CN111521799A - Immunofluorescence kit for detecting esophageal squamous carcinoma patient PD-L1 gene expression through peripheral blood circulation tumor cells - Google Patents

Abstract

The invention provides an immunofluorescence kit for detecting esophageal squamous cell carcinoma patient PD-L1 gene expression through peripheral blood circulation tumor cells, which belongs to the field of molecular biology, and specifically comprises: goat serum, primary antibody suspension consisting of mouse anti-CK, rat anti-CD 45 and rabbit anti-PD-L1 genes, and secondary antibody suspension consisting of fluorescence-labeled goat anti-mouse, fluorescence-labeled goat anti-rat and fluorescence-labeled goat anti-rabbit. The detection method mainly comprises the steps of collecting peripheral blood, processing the peripheral blood, filtering and enriching circulating tumor cells, and detecting the expression condition of the circulating tumor cells PD-L1 gene by an immunofluorescence method.

Description

Immunofluorescence kit for detecting esophageal squamous carcinoma patient PD-L1 gene expression through peripheral blood circulation tumor cells

Technical Field

The invention provides an immunofluorescence kit for peripheral blood circulation tumor cell PD-L1 gene expression of an esophageal squamous carcinoma patient and a detection method, belonging to the technical field of molecular biology.

Background

Esophageal cancer is one of the most common digestive tract malignancies in the world, accounting for about 2% of all malignancies. The pathological types of esophageal cancer mainly include esophageal squamous cell carcinoma (esophageal squamous carcinosoma ESCC) and esophageal adenocarcinoma (esophageal adenocarinoma EADC), and more than about 90 percent of esophageal cancer patients in China are esophageal squamous cell carcinoma. With the continuous improvement of esophageal cancer diagnosis technology, the continuous improvement of operation technology, the application of new preoperative auxiliary chemotherapy and the application of postoperative regular radiotherapy and chemotherapy, the life quality of esophageal cancer patients is gradually improved to a certain extent, but the effect of esophageal cancer treatment is still unsatisfactory on the whole, and the total survival rate of esophageal cancer patients in 5 years in China is only 20-30%.

Immunotherapy with PD-1/PD-L1 as an immune target has brought new eosin for treatment of esophageal squamous carcinoma. The research finds that the immunosuppression is closely related to immune escape and the over-expression of tumor cells PD-L1, and the tumor cells can be combined with PD-1 on the surface of T cells of immune cells through PD-L1 on the surface of the tumor cells to transmit inhibitory signals, so that the T cells cannot recognize the tumor cells and send attack signals to the tumor cells, and the immune escape of the tumor cells is caused. According to the research of literature, the current detection of the esophageal squamous carcinoma circulating tumor cell PD-L1 is not reported at home and abroad. Therefore, the detection of the expression condition of the Circulating Tumor Cell (CTC) PD-L1 has important value for the prognosis of esophageal squamous cell carcinoma and the evaluation of the curative effect of immunotherapy.

Circulating Tumor Cells (CTC) are tumor cells that have been shed from solid tumors into the peripheral blood circulation, and since their discovery in 1989, various methods have been used to detect Circulating tumor cells in the peripheral blood. Recent studies have shown that their detection is of great clinical significance for assessing the prognosis of patients with tumours, especially patients with advanced tumours, and for selecting appropriate individualized treatments. CTC detection is called liquid biopsy of tumor because of its characteristics of minimal invasion, real-time detection, etc.

The immunofluorescence analysis technology is a method for researching the distribution of specific protein antigens in cells by combining an immunological method (antigen-antibody specific binding) with a fluorescence labeling technology. The fluorescence emitted by the fluorescein can be detected under a fluorescence microscope, the fluorescein emits bright fluorescence (yellow green or orange red) under the irradiation of exciting light, the cells or tissues where the fluorescence is located can be seen, and the content is measured by using a quantitative technology, so that the cells are qualitatively and locally analyzed.

Aiming at the current clinical practice, the PD-L1 gene detection specimen of the esophageal squamous carcinoma patient is mainly tumor tissue, is from operation or puncture biopsy, and is difficult to detect for many times or in real time. Therefore, the detection of the expression condition of the PD-L1 gene of the Circulating Tumor Cell (CTC) has important value for the prognosis of esophageal squamous cell carcinoma and the evaluation of the curative effect of immunotherapy.

