Tumor tissue PD-L1 gradient quality control sheet and preparation method and application thereof

Abstract

The invention relates to the technical field of biological pathology detection, and particularly discloses a tumor tissue PD-L1 gradient quality control sheet as well as a preparation method and application thereof. The tumor tissue PD-L1 gradient quality control sheet comprises a slide label area, a staining control area and a tissue patch area, three HIS-PDTX tumor living tissues or hPBMC-PDTX tumor living tissue samples which play a control role and have the PD-L1 staining gradient being negative, the PD-L1 staining gradient being medium expression and the PD-L1 staining gradient being high expression are fixed in the staining control area, and the HIS-PDTX tumor living tissue samples are fixed in the tissue patch area. Meanwhile, traceability and continuous supply of sample sources are ensured; the samples can accurately reflect the distribution condition of PD-L1 in HIS-PDTX tumor living tissue or hPBMC-PDTX tumor living tissue structures, and the accuracy and reliability of the tumor tissue PD-L1 gradient quality control wafer can be improved by using the samples as control.

Description

Technical Field

[0001] The present application relates to the field of biological pathology detection technology, and in particular to a tumor tissue PD-L1 gradient quality control sheet and a preparation method and application thereof. Background Art

[0002] Programmed death-ligand 1 (PD-L1) is a cell surface protein that can be detected in immune cells, including B cells, T cells, macrophages, dendritic cells (DCs), and mast cells. In addition, it is also widely expressed in cancer tissues, such as lung cancer, esophageal cancer, liver cancer, breast cancer, urothelial carcinoma, malignant melanoma, cervical cancer, kidney cancer, intestinal cancer, and ovarian cancer. PD-L1 can inhibit cellular immunity and cytokine signaling pathways by binding to its receptor PD-1, leading to immune tolerance and immune escape. In malignant tumors, tumor cells express PD-L1 at high levels, bind to PD-1 on the surface of immune cells, inhibit the activity of T cells, and escape the attack of immune cells. In addition, PD-L1 can also negatively regulate immune activity by binding to other receptors (such as CD80, B7-1, B7-2, etc.).

[0003] Immunotherapy has shown effectiveness and safety in the treatment of various tumors and has been widely used. Among them, PD-1 / PD-L1 inhibitors have become a hot spot in clinical research. At present, 8 PD-1 / PD-L1 monoclonal antibody drugs have been launched in China, of which 4 are imported PD-1 / PD-L1 inhibitors and 4 are domestic PD-1 inhibitors. With the approval of PD-1 / PD-L1 immune checkpoint inhibitors, PD-L1 immunohistochemistry detection reagents for cancer patients have also been approved as companion diagnosis or supplementary diagnosis according to the indication needs. At the same time, the reagents approved by the US FDA for use with anti-PD-1 antibodies or anti-PD-L1 antibody drugs are all antibody detection reagents for PD-L1 protein. The detection methods are all immunohistochemistry detection of paraffin-embedded tissue (FFPE) samples.

[0004] At present, the interpretation methods used for companion diagnostic PD-L1 detection antibodies mainly include TPS, IC, CPS, TAP, and TC combined with IC. Different types of tumors are suitable for different interpretation methods. For non-small cell lung cancer, the TC interpretation method is used when the 22C3 antibody is used; for triple-negative breast cancer and urothelial carcinoma, the IC interpretation method is used when the SP142 antibody is used; and the SP142 antibody uses a combination of TC and IC for interpretation. When the FDA approved the 22C3 antibody, it stated that the TC interpretation method was used for non-small cell lung cancer, but for gastric adenocarcinoma and gastroesophageal junction adenocarcinoma, the CPS interpretation method was recommended. The CPS interpretation method is also recommended for cervical cancer and urothelial carcinoma. Therefore, accurate interpretation of PD-L1 results requires professional pathologist experience and a lot of training, so promotion and application are relatively difficult. Among them, the interpretation methods of IC, CPS, TC combined with IC and TAP all require the interpretation of the PD-L1 positive staining of immune cells, and the commonly used PD-L1 staining platforms on the market mainly include the AutoStainerLink 48 platform, the Ventana Benchmark Ultra platform and the Leica Bond Max / III platform. These platforms use different secondary antibody systems, resulting in significant differences in the staining results of the same monoclonal antibody on each platform. In order to avoid staining differences, companion diagnostic products on the market usually do not judge the staining intensity, but focus on the positive or negative interpretation of PD-L1. Such a strategy can ensure the consistency of results and minimize the variability between different platforms. However, the control samples of the current PD-L1 antibody detection kit are mostly tumor cell lines or tonsil tissues. The biological characteristics of these control samples are unstable and the sources are limited. Therefore, it is difficult to trace the source of the samples and achieve sustainable supply, and it is impossible to perform dual PD-L1 staining interpretation on tumor cells and immune cells in tumor tissues. In addition, when using immunohistochemistry to detect PD-L1 expression, due to the lack of standard PD-L1 controls, false positive and false negative results of PD-L1 immunohistochemistry staining often occur. When different PD-L1 antibodies are used, batch effects may be observed.

