CN110927392B - Marker for detecting digestive system cancer and application thereof - Google Patents
Marker for detecting digestive system cancer and application thereof Download PDFInfo
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/574—Immunoassay; Biospecific binding assay; Materials therefor for cancer
- G01N33/57407—Specifically defined cancers
- G01N33/57446—Specifically defined cancers of stomach or intestine
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6848—Methods of protein analysis involving mass spectrometry
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
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- C07—ORGANIC CHEMISTRY
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- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70546—Integrin superfamily
- C07K14/70553—Integrin beta2-subunit-containing molecules, e.g. CD11, CD18
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- G01N2333/435—Assays involving biological materials from specific organisms or of a specific nature from animals; from humans
- G01N2333/705—Assays involving receptors, cell surface antigens or cell surface determinants
- G01N2333/70546—Integrin superfamily, e.g. VLAs, leuCAM, GPIIb/GPIIIa, LPAM
- G01N2333/70553—Integrin beta2-subunit-containing molecules, e.g. CD11, CD18
Abstract
The invention discloses a marker for detecting digestive system cancer and application thereof, wherein the marker is a combination of Vimentin and CD11 b. The expression conditions of the marker in different individuals show the unique phenomena of 'ultrahigh expression in peripheral blood of cancer patients, higher expression in peripheral blood of early cancer patients and almost no expression in peripheral blood of healthy volunteers', and the marker can be used for early screening of digestive system tumors such as gastric cancer and dominant population of solid tumor treatment and evaluating treatment curative effect.
Description
Technical Field
The invention belongs to the field of biomedicine, and particularly relates to a marker for cancer detection, in particular to a marker for cancer screening, cancer stage determination, cancer treatment effect evaluation or cancer prognosis evaluation.
Background
China is a country with high incidence of stomach cancer, the incidence rate of the stomach cancer in natural people is about 31.28/10 ten thousand, wherein the incidence rate of the stomach cancer in men is 42.93/10 ten thousand, the incidence rate of the stomach cancer in women is 19.03/10 ten thousand, and the age-standardized incidence rate of the men is twice that of the women; the mortality rate of stomach cancer in China is about 21.48/10 ten thousand, and the morbidity and mortality rate are 2 nd in malignant tumors. Early gastric cancer is asymptomatic, most patients are already in middle and late stages at the time of first diagnosis, and the optimal treatment opportunity is missed. The 5-year survival rate of the gastric cancer in the III and IV stages is lower than 20 percent, and if the early detection and the timely treatment can be realized, the 5-year survival rate of the patient with the early gastric cancer can reach 95 percent, so the early screening of the gastric cancer is particularly important. At present, the gastric cancer screening methods mainly comprise imaging, endoscopic means and serological examination, and various screening methods have the advantages and are not negligible.
Imaging examination is one of the earliest means for cancer examination, which is widely used in the diagnosis of various solid tumors, but lacks specificity and has limited sensitivity in early diagnosis: 1. the upper gastrointestinal barium double contrast radiography is an important method for diagnosing the gastric cancer, and in Japan, the upper gastrointestinal barium meal is a main method for screening the gastric cancer on a large scale, can observe the form of the gastric mucosa and the motion change of the gastric wall, plays a key role in screening the gastric cancer, but easily leaks out flat, non-sunken and other pathological changes. 2. CT is often used for preoperative staging and postoperative follow-up after the confirmation of gastric cancer. The CT arterial staged scanning can directly display the infiltration depth of the gastric cancer to the gastric wall, and can also evaluate the condition of peripheral organs, such as lymph node metastasis, liver or peritoneal metastasis and the like, and is not generally used for screening early gastric cancer. 3. MRI imaging is less studied in the assessment of early stage gastric cancer and, by virtue of its good tissue resolution, is increasingly being applied to the staging of tumors and, as such, is not generally used for screening of early stage gastric cancer. CT and MRI examinations are mostly used to evaluate the progression of gastric cancer or to predict the therapeutic efficacy, and lack the meaning of screening, while the contrast of upper gastrointestinal tract contrast lacks the efficacy due to its poor discrimination against small and early lesions. In addition, artifacts from motion of the upper abdominal gastrointestinal tract also pose a significant challenge to the accuracy of imaging screening for gastric cancer.
Endoscopy and biopsy are the most important and reliable methods for diagnosing gastric cancer. The effectiveness of screening gastric cancer by using an endoscope is obviously higher than that of upper gastrointestinal barium meal. Research shows that the detection rate of the endoscope on early gastric cancer is 2.7-4.6 times of upper gastrointestinal barium meal. One study in japan shows that endoscopy reduces gastric cancer mortality by 67% compared to imaging. In recent years, with the continuous improvement of endoscope equipment, technologies such as a dye endoscope, a magnifying endoscope, narrow-band imaging and the like are gradually added for observing pathological changes, and the accuracy of early gastric cancer and range diagnosis is greatly improved. However, the endoscope is an invasive examination, the cost is high, the discovery of early gastric cancer depends on the experience of an endoscope operator, electronic or chemical dyeing and endoscope amplification equipment, the endoscope is not suitable for large-scale screening of early gastric cancer, and the popularization is poor.