At present, units such as Shandong province first medical university, Shandong province drug research institute combined with Shandong Qixin Biotechnology limited company, Shandong well-known Biotechnology limited company, Jinan Xin Biotechnology limited company, Shandong discovery biotechnology limited company and the like, carry out industrialized popularization on the key technology of detection and identification of circulating tumor cells, the project is a Shandong province major scientific and technological innovation project, the project takes the Shandong province drug research institute in Jinan school of Shandong first medical university as the core, realizes a registration system, relies on the core diagnostic technology of detection and identification of circulating tumor cells, and further registers, identifies and diagnoses a kit, and comprises PD1, PD-L1, ER, PR, Her-2, GPC-3, VEGF, P53, Vimentin, EGFR, RAS, CK, ALK-D5F3, CD20, ALK/EML4, Beta-catendin, E-Cacatenin, EP-CAM, HPV, IDH-1, PSA, PSMA, VEGF, GFAP, cytokeratin, AE1/AE3, estrogen receptor, progestin receptor, BCA-225, CA 125, CEA, EMA, ERCC1, HPV, Ki-67, P53, TOP2A and the like are used as tracers for expression of CTCs, and the identification and diagnosis kit is registered to be an ultrasensitive, ultrafast, high-coverage, low-cost, accurate and specific kit, and industrially popularized by cooperating with Shandong Qicheng Xin Biotech Co., Ltd, Shandong well-known Biotech Co., Ltd, Jinan En Biotech Co., Ltd, Shandong discovery Biotech Co., Ltd registered in Jinan.

Disclosure of Invention

Aiming at the defects that a patient with advanced tumor or recurrent esophageal squamous carcinoma cannot obtain a tissue sample by real-time or repeated puncture and further cannot evaluate the real-time dynamic state of PD-L1 gene of the patient and false positive and false negative easily occur in the existing detection method in the prior art, the invention provides an immunofluorescence kit and a detection method for the expression of PD-L1 gene of peripheral blood circulating tumor cells of the patient with esophageal squamous carcinoma, wherein the Circulating Tumor Cells (CTC) in the peripheral blood of the patient with advanced esophageal squamous carcinoma are obtained by separation of a membrane filtering device, and the expression condition of the PD-L1 gene on the CTC is further detected by using the immunofluorescence technology.

The invention is realized by the following technical scheme:

an immunofluorescence kit for detecting the expression of PD-L1 genes of peripheral blood circulation tumor cells of patients with esophageal squamous carcinoma comprises 45mL of diluent, 1mL of destaining solution, 0.5mL of staining solution A, 1mL of staining solution B, 200 μ L of methanol, 200 μ L of 2% PFA, 100 μ L of 10% goat serum, 100 μ L of primary antibody suspension consisting of mouse anti-CK, rat anti-CD 45 and rabbit anti-PD-L1 genes, 100 μ L of secondary antibody suspension consisting of fluorescence-labeled goat anti-mouse, fluorescence-labeled goat anti-rat and fluorescence-labeled goat anti-rabbit, and a DAPI sealing tablet;

in the primary anti-suspension, the genes of mouse anti-CK, rat anti-CD 45 and rabbit anti-PD-L1 are respectively diluted according to the ratio of 1:100, 1:400 and 1:500, and the total volume is 100 mu L;

and diluting the secondary antibody suspension with fluorescence-labeled goat-anti mouse, fluorescence-labeled goat-anti rat and fluorescence-labeled goat-anti rabbit at a ratio of 1: 500.

Wherein the diluent consists of 1mmol/L EDTA +1mmol of monthly silicate +0.1% BSA +0.2% poloxamer.

Wherein the destaining solution consists of 95% alcohol and 100% xylene according to the volume ratio of 1: 1.

Wherein the staining solution A is a DAB staining solution; the staining solution B is hematoxylin staining solution.