[0005] In summary, the development of a tumor tissue PD-L1 gradient quality control film can display the PD-L1 immunohistochemical staining of tumor cells and immune cells in tumor tissues and perform comprehensive interpretation. This can provide powerful guidance for the accurate interpretation of PD-L1 results and is of great significance for the diagnosis and treatment of cancer. Summary of the invention

[0006] The purpose of this application is to overcome the shortcomings of the above-mentioned prior art and provide a tumor tissue PD-L1 gradient quality control film and its preparation method and application. The tumor tissue PD-L1 gradient quality control film designed in this application can display the staining of PD-L1 in tumor cells and immune cells, provide strong guidance for the accurate interpretation of PD-L1 results, and is of great significance for the diagnosis and treatment of cancer. Through this innovative method, clinical treatment decisions can be better guided, personalized treatment plans can be provided for patients, and it can help improve the prognosis and survival rate of cancer patients. Therefore, the development of such a quality control film has important application prospects and significance in the field of cancer.

[0007] To achieve the above purpose, the technical solution adopted by this application is: The present application provides a method for preparing a tumor tissue PD-L1 gradient quality control sheet, comprising the following steps: S1. Based on the RNA-seq information characteristics of the transcriptome sequencing corresponding to the PD-L1 antibody and the interpretation methods of different PD-L1 antibodies in different tumors, the PDTX tumor biopsy samples that meet the required characteristics are screened out through the sample sequencing information stored in the PDTX tumor biopsy database; S2. Put the CD 34+ HSC cell fluid was transferred into C-NKG mice to construct a C-NKG mouse humanized immune system HIS model, and the PDTX tumor living tissue obtained in step S1 was inoculated into the C-NKG mouse humanized immune system HIS model for passage and amplification to obtain HIS-PDTX tumor living tissue with stable expression of PD-L1; Alternatively, the PDTX tumor biopsy sample obtained in step S1 is inoculated into C-NKG mice, and peripheral blood mononuclear cells are transferred into the mice to obtain hPBMC-PDTX tumor biopsy with stable expression of PD-L1; S3. Fix the HIS-PDTX tumor biopsy tissue or hPBMC-PDTX tumor biopsy tissue obtained in step 2 as a sample of the alternative control area, prepare a wax block, perform staining and detection, screen out tumor biopsy tissues with a tumor cell content of ≥30% and a necrosis ratio of ≤20%, and interpret the PD-L1 expression level of the HIS-PDTX tumor biopsy tissue or hPBMC-PDTX tumor biopsy tissue; S4, preparing wax blocks and slices of the HIS-PDTX tumor biopsy tissue or hPBMC-PDTX tumor biopsy tissue screened in step S3, and attaching the slices to the control area on the adhesive slide with a printed label to obtain a tumor tissue PD-L1 gradient quality control film; The tumor tissue PD-L1 gradient quality control sheet includes a glass slide label area, a staining control area and a tissue patch area, wherein the glass slide label area contains the name and product model of the target antibody PD-L1; the staining control area is fixed with three types of HIS-PDTX tumor biopsy tissue or hPBMC-PDTX tumor biopsy tissue samples, which serve as controls, including negative PD-L1 staining gradients, medium expression PD-L1 staining gradients and high expression PD-L1 staining gradients.

[0008] The mice are triple immunodeficient mice with extremely severe T, B and NK cell deficiency, which are from Saiye Biotechnology Co., Ltd., and the strain name is NOD.Cg-Prkdc scid Iq em1cya / Cya, abbreviated as C-NKG.

[0009] When immunohistochemical staining is required, the tissue sections of the sample to be tested are adhered to the tissue patch area and stained simultaneously with the control tissue.

[0010] In the technical solution of the present application, the present application uses HIS-PDTX technology or hPBMC-PDTX technology to construct a model, and HIS-PDTX tumor biopsy tissue or hPBMC-PDTX tumor biopsy tissue containing tumor cells and immune cells can be obtained as a PD-L1 control sample. When detecting PD-L1 expression by immunohistochemistry, PD-L1 staining of tumor cells and immune cells can be observed at the same time. When using immunohistochemistry to detect PD-L1 expression in samples of multiple cell types, a variety of PD-L1 interpretation methods can be used. Therefore, different antibodies can be selected for PD-L1 staining according to different needs, thereby improving the flexibility and reliability of tumor tissue PD-L1 gradient quality control films, and providing more reliable data support for immunotherapy research.

[0011] The tissue of the staining control area is derived from HIS-PDTX tumor biopsies or hPBMC-PDTX tumor biopsies that stably express PD-L1 and are stored in the tumor biopsies bank. They can be resuscitated and amplified using HIS-PDTX or hPBMC-PDTX technology, so that sufficient tissue can be obtained for control, and the tissue source is stable and controllable. Since these biopsies stably express PD-L1, their quality and reliability can be ensured as staining control areas.

[0012] The HIS-PDTX tumor biopsy tissue or hPBMC-PDTX tumor biopsy tissue samples of the present application are screened by RNA-seq information of the samples stored in the tumor biopsy tissue database, and the control standard is more accurate.

[0013] The control sample of the tumor tissue PD-L1 gradient quality control film in this application is similar to the tissue structure to be tested, can accurately reflect the distribution of PD-L1, and has higher specificity and sensitivity; the control tissue of the tumor tissue PD-L1 gradient quality control film can effectively avoid false positive and false negative results of PD-L1 expression in clinical testing, and can also eliminate the batch effect caused by antibodies produced in different batches.

