CN108507992A - The detection method of circulating tumor cell surface markers PD-L1 - Google Patents

Abstract

The invention discloses a kind of detection methods of circulating tumor cell surface markers PD L1.It includes the following steps：(1) whole blood is handled with erythrocyte cracked liquid, isolates karyocyte, formaldehyde is used in combination to be fixed；(2) tumour immunity fluorescent marker anti-cytokeratin Ab anti CK are first passed through and carry out positive-selecting, it is incubated all cells with PD L1 primary antibodies, then it is incubated with the PD L1 secondary antibodies for being marked with FITC fluorophors, then all cells is marked with nucleus fluorescent dye DAPI；(3) using high-throughput polychrome imaging analysis, the optical filter of CY5, FITC and DAPI are selected, observes channel surface fluorescence color, the final detection realized to circulating tumor cell surface markers PD L1.Detection method is easy, quick, economical, noninvasive, high sensitivity, and specificity is good.

Description

Detection method of circulating tumor cell surface marker molecule PD-L1

technical field

The invention belongs to the technical field of molecular biology, in particular to a method for detecting the surface marker molecule PD-L1 of circulating tumor cells.

Background technique

Programmed death receptor-1 (PD-1) is the main immune checkpoint receptor, by binding to its ligand programmed death ligand-1 (PD-L1), it down-regulates the effector function of T cells, thus contributing to To maintain tolerance to tumor cells. Blocking these pathways by anti-PD-1 and anti-PD-L1 antibodies may help prevent this downregulation and allow T cells to maintain their antitumor properties and ability to mediate tumor cell death. There are currently three main inhibitors of PD-1 and PD-L1 on the market, pembrolizumab (trade name: Keytruda), Nibolumab (trade name: Opdivo) and Atezolizumab (trade name: Tecentriq), which can be used for melanoma, non-small cell Treatment of various cancers such as lung cancer and bladder cancer. However, not all patients can benefit from PD-1/PD-L1 inhibitor treatment, and PD-1/PD-L1 inhibitors can only produce durable tumor control effects in a small number of cancer patients. Therefore, detecting whether a patient has positive expression of PD-L1 can effectively help the patient choose the appropriate drug for treatment. At present, the detection method of PD-1/PD-L1 is mainly based on the detection of cellular protein level. In clinical practice, immunohistochemical method is mainly used, and tumor tissue obtained after surgery or puncture is used for section staining. The results of immunohistochemistry are closely related to the experience of pathologists. In addition to the staining technique, the specificity of the antibody is also particularly important. At present, the detection of PD-L1 in my country is relatively chaotic. First, the staining techniques and conditions are not uniform; second, the staining antibodies are diverse; third, the pathological evaluation standards are not yet unified. The value of the level is reduced. In addition, because immunohistochemistry must obtain tissue for detection, whether it is through surgery or puncture, it is an invasive operation that will cause harm to the patient. Therefore, we are in urgent need of a new non-invasive PD-L1 detection method with stable evaluation methods and standards.

Circulating tumor cells are shed from the primary tumor, enter the blood circulation, settle in distant organs or primary organs, and form metastases. As a hot field of liquid biopsy, CTC detection of circulating tumor cells has gradually emerged in clinical performance in tumor diagnosis, treatment and monitoring. It is currently the most promising means of non-invasive tumor diagnosis and real-time efficacy monitoring, and its clinical application value is extremely significant. Therefore, it will be of great significance to provide a method for detecting PD-L1 on the surface of circulating tumor cells.

Contents of the invention

The purpose of the present invention is to solve the above-mentioned deficiencies in the prior art and provide a detection method for circulating tumor cell surface marker molecule PD-L1, which is simple, fast, economical, non-invasive, high in sensitivity and good in specificity, and can solve the problem of Existing techniques detect deficiencies in PD-L1.

The technical solution of the present invention is specifically introduced as follows.

A method for detecting circulating tumor cell surface marker molecule PD-L1, comprising the following steps:

(1) Treat the whole blood with red blood cell lysate, separate the nucleated cells, and fix them with formaldehyde;

(2) Firstly, positive screening was performed by the tumor immunofluorescence marker cytokeratin antibody anti-CK, and all cells were incubated with PD-L1 primary antibody, and then incubated with PD-L1 secondary antibody labeled with FITC fluorescent group, and then nuclei Fluorescent dye DAPI marks all cells;

(3) Using high-throughput multi-color imaging analysis, select the filters of CY5, FITC and DAPI, observe the fluorescent color on the channel surface, and finally realize the detection of the circulating tumor cell surface marker molecule PD-L1.