Serological screening: 1. serum levels of Pepsinogen (PG) I, PG II, and PG I/II ratio (PGR) reflect functional and morphological changes in different gastric mucosa. In Japan, the high risk group of gastric cancer can be well screened by using 'PG I <70 ug/L and PGR < 3' as the critical values, and an analysis shows that the sensitivity and specificity of gastric cancer patients can be respectively screened to 77.3% and 73.2% by using the critical values. 2. The ABC layering method combines the detection of serum PG I, PG II and serum Hp-IgG antibody to layer the risk of gastric cancer, namely the ABC method. PG positive (+) "PG I <70 ug/mL and PGR < 3" and Hp positive antibody titer ≧ 30U/mL "are defined as: group a PG (-), Hp (-); group B PG (-), Hp (+); group C PG (+), Hp (+); group D PG (+), Hp (-). The prospective study results showed that the RR values of the risk ratios of gastric cancer development in groups B, C and D were 1.1 (95% CI: 0.4-3.4), 6.0 (95% CI: 2.4-14.5) and 8.2 (95% CI: 3.2-21.5), respectively, as compared with group A. 3. Gastrin 17 (Gastrin-17, G-17) uses serum G-17 as another marker of gastric mucosa morphology and function, and is a new means for detection by combining serum PG and G-17. In the high incidence area of gastric cancer in China, a prospective study of combined serum PG I, PG II, PGR, Hp-IgG antibodies and G-17 shows that the reduction of PG I and PGR levels, the reduction of G-17 levels (< 0.5 pmol/L) and the increase of G-17 levels (> 4.7 pmol/L) indicate that the high risk of gastric cancer is existed. Although serological detection is relatively noninvasive, the serological detection is influenced by factors such as the incidence rate of regional stomach cancer, the type of gastric cancer, helicobacter pylori infection and the like, and the screening accuracy is deficient; and false negative easily occurs in patients who are taking proton pump inhibitors and have already been treated with Hp eradication; in addition, related research is carried out in a high incidence area of the gastric cancer, whether the serological index level is influenced by the high incidence rate of the gastric cancer needs to be further proved, and the missed detection risk is high.
Tumor marker detection: the japanese gastric cancer treatment protocol and treatment guidelines both recommend tumor marker examination for gastric cancer, including four items: CEA, CA19-9, CA125 and AFP, while the gastric cancer tumor markers in most hospitals in China comprise CEA, CA19-9, CA24-2 and CA 72-4. Currently, in clinical diagnosis, CEA and CA19-9 are 2 common serum markers for helping to diagnose gastric cancer, and CEA and CA19-9 have high correlation with the advanced stage of gastric cancer. However, CA19-9 proved to be a tumor-specific marker, and its expression level was increased in many hepatobiliary tumors and biliary obstruction diseases. Similar to CA19-9, the specificity of the CA125 and CA50 markers is also low. Research proves that the positive rate of the CA72-4 in the progressive gastric cancer serum is better than that of carcinoembryonic antigen (CEA) and CA19-9, but the positive rate in early gastric cancer is still low. The serum marker cannot have the characteristics of high specificity and high sensitivity at the same time, so that the positive detection rate of the indexes is low. In addition, although most researchers believe that joint detection can be used in clinical detection of gastric cancer, and the disadvantages of low specificity and sensitivity in single detection are avoided, the effect of joint detection is still not ideal at present.
Tadokoro et al.2016 discloses that Vimentin (Vimentin) modulates aggressiveness, a poor prognostic marker in non-small cell lung cancer; yoshihiro et al 2014 disclose that tumor-infiltrating CD11b + antigen presenting cells affect local tumor cell immune cell-cell interactions.
However, none of the above prior arts discloses Vimentin + CD11b + cell proportion, CD16+ cell proportion, and CD11c + cell proportion as a marker of gastric cancer.
Disclosure of Invention
The invention aims to solve the technical problem of overcoming the defects and shortcomings of the existing cancer screening technology, particularly gastric cancer screening technology, and provides a novel noninvasive blood cancer marker. The expression conditions of the marker in different individuals show the unique phenomena of 'ultrahigh expression in peripheral blood of cancer patients, higher expression in peripheral blood of early cancer patients and almost no expression in peripheral blood of healthy volunteers', and the marker can be used for early screening of digestive system tumors such as gastric cancer and dominant population of solid tumor treatment and evaluating treatment curative effect.
In a first aspect the invention provides the use of a marker which is a combination of Vimentin and CD11b in the manufacture of a product for the detection of cancer.
Preferably, the cancer is selected from digestive system cancers. Particularly preferably, the cancer is selected from gastric cancer, esophageal cancer and intestinal cancer.