[0014] As a preferred embodiment of the method for preparing the tumor tissue PD-L1 gradient quality control sheet described in the present application, in the step S1, the TPM normalized value of the transcriptome sequencing corresponding to the PDTX tumor biopsy sample with a negative PD-L1 staining gradient is <5; the TPM normalized value of the transcriptome sequencing corresponding to the PDTX tumor biopsy sample with a medium expression PD-L1 staining gradient is 5~50; the TPM normalized value of the transcriptome sequencing corresponding to the PDTX tumor biopsy sample with a high expression PD-L1 staining gradient is >50.

[0015] The HIS-PDTX tumor biopsy or hPBMC-PDTX tumor biopsy samples of the present application are screened by using the TPM normalized value of the transcriptome sequencing RNA-seq of the samples stored in the tumor biopsy database, which further improves the accuracy of the PD-L1 control tissue and ensures that the selected samples can more accurately represent the expression of PD-L1.

[0016] As a preferred embodiment of the method for preparing the tumor tissue PD-L1 gradient quality control sheet described in the present application, in the step S1, the PD-L1 antibody includes but is not limited to at least one of 22C3 antibody, SP142 antibody, SP263 antibody, WD160 antibody, E1L3N antibody, OR-5E3 antibody, and MXR-003 antibody; however, the PD-L1 antibody is not limited to the above examples, and also includes common PD-L1 antibody types in the art, all of which are within the scope of the present application; The tumor includes but is not limited to at least one of non-small cell lung cancer, urothelial carcinoma, cervical cancer, and triple-negative breast cancer; the tumor is not limited to the above tumor types, and also includes common tumor types in the field, all of which are within the scope of this application; The determination method includes a TPS determination method, a CPS determination method, an IC determination method or a TC+IC determination method.

[0017] As a preferred embodiment of the method for preparing the tumor tissue PD-L1 gradient quality control sheet described in the present application, the 22C3 antibody adopts the TPS interpretation method in non-small cell lung cancer, and the 22C3 antibody adopts the CPS interpretation method in urothelial carcinoma or cervical cancer; The SP142 antibody uses an IC interpretation method in triple-negative breast cancer and urothelial carcinoma; The SP142 antibody adopts the TC+IC interpretation method in non-small cell lung cancer; The SP263 antibody adopts the TC+IC interpretation method in urothelial carcinoma.

[0018] The samples of this application have multiple cell types, so when performing immunohistochemical staining for PD-L1 expression, a variety of methods can be used for interpretation, and different PD-L1 antibodies can be selected for staining as needed. This diverse PD-L1 detection method helps to improve the accuracy and reliability of PD-L1 detection, thereby more accurately evaluating the expression of PD-L1. Multi-dimensional observation and flexible PD-L1 antibody selection are of great significance for more accurate evaluation of PD-L1 expression and provide more reliable data support for immunotherapy research.

[0019] As a preferred embodiment of the method for preparing the tumor tissue PD-L1 gradient quality control sheet of the present application, in step S2, the CD34 + The concentration of HSC cell solution was 5×10 5 cells / ml, injection volume was 5×10 4 cells; The concentration of peripheral blood mononuclear cells was 5×10 6 cells / ml, injection volume was 5×10 5 cells.

[0020] As a preferred embodiment of the method for preparing the tumor tissue PD-L1 gradient quality control sheet of the present application, the step S2 is specifically as follows: C-NKG mice within 3 weeks were irradiated with X-rays at 100 cGy / min for 2.4 min to ablate the myeloablation. Within 4 to 24 hours after myeloablation, the concentration of 5×10 5 CD34 cells / ml + HSC cells; after supplementation with CD34 + In the fourth week after HSC cells were generated, the immune cells in the peripheral blood of mice were detected by flow cytometry every two weeks. + When the expression rate was ≥25%, the humanized immune system HIS model of C-NKG mice was successfully constructed; Cut the PDTX tumor biopsy sample obtained in step S1 into 2×2×2 mm 3 , inoculated into the C-NKG mouse humanized immune system HIS model, and waited for the transplanted tumor to grow to a cumulative volume of 800-1000mm 3 , obtain HIS-PDTX tumor biopsies with stable PD-L1 expression as alternative control area samples; Alternatively, take 5-8 week old C-NKG mice and cut into 2 × 2 × 2 mm 3 PDTX tumor biopsy samples, the transplanted tumor volume accumulates to 500~800mm 3 100 μl of 5×10 6 hPBMC-PDTX tumor biopsies with stable expression of PD-L1 were obtained as alternative control samples.

[0021] By using HIS-PDTX tumor tissue or hPBMC-PDTX tumor biopsy tissue as the control sample of the tumor tissue PD-L1 gradient quality control film, human tumor cells and immune cells can be covered at the same time, so that the PD-L1 staining of tumor cells and immune cells can be observed at the same time when immunohistochemistry is used to detect PD-L1. The control sample of the tumor tissue PD-L1 gradient quality control film is similar to the actual tissue structure to be tested, which can truly reflect the distribution of PD-L1, and has higher specificity and sensitivity.