Among the present invention, the specific reaction conditions of step (2) are as follows:

a. Add PBS solution containing 4-6% skimmed milk powder to the culture dish after cell fixation, and incubate at 4°C in the dark for 40-70min;

b. After absorbing the PBS solution of skimmed milk powder, add PBST along the wall to wash several times;

c. Add tumor immunofluorescent marker cytokeratin antibody anti-CK and PD-L1 primary antibody to the blocking solution. The source of the antibody is abcam company. After mixing evenly, add it to the culture dish along the wall, and incubate overnight at 4°C in the dark;

d. Aspirate the mixed solution, add PD-L1 secondary antibody labeled with FITC fluorescent group to the blocking solution, the source of the antibody is abcam, mix well, add it to the petri dish along the wall, and incubate at 4°C for 40-70min in the dark;

e. Absorb the antibody, wash several times with PBST, add DAPI, and incubate at room temperature in the dark for 20-40min.

In the present invention, in condition b, PBS containing 0.02 wt% Tween-20 in PBST.

In the present invention, in step (3), observe the fluorescent color on the surface of the channel. If it is blue, it is DAPI+; if it is not blue, it is DAPI-; if it is green, it is PD-L1+; if it is not green, it is PD-L1-; CK+, if there is no red color, CK-; according to the fluorescent color, the DAPI+ and CK+ points are considered as circulating tumor cell CTC, if there is PD-L1+ on the circulating tumor cell CTC, then the circulating tumor cell CTC is PD-L1+ CTC.

Compared with the prior art, the present invention combines the detection of CTC and the detection of PD-L1 markers for the first time, by lysing red blood cells in the blood, and using nanotechnology to tile and enrich the remaining nucleated cells (mainly CTCs and lymphocytes) The collection was fixed on the nano-substrate, and then all cells were labeled with the nuclear fluorescent dye DAPI, and then positively screened by the tumor immunofluorescent marker cytokeratin antibody anti-CK. Since the surface of CTC has the specific expression of PD-L1, PD -Incubate all cells with primary antibody L1, then incubate with secondary antibody labeled with FITC fluorophore, and finally scan through high-throughput technology to finally detect PD-L1, a marker molecule on the surface of circulating tumor cells. This detection method is simple and fast , economical, non-invasive, high sensitivity, and good specificity, which solves the shortcomings of the existing technology for detecting PD-L1.

Description of drawings

Figure 1 is a schematic diagram of detecting the positive expression of PD-L1 in circulating tumor cells.

Detailed ways

The present invention will be further described below in conjunction with specific examples.

Example 1

Figure 1 is a schematic diagram of detecting the positive expression of PD-L1 in circulating tumor cells. Figure 1a is the blue fluorescence channel, DAPI is used to mark the nucleus, and the blue color is DAPI+, indicating that it is a complete cell, and the blue color is not DAPI-, which is not a complete cell; Figure 1b is the green fluorescence channel, which is marked with PD-L1 If the protein is green, it is PD-L1+, and if it is not green, it is PD-L1-; Figure 1c is a red fluorescent channel, marking CK protein, if it is red, it is CK+, and if it is not red, it is CK-. Figure 1d shows the synthesis of the three channels.

This example is an example of detecting the expression of PD-L1 on the surface of circulating tumor cells in clinical liver cancer samples, which specifically includes the following steps:

1) Whole blood processing:

a. Add 200 μL of 1× red blood cell lysate to 2 mL of whole blood, place at room temperature for 15 min, and shake evenly during the period.

b. Centrifuge at 200RCF for 5 minutes, aspirate the supernatant and keep the cells.

c. Add 2 mL of 1% FPBS (V(FBS):V(PBS)=1:99) to the centrifuge tube, mix well, centrifuge at 200RCF for 5 minutes, and remove the supernatant.

2) Cell full enrichment:

a. Add 1mL FPBS and mix well, then transfer to the treated petri dish, and place the petri dish in a shaker at 37°C for 45min.

b. After culturing, place the petri dish in a refrigerator at 4°C for 1 min.

3) Cell fixation:

a. Aspirate the FPBS, add 4% formaldehyde to the petri dish and place it at 4°C for 1 min.

b. Suck off formaldehyde, add 1mL of methanol, and place at -20°C for 20min.

c. Aspirate the methanol and wash three times with 2mL PBS each time. (Note: Every time you add liquid, add it along the wall of the culture dish to avoid washing up the cells.