Preferably, said product is used for qualitative or quantitative detection of said marker in a sample to be tested.
Preferably, said product is used for detecting the cell proportion of the positive expression of said marker in a sample to be tested.
Preferably, the sample to be tested is tissue or blood, and the tissue is cancer tissue or tissue beside cancer. More preferably, the sample to be tested is blood. Particularly preferably, the sample to be tested is peripheral blood.
Preferably, the product is detected by using a mass spectrometry flow detection method, an optical flow cytometry detection method, an immunohistochemistry method, an immunofluorescence staining method, an enzyme-linked immunosorbent assay, an immunomagnetic bead method or a PCR method. More preferably, the product is a product detected by mass spectrometry or optical flow cytometry.
Preferably, the cancer detection is selected from the group consisting of cancer screening, cancer stage determination, cancer treatment effect evaluation, cancer prognosis evaluation, and the like. The stage determination of cancer comprises early stage screening of cancer. The evaluation of the cancer treatment effect comprises the evaluation of the cancer treatment effect of chemotherapy, targeted therapy or immunotherapy. The immunotherapy comprises treatment with immune checkpoint inhibitors, such as inhibitors of PD-1, PD-L1, CTLA-4, and the like.
In one embodiment the invention provides the use of a marker in the manufacture of a product for cancer detection, said marker being a combination of Vimentin and CD11 b; the product is used for detecting the cell proportion of the positive expression of the marker in a sample to be detected, the sample to be detected is tissue or blood, and the cancer detection is selected from gastric cancer screening, early gastric cancer screening and gastric cancer treatment effect evaluation or gastric cancer prognosis evaluation.
In a second aspect the invention provides the use of a marker which is a combination of Vimentin and CD11b in the detection of cancer.
A third aspect of the invention provides a method of cancer screening comprising: collecting peripheral blood as a sample to be detected, preparing single cells of the collected sample to be detected, staining and fixing the single cells, marking and detecting, and detecting the cell proportion of positive expression of a marker, wherein the marker is the combination of Vimentin and CD11 b.
In one embodiment of the present invention, when the marker is a combination of Vimentin and CD11b, the ratio of Vimentin + CD11b + cells is less than 1%, the sample to be tested is from a healthy individual; the ratio of Vimentin + CD11b + cells is more than or equal to 1%, and the sample to be tested is from a cancer patient.
The fourth aspect of the invention provides a method for determining the pathological stage of cancer, which comprises the steps of collecting peripheral blood as a sample to be detected, preparing single cells of the collected sample to be detected, staining and fixing the single cells, then marking and detecting, and detecting the cell proportion of positive expression of a marker, wherein the marker is the combination of Vimentin and CD11 b.
In one embodiment of the present invention, when the marker is a combination of Vimentin and CD11b, the ratio of Vimentin + CD11b + cells is greater than or equal to 1% and less than 10%, the sample to be tested is from a patient with early gastric cancer; the ratio of Vimentin + CD11b + cells is more than or equal to 10%, and the sample to be tested is from a gastric cancer patient.
The fifth aspect of the invention provides a method for evaluating the treatment effect and prognosis evaluation of cancer, which comprises the steps of collecting peripheral blood of a cancer patient before and after treatment as a sample to be tested, preparing single cells of the collected sample to be tested, staining and fixing the single cells, marking and detecting, and detecting the proportion of cells positively expressed by a marker, wherein the marker is a combination of Vimentin and CD11 b.
In one embodiment of the present invention, in the method for evaluating the treatment effect and the prognosis evaluation, when the marker is a combination of Vimentin and CD11b, before the gastric cancer patient receives treatment, the ratio of Vimentin + CD11b + cells is more than 10%; after the gastric cancer patient receives treatment, the ratio of Vimentin + CD11b + cells is obviously reduced to below 5%, and the treatment effect of the sample to be detected is good.
A sixth aspect of the present invention provides a method for building a cancer detection model, including: collecting peripheral blood of healthy individuals and cancer patients as samples to be detected, preparing single cells of the collected samples to be detected, staining and fixing the single cells, then marking and detecting, detecting the cell proportion of positive expression of the marker in the samples to be detected, and determining the relevance of the marker and the cancer detection through cluster analysis and/or heat map analysis data. The cluster analysis and the heat map analysis are based on dimension reduction analysis viSNE, and data visualization SPADE is carried out to analyze the intracellular and extracellular expression conditions of the cell population.
Preferably, in the method for establishing the cancer detection model, the marker is a combination of Vimentin and CD11b, CD16 or CD11 c.
Preferably, in the method for establishing a cancer detection model, the cancer is a cancer of the digestive system. More preferably, the cancer is gastric cancer, esophageal cancer or intestinal cancer.
Preferably, in the method for establishing the cancer detection model, the cancer detection is selected from the group consisting of cancer screening, cancer stage determination, cancer treatment effect evaluation and cancer prognosis evaluation.