The method for detecting the PD-L1 gene expression of the peripheral blood circulation tumor cells of the esophageal squamous carcinoma patient by using the kit in a non-diagnosis purpose comprises the following steps:

(1) separating and obtaining peripheral blood of patients with advanced or recurrent esophageal squamous carcinoma who can not obtain tissue specimens by using a membrane filtering device: collecting 5ml of peripheral blood of the median cubital vein of a patient with advanced or recurrent esophageal squamous cell carcinoma, wherein the tissue specimen cannot be obtained;

(2) peripheral blood sample pretreatment: diluting the collected peripheral blood sample by 10 times by using a diluent, and adding polyformaldehyde to fix the peripheral blood sample for 10 minutes after dilution, wherein the fixed final concentration is 0.25%;

(3) and (3) filtering the peripheral blood sample by using a membrane filtration tumor cell separation device, and separating to obtain peripheral blood CTC: adding the pretreated peripheral blood sample into a blood sample container of a membrane filtration tumor cell separation device, and naturally filtering the blood sample by means of gravity;

(4) after the filtration is finished, taking the filter out of the membrane filtration tumor cell separation device, adding 0.5ml of circulating tumor cell staining solution A into the filter, staining for 3min, and washing with PBS buffer solution; after the filtrate is completely filtered, 1ml of staining solution B is added, the dyeing is carried out for 2min, and 1ml of pure water is used for washing for 2 times;

(5) adding 200 μ l of 2% PFA into the filter, fixing at room temperature for 5min, and rinsing with 0.5ml PBS for 3 times, each for 2 min;

(6) adding 200 μ l of precooled methanol into the filter, fixing at 4 ℃ for 15min, taking down the filter membrane, placing on a glass slide, drying, and observing under a microscope to determine whether CTC exists;

(7) and detecting the PD-L1 gene expression condition of the CTC in the peripheral blood by using an immunofluorescence method.

Wherein, the specific method for detecting the PD-L1 gene expression of the CTC in the peripheral blood by using immunofluorescence in the step (7) is as follows:

(1) and (3) decoloring: taking down the filter membrane with CTC from the glass slide, soaking in a decolorizing solution for 4-6 hours, removing the CTC staining solution, and washing with PBS for 2min × 3 times;

(2) and (3) sealing: dropping 100 μ l10% goat serum onto the filter membrane, standing at room temperature for 30min, and removing excess serum (diluting goat serum with PBS);

(3) primary antibody incubation: dripping 100 μ L primary antibody suspension composed of mouse anti-CK, rat anti-CD 45 and rabbit anti-PD-L1 genes on the filter membrane, incubating at 37 deg.C for 1h or overnight at 4 deg.C, and washing with PBS for 3min × 3 times;

(4) and (3) secondary antibody incubation: dripping 100 μ l of secondary antibody suspension composed of fluorescence labeled goat-anti mouse, fluorescence labeled goat-anti rat, and fluorescence labeled goat-anti rabbit onto the filter membrane, incubating at room temperature for 30min, and washing with PBS for 2min × 3 times;

(5) sealing the piece by using a sealing agent containing DAPI, reading the piece and collecting a drawing;

(6) after the light collection was completed, the sections were removed and then stained with giemsa renbergii, which was compared with the IF results.

The device for separating and circulating tumor cells by membrane filtration comprises a filter, a blood sample container, a waste liquid tank and an iron stand, wherein the iron stand is provided with a base, a vertical frame and a support, the blood sample container is arranged at the upper part of the iron stand through the support, the filter is arranged below the blood sample container, the filter is communicated to the waste liquid tank through an infusion apparatus, and the waste liquid tank is arranged on the base.

The filter comprises a filter upper opening, a filter membrane carrying platform and a filter lower opening, and the filter membrane is arranged on the filter membrane carrying platform; the upper port of the filter is connected with a blood sample container, and the lower port of the filter is connected with a waste liquid cylinder through an infusion apparatus.

The filter membrane is made of hydrophobic material, and filter holes with the caliber of 8 microns are uniformly distributed on the filter membrane; tumor cells are typically greater than 15 microns in diameter, while blood cells (including red blood cells, white blood cells) are typically less than 8 microns in diameter, so that when peripheral blood containing CTCs is filtered, the blood cells are filtered by being smaller in diameter than the filter pores, and the CTCs are retained on the filter membrane by being larger in diameter than the filter pores.