4) Blocking and antibody incubation:

a. Add 1 mL of 5% (m/V) skimmed milk powder containing 0.02% Tween-20 (prepared in PBS) to the culture dish, and incubate at 4° C. in the dark for 1 h.

b. After sucking off the skimmed milk powder, add 2 mL of PBST (PBS containing 0.02% Tween-20) along the wall to wash 4 times, 5 min each time.

c. Add 1 μL anti-CK (purchased from abcam company) to 500 μL blocking solution, then add 2 μL PD-L1 primary antibody (purchased from abcam company), mix well, add to the culture dish along the wall, and incubate overnight at 4°C in the dark .

d. Aspirate the mixed solution, add 5 μL of PD-L1 secondary antibody labeled with FITC fluorescent group (purchased from abcam company) to 500 μL of blocking solution, mix well, add to the culture dish along the wall, and incubate at 4 °C for 1 h in the dark.

e. Absorb the antibody, wash it three times with PBST, add 1mL cell nuclear dye DAPI (4,6-bisquined-2-phenylindole), and incubate at room temperature for 30min in the dark.

5) Scanning: Absorb the antibody, wash three times with 2mL PBST, add 1mL PBS to scan.

For the above-mentioned clinical liver cancer samples, high-throughput multi-color imaging analysis was used to select the filters of CY5, FITC and DAPI, and the fluorescence color on the surface of the channel was observed. If the color was blue, it was DAPI+, if it was not blue, it was DAPI-, and if it was green, it was PD-L1+, PD-L1- if no green, CK+ if red, CK- if no red, according to the fluorescence color, the "dot" of DAPI+ and CK+ is considered as CTC, if there is PD-L1+ on the CTC , then the CTC is PD-L1+CTC (Figure 1), otherwise, it is not. The detection method is simple, rapid, economical, non-invasive, high in sensitivity and good in specificity.

The present invention is not limited by the above-mentioned embodiments, and any other changes, modifications, substitutions, combinations, and simplifications that do not deviate from the spirit and principles of the present invention should be equivalent replacement methods and are included in the present invention. within the scope of protection.

Claims (4)

1. A method for detecting circulating tumor cell surface marker molecule PD-L1, comprising the following steps: (1) Treat the whole blood with red blood cell lysate, separate the nucleated cells, and fix them with formaldehyde; (2) Firstly, positive screening was performed by the tumor immunofluorescence marker cytokeratin antibody anti-CK, and all cells were incubated with PD-L1 primary antibody, and then incubated with PD-L1 secondary antibody labeled with FITC fluorescent group, and then nuclei Fluorescent dye DAPI marks all cells; (3) Using high-throughput multi-color imaging analysis, select the filters of CY5, FITC and DAPI, observe the fluorescent color on the channel surface, and finally realize the detection of the circulating tumor cell surface marker molecule PD-L1. 2. detection method according to claim 1, is characterized in that, the concrete reaction condition of step (2) is as follows: a. Add PBS solution containing 4-6% skimmed milk powder to the culture dish after cell fixation, and incubate at 4°C in the dark for 40-70min; b. After absorbing the PBS solution of skimmed milk powder, add PBST along the wall to wash several times; c. Add tumor immunofluorescent marker cytokeratin antibody anti-CK and PD-L1 primary antibody to the blocking solution. The source of the antibody is abcam company. After mixing evenly, add it to the culture dish along the wall, and incubate overnight at 4°C in the dark; d. Aspirate the mixed solution, add PD-L1 secondary antibody labeled with FITC fluorescent group to the blocking solution, the source of the antibody is abcam company, mix well, add to the culture dish along the wall, and incubate at 4°C for 40-70min in the dark; e. Absorb the antibody, wash several times with PBST, add DAPI, and incubate at room temperature in the dark for 20-40min. 3. The detection method according to claim 2, characterized in that, in condition b, PBS containing 0.02wt% Tween-20 in PBST. 4. The detection method according to claim 1, characterized in that, in step (3), observe the fluorescent color on the surface of the channel, if it is blue, it is DAPI+, if it is not blue, it is DAPI-, if it is green, it is PD-L1+, If it does not show green, it means PD-L1-, if it shows red, it means CK+, and if it does not show red, it means CK-; according to the fluorescent color, the point of DAPI+ and CK+ is regarded as circulating tumor cell CTC, if there is PD-L1+ on the circulating tumor cell CTC , then the circulating tumor cell CTC is PD-L1+CTC.