Preferably, in the method for establishing a cancer detection model, the correlation is selected from the group consisting of:
(1) when the cancer detection is cancer screening: the proportion of Vimentin + CD11b + cells is less than 1%, and the sample to be detected is from a healthy individual; the ratio of Vimentin + CD11b + cells is more than or equal to 1%, and the sample to be detected is from a cancer patient;
(2) when the cancer detection is determined as the stage of the cancer: the ratio of Vimentin + CD11b + cells is more than or equal to 1% and less than 10%, and the sample to be detected is from a patient with early cancer; the ratio of Vimentin + CD11b + cells is more than or equal to 10%, and the sample to be tested is from a cancer patient;
(3) when the cancer detection is the evaluation of the cancer treatment effect or the cancer prognosis evaluation: after the cancer patient receives the treatment, the Vimentin + CD11b + cell percentage is reduced to be below 5%, and the treatment effect of the sample to be detected is good.
The term CyTOF as used herein refers to Mass Cytometry (Mass Cytometry).
The term Vimentin as used herein refers to Vimentin.
The term Vimentin + refers to Vimentin positive expression, and Vimentin-refers to Vimentin negative expression; CD11b + refers to CD11b positive expression, CD11 b-refers to CD11b negative expression; CD16+ refers to CD16 positive expression, CD 16-refers to CD16 negative expression; CD11c + refers to CD11c positive expression, CD11 c-refers to CD11c negative expression.
Drawings
FIG. 1 is a cluster analysis plot and heat map of different cell populations in paired cancer tissues, paracarcinoma tissues and peripheral blood samples from patients with gastric cancer using CyTOF: FIG. 1a shows cancer tissue of a gastric cancer patient, FIG. 1b shows para-carcinoma tissue of a gastric cancer patient, FIG. 1c shows blood of a gastric cancer patient, and FIG. 1d shows blood of a healthy volunteer; the phenotypes represented by the numbers 1-20 cell populations in the figure are shown in the following table:
FIG. 2 is differential cell analysis of gastric cancer patients versus healthy volunteers: panel a is tissue from a gastric cancer patient, panel b is para-carcinoma tissue from a gastric cancer patient, panel c is blood from a gastric cancer patient, wherein the number 5 is Vimentin + CD11b + cell population, and panel d is blood from a healthy volunteer.
FIG. 3 shows the ratio of Vimentin +/CD11b-, Vimentin-/CD11b +, Vimentin +/CD11b + cells in the peripheral blood of healthy individuals, patients with early gastric cancer, and patients with gastric cancer.
FIG. 4 is a flow-through analysis of cell expression of Vimentin +/CD11b-, Vimentin-/CD11b +, Vimentin +/CD11b + in peripheral blood of healthy individuals, patients with early gastric cancer, and patients with gastric cancer: FIG. a shows that the ratio of Vimentin +/CD11b + cells is less than 1%, and the sample to be tested is from a healthy individual; FIG. b shows that the ratio of Vimentin +/CD11b + cells is greater than or equal to 1% and less than 10%, and the sample to be tested is from a patient with stomach early cancer; FIG. c shows that the ratio of Vimentin +/CD11b + cells is 10% or more, and the specimen to be tested is derived from a patient with gastric cancer.
FIG. 5 shows the ratio of Vimentin +/CD11b-, Vimentin-/CD11b +, Vimentin +/CD11b + cells in the peripheral blood before and after treatment of gastric cancer patients.
FIG. 6 is a flow-based analysis of results of expression of Vimentin +/CD11b-, Vimentin-/CD11b +, Vimentin +/CD11b + cells in peripheral blood before and after treatment of gastric cancer patients: FIG. a shows that before treatment, Vimentin +/CD11b + cells are present at a ratio of 10% or more; panel b shows that Vimentin +/CD11b + cell content decreased significantly to below 5% after treatment.
FIG. 7a shows the single-expression levels of 37 biomarkers in the population at 100% maximum on the horizontal axis, wherein Healthedono-pbmc represents healthy population-peripheral blood, cancer-pbmc represents gastric cancer-peripheral blood, cancer-ca represents gastric cancer-cancer tissue, and cancer-nor represents gastric cancer-paracancerous tissue.
FIG. 7b is the fraction of the total single-expression of partial-term biomarkers up to 25% on the horizontal axis, wherein healthdenor-pbmc represents healthy population-peripheral blood, cancer-pbmc represents gastric cancer-peripheral blood, cancer-ca represents gastric cancer-cancer tissue, and cancer-nor represents gastric cancer-paracancerous tissue.
FIG. 7c is a screening of intracellular and extracellular markers with positive expression: the indexes in 7a and 7b are expressed by a scatter diagram, the vertical axis is fixed as Vimentin, the horizontal axis is other 36 types of indexes, CD11b, CD45, HLA-ABC, CD16, CD11c and PD-L1 in the graph have positive expression, but only CD11b-Vimentin is taken as the horizontal and vertical coordinates, the cell groups (Vimentin + CD11b + group and Vimentin + CD11 b-group) are obvious, and when the Vimentin is taken as a screening index, the CD11b has unique sorting significance.