Advantageous effects

(1) The detection method provided by the invention can detect the PD-L1 gene expression condition of a late stage or recurrent esophageal squamous cell carcinoma patient without obtaining a tissue sample by puncture biopsy, and can realize real-time dynamic detection by utilizing a minimally invasive technology.

(2) The method provided by the invention has the advantages of good separation of circulating tumor cells, capability of avoiding the interference of blood cells, capability of avoiding false positive results caused by edge effect possibly generated in the dyeing process, good stability, reduction of cell loss and improvement of detection accuracy.

Drawings

FIG. 1 is a schematic structural view of a membrane filtration apparatus according to the present invention;

FIG. 2 is a schematic sectional view showing the structure of a filter of the membrane filtration apparatus of the present invention;

FIG. 3 is a schematic view showing the structure of a filter membrane of the membrane filtration apparatus of the present invention;

FIG. 4 is a diagram of an image of circulating tumor cells isolated from peripheral blood of a patient with esophageal squamous carcinoma;

FIG. 5 is an immunofluorescence staining image of PD-L1 gene in peripheral blood circulating tumor cells of patients with advanced esophageal squamous carcinoma, wherein A is merge, B shows target gene expression (red), C shows CK (green), and D shows CD45 (blue).

In the figure: 1 iron stand, 2 blood sample containers, 3 filters, 4 transfusion devices, 5 waste liquid jars, 6 filter upper ports, 7 filter membranes, 8 filter membrane platforms, 9 filter lower ports, 10 filter holes, 11 bases, 12 vertical frames and 13 supports.

Detailed Description

The invention is elucidated below with reference to the figures and embodiments.

The specific specification of the immunofluorescence kit used in the embodiment of the invention is shown in table 1:

TABLE 1

The primary antibody suspension consists of mouse anti-CK, rat anti-CD 45 and rabbit anti-PD-L1 genes, the mouse anti-CK, rat anti-CD 45 and rabbit anti-PD-L1 genes are respectively diluted by BD wash buffer according to the ratio of 1:100, the ratio of 1:500 and the ratio of 1:400, and 10 mu L of mouse anti-CK, 50 mu L of rat anti-CD 45 and 40 mu L of rabbit anti-PD-L1 genes are taken to form the primary antibody suspension after dilution;

the secondary antibody suspension consists of a fluorescence-labeled goat-Anti mouse, a fluorescence-labeled goat-Anti rat and a fluorescence-labeled goat-Anti rabbit, which are respectively Alexa Fluor 546 gat Anti-mouse, Alexa Fluor 488 gat Anti-rat and Alexa Fluor 647 gat Anti-rabbitt sold in the market, and equal amounts of the three fluorescence-labeled secondary antibodies are respectively diluted by BD washbuffer according to a ratio of 1:500 and mixed uniformly to obtain the secondary antibody suspension.

The method is applied to the embodiment of separating, obtaining and identifying the peripheral blood circulation tumor cells of 10 cases of esophageal squamous carcinoma patients (15 cases of normal human samples are simultaneously detected as negative controls).

Example 1

Firstly, separating and acquiring CTCs in peripheral blood of patients with advanced or recurrent esophageal squamous carcinoma who cannot obtain tissue specimens by using a membrane filtration device, and determining whether the CTCs exist:

collecting 5ml of fasting 8-12 hours fasting blood from the median cubital vein, diluting peripheral blood with 45ml of diluent, and then adding 3ml of 4% paraformaldehyde to fix the diluted blood sample for 10 minutes;

at fixed intervals, a membrane filtration device was assembled: as shown in fig. 1, 2 and 3, the filter device comprises a filter 3, a filter membrane 7, a blood sample container 2, a waste liquid tank 5 and an iron stand 1;

wetting the filter 3 with 10ml of PBS, then adding the fixed peripheral blood sample into the blood sample container 2 of the membrane filtration device, allowing it to naturally filter by gravity, and the CTC being trapped on the filter membrane 7;

