited thereto.

[0028] Example 1

[0029] 50-100 mL of ascites specimens from patients with colorectal cancer were collected, centrifuged and separated, and then resuspended in colorectal cancer-specific PTC culture medium and incubated at 10 5 / cm 2 The cells were inoculated at a density of 100 μg / mL in a low-adsorption surface culture dish and cultured at 37°C and 5% CO2. The culture medium was changed every 2-3 days. The obtained PTC model was fixed with 4% paraformaldehyde, embedded in paraffin and sliced. PD-L1 was detected using Dako's 22C3 antibody, and the cell nuclei were counterstained with hematoxylin, and the slides were sealed for microscopic examination to examine the expression of PD-L1 in the patient's ascites-derived PTC model.

[0030] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A method for using patient-derived tumor-like cell cluster (PTC) from ascites to assist in identifying PD-L1 expression in patients with colorectal cancer, wherein: The method for assisting in identifying PD-L1 expression in colorectal cancer patients only includes identification based on PTC. It is characterized by comprising the following steps: Step 1: Collect experimental ascites specimens from colorectal cancer patients and construct a PTC model; Step 2: Based on the culture results of the PTC model, tumor microspheres are collected and made into paraffin sections; Step 3: Based on PTC paraffin sections, PD-L1 expression was identified by immunohistochemistry.

2. The method according to claim 1, characterized in that The experimental specimens in step 1 include but are not limited to ascites samples from patients with colorectal cancer.

3. The PTC construction method according to claim 1 or 2, characterized in that: The following steps are involved: Step 1: Using enzymatic hydrolysis, the experimental specimens of colorectal cancer patients are dissociated into single cells; Step 2: Cultivate the dissociated specimen in a PTC culture medium specifically for colorectal cancer. The tumor cells in the specimen self-assemble with other component cells to form tumor microspheres to obtain a PTC model.

4. The method according to claim 1, characterized in that The paraffin section in step 2 comprises the following steps: Step 1: Collect tumor microspheres in the PTC model by centrifugation and fix them with 4% paraformaldehyde; Step 2: embed the fixed tumor microspheres in paraffin and slice them into 5um thick paraffin sections.

5. The method according to claim 1, characterized in that In the immunohistochemistry in step 3, the antibody used is 22C3, the detection platform is DAKO AutoStainer Link 48 platform, and the scoring method is CPS.