FIG. 8 shows the expression levels of the target biomarkers Vimentin, CD11b, CD16 and CD11c in various specimens, wherein, the health denor-pbmc represents healthy people-peripheral blood, the cancer-pbmc represents gastric cancer-peripheral blood, the cancer-ca represents gastric cancer-cancer tissue and the cancer-nor represents gastric cancer-para-cancer tissue.
FIG. 9a shows the suitability of two indicators, Vimentin-CD11b and Vimentin-CD16, wherein, healydonor-pbmc represents healthy people-peripheral blood, cancer-pbmc represents gastric cancer-peripheral blood, cancer-ca represents gastric cancer-cancer tissue, and cancer-nor represents gastric cancer-paracarcinoma tissue.
FIG. 9b is a verification of the applicability of two indicators, Vimentin-CD11c and CD11b-CD16, wherein, healydonor-pbmc represents healthy people-peripheral blood, cancer-pbmc represents gastric cancer-peripheral blood, cancer-ca represents gastric cancer-cancer tissue, and cancer-nor represents gastric cancer-paracarcinoma tissue.
FIG. 10 shows that the Vimentin + CD11b + cell population had a differential distribution among the different types of specimens, where gc-ca represents gastric cancer-cancer tissue, gc-nor represents gastric cancer-paracancer tissue, gc-pbmc represents gastric cancer-peripheral blood, and hd-pbmc represents healthy population.
Detailed Description
The invention is further illustrated by the following figures and specific examples in conjunction with the description. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Experimental procedures, in which specific conditions are not indicated in the examples below, are generally carried out according to conditions conventional in the art or as recommended by the manufacturer. Unless otherwise specified, all are conventional methods. Unless defined otherwise, technical and scientific terms used herein have the same meaning as is familiar to those skilled in the art.
Example 1 detection of percentage of positively expressed cells of Vimentin, CD11b, CD16, CD11c in paired cancer tissues, paracarcinoma tissues and peripheral blood samples of gastric cancer patients using CyTOF
1. 35 samples of paired cancer tissues, tissues adjacent to the cancer and peripheral blood from patients who had undergone surgical resection of gastric cancer were collected, and 20 peripheral blood samples from healthy volunteers were collected.
2. Preparation of single cells:
2.1 preparation of Single cells in peripheral blood
(1) Taking 2ml of peripheral blood, balancing and centrifuging at room temperature, sucking serum, adding 1:1 PBS into the residual blood cells, and uniformly mixing;
(2) adding 2ml of Ficoll lymphocyte separation solution into another 15ml of centrifuge tube, and slowly adding the whole blood diluted in the step (1) onto the Ficoll by using a disposable straw to keep the whole blood in a suspended state;
(3) balancing the centrifugal tube in the step (2) at 4 ℃, performing gradient centrifugation, sucking out the middle white cloudy cell layer (namely PBMC cells), and transferring the middle white cloudy cell layer into a new 15ml centrifugal tube;
(4) adding PBS to 8ml into the centrifuge tube containing the PBMC cells in the step (3), balancing, centrifuging, and removing supernatant to obtain PBMC cell sediment at the bottom of the centrifuge tube;
(5) taking 1ml of serum-free freezing medium under aseptic condition, after suspending the PBMC cell sediment in the step (4), transferring the PBMC cell sediment to a freezing tube, and storing the PBMC cell sediment in a freezing condition at-80 ℃ for later use.