[0022] As a preferred embodiment of the method for preparing the tumor tissue PD-L1 gradient quality control sheet described in the present application, in step S3, the PDTX tumor living tissue obtained in step S1 is cut into 2×2×2 mm 3 , inoculated into the C-NKG mouse humanized immune system HIS model obtained in step S2, and then propagated until the transplanted tumor grows to a cumulative volume of 800-1000mm 3 , obtain HIS-PDTX tumor biopsy tissue with stable expression of PD-L1, or cut the PDTX tumor biopsy tissue obtained in step S1 into 2×2×2mm 3 , inoculated into C-NKG mice, and peripheral blood mononuclear cells were transferred into tumor-bearing C-NKG mice to obtain hPBMC-PDTX tumor living tissue with stable expression of PD-L1.

[0023] The tissue of the staining control area of ​​this application comes from HIS-PDTX tumor biopsy tissue or hPBMC-PDTX tumor biopsy tissue samples with stable expression of PD-L1 stored in the tumor biopsy sample library. Using HIS-PDTX or hPBMC-PDTX technology, these samples can be successfully revived and amplified to obtain sufficient tissue for use as a control. This method can ensure the stability and controllability of the tissue source and provide reliable tissue samples for the staining control area. By using these samples as controls, we can more accurately evaluate the expression of PD-L1 and provide reliable results and data support for the research.

[0024] As a preferred embodiment of the method for preparing the tumor tissue PD-L1 gradient quality control sheet described in the present application, in the step S3, the staining includes HE staining or immunohistochemistry staining IHC.

[0025] The present application provides a tumor tissue PD-L1 gradient quality control sheet prepared by the preparation method of the above-mentioned tumor tissue PD-L1 gradient quality control sheet, and the tumor tissue PD-L1 gradient quality control sheet contains lymphocytes with positive PD-L1 expression.

[0026] The tumor tissue PD-L1 gradient quality control film designed in this application has a control sample with consistent biological characteristics and stability, while ensuring the traceability and continuous supply of the sample source; this application selects real HIS-PDTX tumor biopsy tissue or hPBMC-PDTX tumor biopsy tissue samples as control samples for the tumor tissue PD-L1 gradient quality control film; the tumor biopsy tissue prepared in this application contains lymphocytes that are positive for PD-L1 expression. These samples have real immune microenvironment and heterogeneity characteristics, and can accurately reflect the distribution of PD-L1 in the HIS-PDTX tumor biopsy tissue or hPBMC-PDTX tumor biopsy tissue structure. By using these samples as controls, the accuracy and reliability of the tumor tissue PD-L1 gradient quality control film can be improved; The control sample of the selected tumor tissue PD-L1 gradient quality control film is a human HIS-PDTX tumor biopsy tissue or hPBMC-PDTX tumor biopsy tissue sample, which has a human immune microenvironment. When using immunohistochemistry to detect PD-L1, the PD-L1 expression of tumor cells and immune cells in tumor tissue can be detected. Therefore, a variety of PD-L1 immunohistochemistry interpretation methods can be selected as needed. This flexibility reduces the selection restrictions for detecting PD-L1 antibodies; clinically, there will be problems of false positives and false negatives in PD-L1 immunohistochemistry staining. The tumor tissue PD-L1 gradient quality control film provided in this application has stable PD-L1 expression and can avoid the problems of false positives and false negatives in immunohistochemistry. At the same time, batch effects caused by different antibodies are avoided.

[0027] The present application also provides the use of the above-mentioned tumor tissue PD-L1 gradient quality control film in detecting PD-L1 clinical samples, wherein the clinical samples are non-living tissue samples.

[0028] Compared with the prior art, this application has the following beneficial effects: The present application provides a tumor tissue PD-L1 gradient quality control film and its preparation method and application. The tumor tissue PD-L1 gradient quality control film of the present application provides a control sample with consistent biological characteristics and stability, while ensuring the traceability and continuous supply of the sample source; the present application selects real HIS-PDTX tumor biopsy tissue or hPBMC-PDTX tumor biopsy tissue samples as control samples for the tumor tissue PD-L1 gradient quality control film; the tumor biopsy tissue prepared in the present application contains lymphocytes with positive PD-L1 expression. These samples have real immune microenvironment and heterogeneity characteristics, and can accurately reflect the distribution of PD-L1 in the HIS-PDTX tumor biopsy tissue or hPBMC-PDTX tumor biopsy tissue structure. By using these samples as controls, the accuracy and reliability of the tumor tissue PD-L1 gradient quality control film can be improved; when using immunohistochemistry to detect PD-L1, the PD-L1 expression of tumor cells and immune cells in tumor tissue can be detected, so a variety of PD-L1 immunohistochemistry interpretation methods can be selected as needed, and this flexibility reduces the selection restrictions on the detection of PD-L1 antibodies. The PD-L1 detection control substance provided in the present application has stable PD-L1 expression, which can avoid the problems of false positive and false negative in immunohistochemistry. At the same time, it avoids the batch effect caused by different antibodies. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 A flowchart of modeling, recovery, amplification, and sampling of HIS-PDTX tumor biopsy tissue in step 2 of Example 1; Figure 2 This is a flow chart of hPBMC-PDTX tumor biopsy modeling, recovery, expansion, and sampling in step 2 of Example 1; Figure 3 HE staining of tumor tissue and immunohistochemical staining of PD-L1 in tumor tissue; Figure 4 This is a schematic diagram of the PD-L1 gradient quality control film of tumor tissue; Figure 5 This is the immunohistochemical staining image of the tumor tissue PD-L1 gradient quality control film prepared in Example 2. DETAILED DESCRIPTION

[0030] In order to better illustrate the purpose, technical solutions and advantages of the present application, the present application will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0031] In the following examples, the experimental methods used are conventional methods unless otherwise specified, and the materials, reagents, etc. used are all commercially available unless otherwise specified.