the tumor cells are typically larger than 15 microns in diameter, while the blood cells (including red blood cells, white blood cells) are typically smaller than 8 microns in diameter, so that when peripheral blood containing CTCs is filtered, the blood cells can be filtered by being smaller than filter pores 10, while the CTCs are retained on filter membrane 7 by being larger than filter pores 10. It should be noted here that the diluent plays a role in detackifying and dispersing, and the laurate and the poloxamer are used in combination to ensure that the blood cells and the CTCs are not adhered and are sufficiently dispersed in the diluent, so that the blood cells and the CTCs are effectively trapped by the filter membrane 7.

After the filtration is finished, taking the filter 3 from the filter device, opening and removing the upper opening 6 of the filter, adding 0.5ml of circulating tumor cell staining solution A into the filter, staining for 3min, and washing with PBS buffer solution; filtering the filtrate completely, adding solution B, 1ml, staining for 2min, and pure water 1ml, washing filter 3 with PBS buffer solution, taking down filter membrane 7 with ophthalmic forceps with cell surface facing upwards, and placing on glass slide;

the filters were dried and observed under a microscope to determine the presence of CTCs, with the results shown in table 2.

By observation, no CTCs were detected in 15 healthy volunteers; except that 2 patients with esophageal squamous carcinoma did not detect CTC (1 patient with late esophageal squamous carcinoma +1 patient with recurrent esophageal squamous carcinoma), CTC was detected in the other 8 patients (Table 1), and the positive rate of the detection was 80%.

TABLE 2 results of CTC assay in examples

Secondly, detecting the PD-L1 gene expression condition of the CTC by using an immunofluorescence technique:

taking down the filter membrane 7 carrying the CTC on the glass slide from the glass slide, soaking in a decoloration solution which is uniformly mixed by 95% alcohol and 100% dimethylbenzene according to the volume ratio of 1:1 for 4-6 hours, removing the CTC staining solution, and washing with PBS for 2min multiplied by 3 times; dripping 100 μ l10% goat serum, standing at room temperature for 30min, sucking off excessive serum, dripping 100 μ l primary antibody suspension onto the filter membrane, incubating at 37 deg.C for 1h, and washing with PBS for 3min × 3 times; then, 100 mul of secondary antibody suspension is dripped on the filter membrane, and the mixture is incubated for 30min at room temperature, and then washed for 2min multiplied by 3 times by PBS; sealing the piece by using a sealing agent containing DAPI, reading the piece and collecting a drawing; after the light collection was completed, the sections were removed and then stained with giemsa renbergii, which was compared with the IF results.

FIG. 5 is an immunofluorescence staining image of peripheral blood circulating tumor cells of a patient with advanced esophageal squamous carcinoma, which shows that the tumor cells have large cell size and abnormal karyoplasmic ratio according to the immunological and morphological performances, and the immunological performances are typical CTCs.

The detected circulating tumor cells are verified to be PD-L1 by applying immunofluorescence and compared with the result of the esophageal squamous carcinoma general specimen PD-L1, the difference is observed, the target treatment of the esophageal squamous carcinoma is guided mainly aiming at patients with negative general specimen PD-L1 and positive circulating tumor cell expression, and a new idea is provided for the target treatment of the esophageal squamous carcinoma.

Claims (6)

1. An immunofluorescence kit for detecting the expression of PD-L1 genes of peripheral blood circulation tumor cells of patients with esophageal squamous carcinoma is characterized by comprising 45mL of diluent, 1mL of destaining solution, 0.5mL of staining solution A, 1mL of staining solution B, 200 μ L of methanol, 200 μ L of 2% PFA, 100 μ L of 10% goat serum, 100 μ L of primary antibody suspension consisting of mouse anti-CK, rat anti-CD 45 and rabbit anti-PD-L1 genes, 100 μ L of secondary antibody suspension consisting of fluorescence-labeled goat anti-mouse, fluorescence-labeled goat anti-rat and fluorescence-labeled goat anti-rabbit, and 100 μ L of DAPI encapsulated tablets;

in the primary anti-suspension, the genes of mouse anti-CK, rat anti-CD 45 and rabbit anti-PD-L1 are respectively diluted according to the ratio of 1:100, 1:400 and 1:500, and the total volume is 100 mu L;

and diluting the secondary antibody suspension with fluorescence-labeled goat-anti mouse, fluorescence-labeled goat-anti rat and fluorescence-labeled goat-anti rabbit at a ratio of 1: 500.