2.2 preparation of single cells from cancer tissue or tissue adjacent to cancer:
(1) taking cancer tissues or tissues beside the cancer in a plate, washing with PBS buffer solution for three times, and removing peripheral fat, connective tissues, blood and the like;
(2) shearing the tissue in the step (1) into small pieces of 1-2mm by using sterile ophthalmic surgical scissors, washing the small pieces of 1-2mm with PBS (phosphate buffer solution) for three times, and transferring the small pieces of 1-2mm to a50 ml centrifuge tube;
(3) adding 0.25% pancreatin solution 6 times the amount of tissue in step (2), digesting at 37 deg.C for 30min, and lightly shaking once every 5 min;
(4) adding 5ml of serum-containing culture medium in the step (3), standing for 2-3min, and transferring the suspension into a new centrifuge tube;
(5) filtering the suspension of step (4) with 200/300 mesh nylon net for 2 times;
(6) Centrifuging the suspension filtered in the step (5) at 1000rpm for 5-10min, and removing the supernatant;
(7) adding 5ml of PBS buffer solution in the step (6), gently dispersing the cells, centrifuging again, and removing the supernatant;
(8) taking 1ml of serum-free freezing medium under aseptic condition, after cell precipitation in the step (7) is carried out, transferring the cell precipitation to a freezing tube, and storing the cell precipitation in a freezing condition of-80 ℃ for later use;
cell staining fixation for CyTOF assay:
(1) taking out the single cell frozen stock solution in the freezing condition of the temperature of 2.1 (5) or 2.2 (8) -80 ℃, preheating to a fluid state, and transferring to a centrifuge tube;
(2) adjusting the single cells in the step (1) to be less than 1 × 107 cells/ml, washing the single cells once with PBS, centrifuging the single cells at 500g for 5min, and resuspending the single cells with 1ml of PBS;
(3) adding 1 mul of 0.5 mu M cisplatin into the sample in the step (2) and dyeing for 2min at normal temperature;
(4) adding 2-5 times of Cell stabilizing Buffer (2-5ml) into the sample obtained in the step (3) to stop dyeing, and centrifuging at room temperature for 5min at 500 g;
(5) adding 1ml of Cell stabilizing Buffer into the sample obtained in the step (4) for resuspension, adding 1ml of 3.2% PFA (PBS for dilution) for fixation, and standing at room temperature for 10 min;
(6) adding 4ml of ice-cold Cell stabilizing Buffer into the sample in the step (5), centrifuging at the temperature of 4 ℃ and 600g for 5min, removing supernatant, and uniformly flicking to obtain 50 mu l of residual liquid;
(7) adding 1ml of 10% DMSO into the sample obtained in the step (6), and putting the mixture on ice; counting, adjusting to 2-3X 106/0.5ml, and freezing and storing at-80 ℃.
4. Sample labeling and detection for CyTOF detection:
(1) fixing the frozen cells by PFA, thawing the cells on ice or in a cold water bath, shaking the resuspended cells, adding 2ml of cell stabilizing Buffer, and centrifuging at 600g for 5 min;
(2) carefully sucking up the liquid in the flow tube in the step (1), adding 50ul Blocking Mix for resuspension, adding 50ul cocktail into each tube, uniformly mixing, and keeping the temperature for 30 min;
(3) adding 2ml of Cell stabilizing Buffer into the sample in the step (2), centrifuging for 5min at the normal temperature of 500g, and sucking supernatant;
(4) repeating the steps (2) and (3) and washing once more;
(5) adding 1ml of Nuclear Antigen stabilizing Buffer into the sample obtained in the step (4), gently shaking, and incubating for 30min at room temperature;
(6) adding 2ml of Nuclear Antigen stabilizing Perm into the sample obtained in the step (5), washing the cells, centrifuging 600-800 g, and discarding the supernatant;
(7) repeating the steps (5) and (6) and washing once more;
(8) resuspending the precipitate with the remaining solution in the tube of step (7), shaking gently, adding intracellular antibody mixture (50 μ l) directly, and staining at room temperature for 30 min;
(9) adding 2ml of Maxpar Cell stabilizing Buffer into the sample obtained in the step (8), washing for 2 times, shaking the sample to dissolve the precipitate, preparing a Cell interaction solution at the same time, and sucking a supernatant after the centrifugation is finished;
(10) adding 100ul PBS (phosphate buffer solution) without Ca2+ Mg2+ into the sediment obtained in the step (9), and uniformly mixing the mixture by vortexing for 5 seconds to fully suspend the cell sediment;
(11) holding the orifice of the flow tube, shaking and mixing uniformly, adding 1ml of Cell interaction solution dropwise, adding 2ml of Cell stabilizing Buffer, centrifuging for 5min at 800g, and removing supernatant;
(12) adding 2ml of deionized water into the precipitate in the step (11) for resuspension, centrifuging at 800g for 5min, and removing a supernatant; washing was repeated twice;
(13) 1ml of water containing 10% EQ beads was added to the pellet from step (12) and resuspended, and the sample was placed on ice and ready for loading.
5. Mass spectrum flow type detection on a computer:
after tissue and blood single cells are prepared, Vimentin + CD11b + cell proportion, CD16+ cell proportion and CD11c + cell proportion are detected by using CyTOF.
6. Mass spectrometry data analysis:
the cluster analysis and the heat map are based on dimension reduction analysis viSNE, and data visualization SPADE analysis is carried out on the in-vitro and in-vitro expression conditions of the cell population, as shown in figure 1 and figure 2, the Vimentin + CD11b + cell proportion, the CD16+ cell proportion and the CD11c + cell proportion are expressed in 'stomach cancer tissues, low expression in cancer side tissues, ultrahigh expression in peripheral blood of a stomach cancer patient and almost no expression in peripheral blood of healthy volunteers'; vimentin + CD11b + cells were significantly differentially expressed in each type of sample.
Example 2 application of the ratio of Vimentin, CD11b, CD16 and CD11c positive expression cells in gastric cancer screening and gastric cancer pathological stage
1. Blood sampling
2ml of whole blood of a subject to be tested is collected, stored in a vacuum blood collection tube using EDTA as an anticoagulant, and the sample is sent to a laboratory as soon as possible after blood drawing (the time from blood drawing to PBMCs preparation cannot exceed 24 hours).