[0032] Example 1. Screening of HIS-PDTX tumor biopsies or hPBMC-PDTX tumor biopsies with stable expression of PD-L1 Step 1: Screening of tumor tissue: To date, immune cell scoring has been limited to clone SP142 in atezolizumab-treated triple-negative breast cancer and urothelial carcinoma. The score is defined as the proportion of tumor area (intratumoral and adjacent peritumoral stroma, excluding necrosis) occupied by immune cells with PD-L1 staining of any intensity. The CPS score is defined as the combined positive score calculated by measuring PD-L1 staining of tumor cells and tumor-associated immune cells (lymphocytes, histiocytes), divided by the total number of tumor cells, and then multiplied by 100. This interpretation method can be used for cervical cancer and urothelial carcinoma with the 22C3 antibody, and the TPS interpretation method, that is, PD-L1 positive staining of tumor cells, is used in non-small cell lung cancer. The immune cell (IC) interpretation method can be used with the SP142 antibody in triple-negative breast cancer and urothelial carcinoma; combined tumor cell (TC) and immune cell (IC) scoring using SP142 staining for atezolizumab-treated non-small cell lung cancer, or SP263 staining for durvalumab-treated urothelial carcinoma.

[0033] Table 1 Antibodies and PD-L1 interpretation methods in different tumors According to the transcriptome sequencing (RNA-seq) information characteristics corresponding to PD-L1, the sample sequencing information stored in the PDTX tumor biopsy database built by Puenrui was used to screen out biopsy samples that met the following feature requirements, as shown in Table 2.

[0034] Table 2 RNA-seq information features Step 2: HIS-PDTX tumor biopsy modeling recovery, expansion, and sampling: (1) Fresh umbilical cord blood was delivered to the laboratory within 24 hours after blood collection. The blood was then diluted 1:3 with phosphate buffered saline (PSB) and mononuclear cells were isolated by density gradient centrifugation on Ficoll medium.

[0035] (2) The monocytes are expressed via CD34 + CD34 isolated by microbead kit + HSC cell fluid, CD34 + The HSC cell solution was adjusted to a concentration of 5×10 5 cells / ml, and then the 5×10 5 CD34 cells / ml + HSC cell fluid was stored at 2-8°C until injection.

[0036] (3) C-NKG mice within 3 weeks of age were selected (100 cGy / min×2.4 min, X-ray irradiation, myeloablation) and injected with 5×10 5 CD34 cells / ml + HSC cells, injected in 100 μl (5×10 4 cells).

[0037] (4) In the supplementation of CD34 + From the fourth week after HSC cells were generated, the immune cells in the peripheral blood of mice were detected by flow cytometry. The interval was two weeks. + When the expression level was ≥25%, the construction of the humanized immune system HIS model of C-NKG mice was determined to be successful.

[0038] (5) While constructing the C-NKG mouse humanized immune system HIS model, the PDTX tumor biopsy tissue that meets the requirements after screening was cut into 2×2×2 mm 3 , inoculated into the subcutaneous tissue of the back of C-NKG mice. Tumor tissue samples that were passed to P5 and had a stable growth rate were inoculated into the subcutaneous tissue of the C-NKG mouse humanized immune system HIS model.

[0039] (6) After inoculation, the animal status was observed daily, the tumor volume was measured and the weight of the tumor-bearing mice was weighed once a week, and the tumor volume V (mm 3 )= (a×b 2 ) / 2, a: long diameter of the tumor, b: short diameter of the tumor.

[0040] (7) The volume of the tumor to be transplanted has accumulated to 80 mm 3 The cells were dissected out, cut and randomly inoculated (P1 generation) into the subcutaneous tissue of the C-NKG mouse humanized immune system HIS model for passage and expansion.

[0041] (8) The volume of the transplanted tumor grows to 800-1000 mm 3 , dissected out and used as alternative control area samples for paraffin embedding and section preparation. Figure 1 shown.

[0042] or, Step 2: hPBMC-PDTX tumor biopsy modeling recovery, expansion, and sampling: (1) Cut the selected PDTX tumor tissue into 2×2×2mm 3 , inoculated subcutaneously into the back of C-NKG mice.

[0043] (2) Fresh peripheral blood was delivered to the laboratory within 24 hours after blood collection, diluted (peripheral blood: phosphate buffer or RPMI1640 = 1:2), and peripheral blood mononuclear cells were isolated by density gradient centrifugation on Ficoll medium.

[0044] (3) Count the peripheral blood mononuclear cells above and then adjust the peripheral blood mononuclear cell concentration to 10×10 6 / ml.

[0045] (4) When the tumor volume in C-NKG mice reaches 500-800 mm 3 Afterwards, peripheral blood mononuclear cells were injected via the tail vein with an injection volume of 100 μl (5×10 5 cells).

[0046] (5) The samples were removed by dissection and used as alternative control samples for paraffin embedding and sectioning. Figure 2 shown.