2. The kit according to claim 1, characterized in that said diluent consists of 1mmol/L EDTA +1mmol of monthly silicate +0.1% BSA +0.2% poloxamer.

3. The kit of claim 1, wherein the destaining solution is comprised of 95% alcohol to 100% xylene in a volume ratio of 1: 1.

4. The kit according to claim 1, wherein the staining solution A is DAB staining solution; the staining solution B is hematoxylin staining solution.

5. A method for detecting the expression of PD-L1 gene in peripheral blood circulating tumor cells of an esophageal squamous carcinoma patient with the use of the kit of any one of claims 1 to 4 for non-diagnostic purposes, which comprises the following steps:

(1) separating and obtaining peripheral blood of patients with advanced or recurrent esophageal squamous carcinoma who can not obtain tissue specimens by using a membrane filtering device: collecting 5ml of peripheral blood of the median cubital vein of a patient with advanced or recurrent esophageal squamous cell carcinoma, wherein the tissue specimen cannot be obtained;

(2) peripheral blood sample pretreatment: diluting the collected peripheral blood sample by 10 times by using a diluent, and adding polyformaldehyde to fix the peripheral blood sample for 10 minutes after dilution, wherein the fixed final concentration is 0.25%;

(3) and (3) filtering the peripheral blood sample by using a membrane filtration tumor cell separation device, and separating to obtain peripheral blood CTC: adding the pretreated peripheral blood sample into a blood sample container of a membrane filtration tumor cell separation device, and naturally filtering the blood sample by means of gravity;

(4) after the filtration is finished, taking the filter out of the membrane filtration tumor cell separation device, adding 0.5ml of circulating tumor cell staining solution A into the filter, staining for 3min, and washing with PBS buffer solution; after the filtrate is completely filtered, 1ml of staining solution B is added, the dyeing is carried out for 2min, and 1ml of pure water is used for washing for 2 times;

(5) adding 200 μ l of 2% PFA into the filter, fixing at room temperature for 5min, and rinsing with 0.5ml PBS for 3 times, each for 2 min;

(6) adding 200 μ l of precooled methanol into the filter, fixing at 4 ℃ for 15min, taking down the filter membrane, placing on a glass slide, drying, and observing under a microscope to determine whether CTC exists;

(7) and detecting the PD-L1 gene expression condition of the CTC in the peripheral blood by using an immunofluorescence method.

6. The method of claim 5, wherein the step (7) of using immunofluorescence to detect the expression of PD-L1 gene in CTC in peripheral blood comprises the following steps:

(1) and (3) decoloring: taking down the filter membrane with CTC from the glass slide, soaking in a decolorizing solution for 4-6 hours, removing the CTC staining solution, and washing with PBS for 2min × 3 times;

(2) and (3) sealing: dropping 100 μ l10% goat serum onto the filter membrane, standing at room temperature for 30min, and removing excess serum (diluting goat serum with PBS);

(3) primary antibody incubation: dripping 100 μ L primary antibody suspension composed of mouse anti-CK, rat anti-CD 45 and rabbit anti-PD-L1 genes on the filter membrane, incubating at 37 deg.C for 1h or overnight at 4 deg.C, and washing with PBS for 3min × 3 times;

(4) and (3) secondary antibody incubation: dripping 100 μ l of secondary antibody suspension composed of fluorescence labeled goat-anti mouse, fluorescence labeled goat-anti rat, and fluorescence labeled goat-anti rabbit onto the filter membrane, incubating at room temperature for 30min, and washing with PBS for 2min × 3 times;

(5) sealing the piece by using a sealing agent containing DAPI, reading the piece and collecting a drawing;

(6) after the light collection was completed, the sections were removed and then stained with giemsa renbergii, which was compared with the IF results.

Images