2. Peripheral blood single cell preparation.
Single cells were prepared according to the method for preparing single cells of peripheral blood in example 1.
3. Cell staining for optical flow assay:
(1) taking out the PBMC cell frozen stock solution in the freezing condition of-80 ℃, and preheating to a fluid state;
(2) transferring the PBMC cells obtained in the step (1) into a centrifuge tube, adding MACS for resuspension, transferring into a flow tube, balancing and centrifuging at room temperature, and removing supernatant to obtain bottom cell sediment;
(3) adding 100ul MACS into a flow tube, resuspending and precipitating PBMC cells, and sequentially adding 1ul vimentin flow fluorescent antibody, 1ul CD11b flow fluorescent antibody and 1ul CD16 flow fluorescent antibody under aseptic conditions;
(4) putting the sample obtained in the step (3) into a dark condition at 4 ℃, and incubating for 15-20 min;
(5) taking out the flow tube in the step (4), adding 1ml of MACS to fully wash the cells, carrying out balancing and centrifugation again, and removing the supernatant;
(6) 100ul of MACS resuspended antibody stained cells were added to the sample from step (5) and ready for up-test.
4. And (3) installing an optical flow detection machine:
(1) placing a sample to be detected into a sample tube, pushing the sample into a flow chamber under the action of gas pressure, filling sheath liquid in the flow chamber, and under the action of the sheath liquid, enabling cells to be arranged in a single row to be sprayed out from a nozzle opening of the flow chamber and to be surrounded by the sheath liquid to form a cell liquid column;
(2) fluorescent signals and scattered light signals emitted by cells are received by a fluorescent photomultiplier, are converted into electronic signals through integral amplification and inversion, are amplified and processed and then are stored in a computer, and the computer system performs collection, storage and imaging and then performs multi-parameter statistical analysis by using related software.
5. And (4) analyzing results:
according to the results of optical flow analysis shown in fig. 3 and 4, the ratio of Vimentin + CD11b + cells is less than 1%, and the sample to be tested is from a healthy individual; the proportion of Vimentin + CD11b + cells is more than or equal to 1% and less than 10%, and the sample to be detected is from a patient with stomach early cancer; the ratio of Vimentin + CD11b + cells is more than or equal to 10%, and the sample to be tested is from a gastric cancer patient.
Example 3 application of Vimentin, CD11b, CD16 and CD11c positive expression cell ratios in evaluation of gastric cancer treatment effect and gastric cancer prognosis evaluation
1.2 ml of whole blood of a gastric cancer patient to be detected before and after treatment is collected and stored by using a vacuum blood collection tube with EDTA as anticoagulant. After blood withdrawal, the samples were sent to the laboratory as soon as possible (no more than 24 hours from blood withdrawal to preparation of PBMCs);
2. flow cytometry was performed according to the method of example 2;
3. and (4) analyzing results:
according to the flow analysis results shown in fig. 5 and fig. 6, before the gastric cancer patients receive treatment, the ratio of Vimentin + CD11b + cells is more than 10%; after the gastric cancer patient receives treatment, the ratio of Vimentin + CD11b + cells is obviously reduced to be less than 5%; changes in Vimentin + CD11b + cell ratios before and after treatment were of significant significance.
In examples 1, 2 and 3, the ratio of Vimentin + CD11b + cells, the ratio of CD16+ cells and the ratio of CD11c + cells are mainly detected by mass flow cytometry and optical flow cytometry, but conventional methods such as immunohistochemistry, immunofluorescence staining, enzyme-linked immunosorbent assay, immunomagnetic bead method and PCR can be used for detection and judgment of the marker, and the methods are all operable. Furthermore, the optical flow technology is recommended as the first choice for the patent to be implemented, taking into consideration the difficulty of operation, the popularity of detection means and the cost-effectiveness ratio.
Example 4 analysis of biomarker expression levels under Single-index, pairwise combination-index statistics
In order to explore and simplify the target cell population, the expression content of the biomarker is analyzed under the statistics of single index and pairwise combined index of various overall samples.
As shown in fig. 7a, 7b, and 7c, the expression of CXCR5, CD11b, CD8, CD3, CD4 in the cancer blood-normal blood was significantly different in the single index statistics (p <0.05 in t test); the expression of CD11b, HLA _ DR, CD16, CD11c, granzyme b was significantly different in cancer blood-cancer tissues; the expression of CD11b, CD11c, CD16, CD45 and CD117 has a significant difference in cancer blood-cancer side, the expression in cancer tissue-cancer side has certain similarity, and no biomarker with significant difference is detected. The expression of CD11b was significantly different in all three groups (cancer blood-normal blood, cancer blood-cancer tissue, and cancer tissue-paracancer).