[0047] Step 3: Fixation and paraffin block preparation of PDTX tumor biopsies: (1) Tissue fixation: Add HIS-PDTX tumor biopsy tissue or hPBMC-PDTX tumor biopsy tissue into 10 times the volume of 10% neutral formaldehyde solution and fix at room temperature for 12-24 hours.

[0048] (2) Tissue collection: Cut the fixed tissue into tissue blocks ≤1.5cm×1.5cm×0.5cm, place them in an embedding box with a printed label, and then place them in a tissue dehydration basket.

[0049] (3) Tissue dehydration: Immerse the tissue in different concentration gradients of ethanol for tissue dehydration: 70% ethanol for 1 hour → 80% ethanol for 2 hours → 85% ethanol for 70 minutes → 95% ethanol for 1 hour → 95% ethanol for 50 minutes → anhydrous ethanol for 1 hour → anhydrous ethanol for 50 minutes.

[0050] (4) Tissue clearing: After dehydration with graded ethanol, immerse the tissue in xylene for clearing: xylene I for 30 min → xylene II for 25 min.

[0051] (5) Tissue wax immersion: Immerse the transparent tissue into melted paraffin for tissue wax immersion: Paraffin I 1h → Paraffin II 1h → Paraffin III 1h.

[0052] (6) Tissue embedding: Tissue embedding is performed in a tissue embedding machine. Drop a small amount of melted paraffin into the preheated stainless steel embedding mold. After the paraffin at the bottom solidifies slightly, place the tissue at the bottom of the mold, adjust the position of the tissue, press the tissue lightly so that the tissue is on the same plane, place the embedding box on the mold, continue to fill it with wax liquid, transfer it to a pre-cooled freezing table to cool for 30 minutes, remove the wax block, and trim off the excess paraffin.

[0053] Step 4: HE staining: (1) Place the wax block into the slot of the microtome and slice it. Cut out 4 μm white slices and place them in a water bath at about 45°C until the slices are fully expanded.

[0054] (2) After unfolding, adhere the tissue to a glass slide.

[0055] (3) After slightly drying, the slices can be baked. Generally, the slices are baked in a baking machine at about 60°C for 15-30 minutes. The baking time can be extended for special samples.

[0056] (4) After baking, place the slides in the Leica ST5010 automatic stainer for staining.

[0057] (5) The reagent sequence and staining time for Leica ST5010 automatic staining machine are as shown in Table 3: Table 3 (6) After staining, the sections were moved to the Leica CV5030 coverslip machine for sealing.

[0058] (7) Observation under a microscope: Samples with tumor cell ratio <30% and necrosis ratio >20% were eliminated. Figure 3 The following are examples of HE staining results of tumor tissues. a is a schematic diagram of an unqualified tumor sample, with a tumor cell content of <30% and a necrosis ratio of >20%; b is a schematic diagram of a qualified tumor sample, with a tumor cell content of ≥30% and a necrosis ratio of ≤20%.

[0059] Step 5: Immunohistochemistry (IHC): (1) Tissue sectioning: Place the wax block obtained above on a cold stage for 20 minutes, and then place it on a microtome for sectioning. First, trim the wax block: In the trimming mode, the thickness of the trimmed slices can be adjusted to 15-20μm. After trimming the wax block until all tissues are exposed, adjust the slice thickness to about 4μm, and then section. The slices must be complete, uniform, without knife marks, wrinkles, and cracks.

[0060] (2) Spreading the slice: Use a brush to gently pick up the slice, and use tweezers to move it to the water surface in the slide spreader to spread it (the temperature is 45°C. During the process of transferring the slice to the water surface of the slide spreader, the slice can be rotated but not flipped).

[0061] (3) Fishing out the slice: Place the unfolded slice on a glass slide. Hold the frosted area of ​​the slide with your right hand when fishing out the slice. Try to avoid bubbles between the slice and the slide. Tilt the slide to 45 degrees to allow excess water on the slice to flow off.

[0062] (4) Baking: Place the slide on a slide baking machine and bake at 60°C for 60 minutes to make tissue paraffin sections.

[0063] (5) Dewaxing: After baking, remove the paraffin sections and quickly transfer them to xylene: xylene I for 5 min → xylene II for 5 min → xylene III for 5 min.

[0064] (6) Hydration: Remove the slices from xylene, drain and transfer to ethanol with different concentration gradients for hydration: anhydrous ethanol I for 5 min → anhydrous ethanol II for 5 min → 95% ethanol for 5 min → 75% ethanol for 5 min, and then wash with water.

[0065] (7) Antigen repair: Add an appropriate amount of antigen repair solution (Shenzhen DaMeng, Guangdong Shenxiebei 20221796) into the staining jar for heat repair. Boil for 10 minutes. After natural cooling, rinse with PBS buffer three times, each time for 3 minutes.

[0066] (8) Blocking: Shake off excess PBS buffer from the slices, add endogenous peroxidase blocking reagent, drip 100 μl onto the tissue, incubate at 37°C for 10 min, and rinse with PBS buffer three times, 3 min each time.

[0067] (9) Primary antibody incubation: Shake off excess PBS buffer from the sections, add 100 μl of diluted PD-L1 antibody, incubate at 37°C for 1 h, and rinse with PBS buffer three times, 3 min each time.

[0068] (10) Secondary antibody incubation: Shake off excess PBS buffer from the sections, add 100 μl of secondary antibody and incubate at room temperature for 20 min. Rinse with PBS buffer three times, 3 min each time.

[0069] (11) BAB color development: Shake off excess PBS buffer from the slices, add 100 μl of DAB mixture (liquid A:liquid B = 1:1), incubate for 5-8 min, and wash with water to terminate color development.

[0070] (12) Re-staining the nucleus: shake off excess liquid from the slices, add 100 μl of hematoxylin staining solution, incubate for 20-60 seconds (the staining time can be adjusted according to actual conditions), and rinse with running water for 5 minutes.

[0071] (13) Differentiation (if necessary, depending on the properties of hematoxylin): Immerse the slices in differentiation solution for 2-5 seconds, then quickly remove the slices and rinse the slides with tap water for 5 minutes.

[0072] (14) Dehydration and transparency: Shake off the water used to rinse the slices, and dehydrate with gradient ethanol in the following order: 75% ethanol: 5 min → 95% ethanol: 5 min → anhydrous ethanol: 5 min. Place the slices taken out from anhydrous ethanol in xylene I for 5 min → xylene II for 5 min → xylene III for 5 min for transparency (this process is carried out in a fume hood).

[0073] (15) Neutral gum sealing: Take out the slices and place them in a fume hood. Before the xylene evaporates, add a drop of neutral gum and cover with a coverslip (this process is carried out in a fume hood).

[0074] (16) Observation under a microscope: Select tissue samples with PD-L1 results as negative, medium expression, and high expression, respectively.

[0075] Example 2: A PDTX tumor tissue tumor tissue PD-L1 gradient quality control sheet and its preparation method (1) Preparation of wax blocks: Use a tissue chip array to remove 1 mm of HIS-PDTX tumor biopsy tissue or hPBMC-PDTX tumor biopsy tissue that stably expresses PD-L1 (negative, medium expression, and high expression) and place it in a precast wax block. Place the wax block with tissue into a stainless steel bottom mold, paying attention to the direction: the cut surface faces down, and place it in a 70°C oven for 30-60 minutes. When the wax block is completely transparent, remove the wax block. Place the tissue embedding box on top of the stainless steel mold, continue to inject wax until the bottom of the box is completely immersed, and place it to cool.

[0076] (2) Tissue sectioning: Place the wax block on a cold stage for 20 minutes, then place it on a microtome for sectioning. Trim the wax block first: In the trimming mode, the thickness of the trimmed slices can be adjusted to 15-20 μm. After trimming the wax block until all three tissues are exposed and on the same plane, adjust the slice thickness to about 4 μm, and then section. The slices must be complete, uniform, without knife marks, wrinkles, or cracks.

[0077] (3) Spreading the slice: Use a brush to gently pick up the slice, and use tweezers to transfer the slice to the water surface in the slice spreader and spread it (temperature 45°C. During this process, it is necessary to ensure that the slice is not flipped).

[0078] (4) Fishing out the slice: Place the fully unfolded slice on an adhesive slide. When fishing out the slice, hold the frosted area of ​​the slide with your right hand and try to avoid bubbles between the slice and the slide. After fishing out the slice, tilt the slide 45 degrees to allow excess water on the slice to flow out.

[0079] This embodiment provides a tumor tissue PD-L1 gradient quality control sheet, which includes a slide label area, a staining control area and a tissue patch area. Figure 4 The slide label area contains the name and product model of the target antibody PD-L1 corresponding to the immunohistochemical slide with control; the staining control area is fixed with 3 kinds of PDTX tumor living tissue samples with different PD-L1 staining gradients of negative, medium expression and high expression as controls; the staining control area is divided into 3 independent blocks from top to bottom, which are the staining control areas with negative PD-L1 staining gradient, medium expression PD-L1 staining gradient and high expression PD-L1 staining gradient; when immunohistochemical staining is required, the tissue section of the sample to be tested is adhered to the tissue patch area.

[0080] In some specific embodiments, the control tissue is stained simultaneously with the test tissue during the immunohistochemical staining process. Figure 5 The immunohistochemical staining images of the PD-L1 gradient quality control film of tumor tissue. a, b, and c are the immunohistochemical staining images of tumor tissue sections with negative, medium expression, and high expression of PD-L1, respectively.

[0081] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present application rather than to limit the scope of protection of the present application. Although the present application has been described in detail with reference to the preferred embodiments, ordinary technicians in this field should understand that the technical solution of the present application can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present application.