Furthermore, based on the results of high-dimensional flow analysis, the Vimentin + CD11b + CD16+ CD11c + cell population was taken as the research target, and the expression contents of Vimentin, CD11b, CD16 and CD11c in various samples were respectively counted, as shown in fig. 8, it was found that only CD11b was significantly different, and the special expression of "the presence in gastric cancer tissues, the presence in a small amount in paracancer tissues, and the presence in a large amount in peripheral blood of gastric cancer patients" was observed.
In order to simplify and optimize detection indexes and search classification indexes with significance, pairwise index analysis is carried out on four indexes, and the fact that only when Vimentin and CD11b are subjected to combined analysis, the statistical result similar to high-dimensional flow analysis is found, and the statistical result has difference; in other pairwise combination verification of the four types of indicators, as shown in fig. 9a and 9b, the unique pattern of "expression in cancer tissue, small amount of expression in tissue adjacent to cancer, and cancer blood enrichment" cannot be detected.
In contrast, as shown in fig. 10, the Vimentin + CD11b + cell population (cancer 10.37%, peripheral blood 69.47%) was very low in the normal tissues of gastric cancer (4.79%), and was almost absent in healthy volunteers (0.05%). And (3) carrying out pairwise sample t test on the ratio of the 4 groups of cell populations, wherein the test result shows that the ratio of Vimentin + CD11b + cell populations of cancer tissues-cancer blood, paracancer-cancer blood and cancer blood-normal blood has significant difference (p is less than 0.05).
The foregoing examples are provided for the purpose of illustrating the method of the present invention, and the invention is not to be limited to the details of the foregoing embodiments. It will be apparent to those skilled in the art that modifications and variations can be made in the present invention without departing from the principles thereof, and such modifications and variations will fall within the scope of the appended claims.
Claims (10)
1. The application of the marker in preparing a product for detecting digestive system cancer is characterized in that the marker is a combination of Vimentin and CD11b, the product is used for detecting the cell proportion of positive expression of the marker in a sample to be detected, and the sample to be detected is peripheral blood.
2. The use of claim 1, wherein the cancer is gastric cancer, esophageal cancer or intestinal cancer.
3. The use according to claim 1, wherein said product is used for qualitative or quantitative detection of said marker in a sample to be tested.
4. The use of claim 1, wherein the product is a product detected using mass spectrometry, optical flow cytometry, immunohistochemistry, immunofluorescent staining, enzyme linked immunosorbent assay, immunomagnetic bead assay, or PCR.
5. The use of claim 1, wherein the detection of cancer in the digestive system is selected from the group consisting of digestive system cancer screening, digestive system cancer staging, digestive system cancer treatment efficacy assessment, and digestive system cancer prognosis assessment.
6. The use of claim 5, wherein the determination of the stage of the digestive system cancer is selected from the group consisting of early stage screening of digestive system cancer; the evaluation of the treatment effect of the cancer of the digestive system is the evaluation of the treatment effect of the cancer of the digestive system selected from chemotherapy, targeted therapy or immunotherapy.
7. A method for establishing a digestive system cancer detection model is characterized by comprising the following steps: collecting peripheral blood of healthy individuals and digestive system cancer patients as samples to be detected, preparing single cells of the collected samples to be detected, staining and fixing the single cells, marking and detecting, detecting the cell proportion of positive expression of a marker in the samples to be detected, and determining the detection relevance of the marker and the digestive system cancer by cluster analysis and/or heat map analysis data, wherein the marker is the combination of Vimentin and CD11 b.
8. The method of claim 7, wherein the cancer is gastric cancer, esophageal cancer, or intestinal cancer.
9. The method for constructing a model for detecting cancer in the digestive system according to any one of claims 7 to 8, wherein the detection of cancer in the digestive system is selected from the group consisting of screening cancer in the digestive system, determining the stage of cancer in the digestive system, evaluating the therapeutic effect of cancer in the digestive system, and evaluating the prognosis of cancer in the digestive system.
10. The method of claim 9, wherein the correlation is selected from the group consisting of:
(1) when the digestive system cancer is detected as the digestive system cancer screening: the proportion of Vimentin + CD11b + cells is less than 1%, and the sample to be detected is from a healthy individual; the ratio of Vimentin + CD11b + cells is more than or equal to 1%, and the sample to be detected is from a cancer patient;
(2) when the digestive system cancer is detected to determine the stage of the digestive system cancer: the ratio of Vimentin + CD11b + cells is more than or equal to 1% and less than 10%, and the sample to be detected is from a patient with early cancer; the ratio of Vimentin + CD11b + cells is more than or equal to 10%, and the sample to be tested is from a cancer patient;
(3) when the digestive system cancer is detected as the digestive system cancer treatment effect evaluation or the cancer prognosis evaluation: after the cancer patient receives the treatment, the Vimentin + CD11b + cell percentage is reduced to be below 5%, and the treatment effect of the sample to be detected is good.
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