Claims

1. A method for preparing a tumor tissue PD-L1 gradient quality control sheet, characterized in that: The following steps are involved: S1. Based on the RNA-seq information characteristics of the transcriptome sequencing corresponding to the PD-L1 antibody and the interpretation methods of different PD-L1 antibodies in different tumors, the PDTX tumor biopsy samples that meet the characteristic requirements are screened out through the sample sequencing information stored in the PDTX tumor biopsy database; S2. Put the CD 34+ HSC cell fluid was transferred into C-NKG mice to construct a C-NKG mouse humanized immune system HIS model, and the PDTX tumor living tissue obtained in step S1 was inoculated into the C-NKG mouse humanized immune system HIS model for passage and amplification to obtain HIS-PDTX tumor living tissue with stable expression of PD-L1; Alternatively, the PDTX tumor biopsy sample obtained in step S1 is inoculated into C-NKG mice, and peripheral blood mononuclear cells are transferred into the mice to obtain hPBMC-PDTX tumor biopsy with stable expression of PD-L1; S3, fix the HIS-PDTX tumor biopsy tissue or hPBMC-PDTX tumor biopsy tissue obtained in step S2 as a sample of the alternative control area, prepare a wax block, stain and detect, screen out tumor biopsy tissues with tumor cells ≥30% and necrosis ratio ≤20%, and interpret the PD-L1 expression level of the HIS-PDTX tumor biopsy tissue or hPBMC-PDTX tumor biopsy tissue; S4, preparing wax blocks and slices of the HIS-PDTX tumor biopsy tissue or hPBMC-PDTX tumor biopsy tissue screened in step S3, and attaching the slices to the control area on the adhesive slide with a printed label to obtain a tumor tissue PD-L1 gradient quality control film; The tumor tissue PD-L1 gradient quality control sheet includes a glass slide label area, a staining control area and a tissue patch area, wherein the glass slide label area contains the name and product model of the target antibody PD-L1; the staining control area is fixed with three types of HIS-PDTX tumor biopsy tissue or hPBMC-PDTX tumor biopsy tissue samples, which serve as controls, including negative PD-L1 staining gradients, medium expression PD-L1 staining gradients and high expression PD-L1 staining gradients.

2. The method for preparing the tumor tissue PD-L1 gradient quality control sheet according to claim 1, characterized in that: In step S1, the TPM normalized value of the transcriptome sequencing corresponding to the PDTX tumor biopsy sample with a negative PD-L1 staining gradient is <5; the TPM normalized value of the transcriptome sequencing corresponding to the PDTX tumor biopsy sample with a medium expression PD-L1 staining gradient is 5-50; the TPM normalized value of the transcriptome sequencing corresponding to the PDTX tumor biopsy sample with a high expression PD-L1 staining gradient is >50.

3. The method for preparing the tumor tissue PD-L1 gradient quality control sheet according to claim 1, characterized in that: In step S1, the PD-L1 antibody includes but is not limited to at least one of 22C3 antibody, SP142 antibody, SP263 antibody, WD160 antibody, E1L3N antibody, OR-5E3 antibody, and MXR-003 antibody; The tumor is not limited to at least one of non-small cell lung cancer, urothelial carcinoma, cervical cancer, and triple-negative breast cancer; The determination method includes a TPS determination method, a CPS determination method, an IC determination method or a TC+IC determination method.

4. The method for preparing the tumor tissue PD-L1 gradient quality control sheet according to claim 3, characterized in that: The 22C3 antibody is analyzed by TPS in non-small cell lung cancer, and the 22C3 antibody is analyzed by CPS in urothelial carcinoma or cervical cancer; The SP142 antibody uses an IC interpretation method in triple-negative breast cancer and urothelial carcinoma; The SP142 antibody adopts the TC+IC interpretation method in non-small cell lung cancer; The SP263 antibody adopts the TC+IC interpretation method in urothelial carcinoma.

5. The method for preparing the tumor tissue PD-L1 gradient quality control sheet according to claim 1, characterized in that: In step S2, the CD34 + The concentration of HSC cell solution was 5×10 5 cells / ml, injection volume was 5×10 4 cells; The concentration of peripheral blood mononuclear cells was 5×10 6 cells / ml, injection volume was 5×10 5 cells.

6. The method for preparing the tumor tissue PD-L1 gradient quality control sheet according to claim 1, characterized in that: The step S2 is specifically as follows: C-NKG mice within 3 weeks were irradiated with X-rays at 100 cGy / min for 2.4 min to ablate the myeloablation. Within 4 to 24 hours after myeloablation, the concentration of 5×10 5 CD34 cells / ml + HSC cells; after supplementation with CD34 + Starting from the fourth week after HSC cells were generated, immune cells in the peripheral blood of mice were detected by flow cytometry every two weeks. + When the ratio is ≥25%, the humanized immune system HIS model of C-NKG mice was successfully constructed. The PDTX tumor biopsy samples obtained in step S1 were cut into 2×2×2 mm 3 , inoculated into the C-NKG mouse humanized immune system HIS model, and waited for the transplanted tumor to grow to a cumulative volume of 800-1000mm 3 , obtain HIS-PDTX tumor biopsies with stable PD-L1 expression as alternative control area samples; Alternatively, take 5-8 week old C-NKG mice and cut into 2 × 2 × 2 mm 3 PDTX tumor biopsy samples, the transplanted tumor volume accumulates to 500~800mm 3 100 μl of 5×10 6 hPBMC-PDTX tumor biopsies with stable expression of PD-L1 were obtained as alternative control samples.

7. The method for preparing the tumor tissue PD-L1 gradient quality control sheet according to claim 1, characterized in that: In the step S3, the staining includes HE staining or immunohistochemistry staining (IHC).

8. The tumor tissue PD-L1 gradient quality control sheet prepared by the method for preparing the tumor tissue PD-L1 gradient quality control sheet according to any one of claims 1 to 7, characterized in that: The tumor tissue PD-L1 gradient quality control film contains lymphocytes that are positive for PD-L1 expression.

9. Use of the tumor tissue PD-L1 gradient quality control sheet according to claim 8 in detecting PD-L1 clinical samples, characterized in that: The clinical sample is a non-living tissue sample.

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