CN116555430A - Detection panel for peritoneal metastasis evaluation of gastric cancer patient, application thereof and kit - Google Patents
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- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
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- C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
- C12Q1/6886—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material for cancer
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Abstract
The present disclosure relates to a detection panel for peritoneal metastasis evaluation of gastric cancer patients, as well as the use of such detection panel for preparing a reagent or composition and for constructing a risk prediction model for peritoneal metastasis of gastric cancer patients, and also includes a kit for peritoneal metastasis evaluation of gastric cancer patients. The detection panel and kits, models, etc. referred to in this disclosure are based on gene detection probes comprising the SPP1, CD14, S100A9, MAFB, APOE, FN1, CTSL, RNASE1, FCGR3A genes. Dendritic cells with monocyte characteristics can be evaluated by measuring the expression level of the genes, and the risk and prognosis of peritoneal metastasis of gastric cancer patients can be further evaluated based on the dendritic cells, so that a practical and reliable basis is provided for the decision of clinical treatment schemes.
Description
Technical Field
The disclosure relates to the field of gastric cancer clinical medicine, in particular to a detection panel for peritoneal metastasis evaluation of gastric cancer patients, and application and a kit thereof.
Background
Gastric cancer is taken as a malignant tumor, and is the second most frequent in the morbidity and mortality of malignant tumors in China. Although the diagnosis and treatment level of malignant tumors have been greatly improved, the treatment and prognosis of gastric cancer still cannot be effectively improved, and particularly the prognosis still has a higher recurrence possibility.
The peritoneum is the most common recurrent site after radical gastric cancer treatment and is also an important cause of short survival time of patients with advanced gastric cancer. Peritoneal metastasis has been established in 10% to 20% of gastric cancer patients at the time of diagnosis, while peritoneal metastasis still occurs in 40% to 60% of patients after radical gastric cancer. Prognosis of patients with peritoneal metastasis from gastric cancer is extremely poor and their tumor malignancy is higher.
At present, the related pathophysiological mechanism of gastric cancer peritoneal metastasis is still unclear, and great difficulty is brought to diagnosis and treatment of patients. Can realize early diagnosis of gastric cancer peritoneal metastasis, further guide clinical decision and have important significance for prognosis evaluation of gastric cancer peritoneal metastasis patients.
Currently in clinical practice, the assessment of preoperative peritoneal metastasis in gastric cancer patients is largely dependent on imaging diagnosis and serum marker detection. Among them, X-ray computed tomography (computed tomography, CT) is the main imaging examination means for peritoneal metastasis of gastric cancer. However, the sensitivity of CT diagnosis is related to the size of the metastasis, and it is difficult to detect peritoneal metastasis that is positive in cytological examination and invisible to the naked eye. Carcinoembryonic antigen (CEA) is considered a potential serum marker for monitoring peritoneal metastasis of gastric cancer, but its sensitivity and specificity are too low to meet clinical needs. Laparoscopic exploration can improve the accuracy of gastric cancer staging and effectively evaluate gastric cancer peritoneal metastasis, but laparoscopic exploration is a invasive examination and still has a false negative rate as high as 10.6% to 17.2%. The cytological examination of the peritoneal irrigation solution is a gold standard for diagnosing gastric cancer peritoneal metastasis at present, but the detection accuracy is greatly limited due to the fact that the number of free cancer cells in the peritoneal cavity is small, sampling is incomplete in the peritoneal lavage operation process and the like. The emerging liquid biopsy technology predicts the peritoneal metastasis of gastric cancer by detecting the number of gastric cancer circulating tumor cells in peripheral blood, but the technology is still in a research stage and cannot be applied to clinical transformation. There are studies on predicting prognosis of patients with peritoneal metastasis from gastric cancer using artificial intelligence image processing or based on the radiological characteristics of gastric cancer imaged by computed tomography, but the results are highly heterogeneous.
In view of the above, there is still a lack of tools that can diagnose and evaluate gastric cancer peritoneal metastasis effectively at an early stage.
Disclosure of Invention
The present disclosure aims to solve at least one of the problems existing in the prior art, and more precisely, to provide a gene detection panel, a kit, an evaluation method, an apparatus, a drug, or the like that helps to more precisely recognize the progress of peritoneal metastasis of gastric cancer.
To solve the above-described problems, a first aspect of the present disclosure provides a detection panel for peritoneal metastasis evaluation of gastric cancer patients.
In a second aspect the present disclosure provides the use of the detection panel described above for the preparation of a reagent or composition.
In a third aspect of the present disclosure, use of the above detection panel for constructing a risk prediction model for peritoneal metastasis from gastric cancer is provided.
In a fourth aspect, the present disclosure provides a kit for peritoneal metastasis evaluation of gastric cancer patients.
In a fifth aspect, the present disclosure provides a targeted drug using each gene in the detection panel as a therapeutic target.
The first aspect of the present disclosure provides a detection panel comprising detection probes for the following 9 genes:
SPP1、CD14、S100A9、MAFB、APOE、FN1、CTSL、RNASE1、FCGR3A。
based on clustering and grouping of Dendritic Cells (DCs), the DC cells can be divided into 3 subpopulations, defined as DC1, DC2 and monoDC cell populations (DC subpopulations with monocytic properties), respectively; that is, there are heterogeneous cell subsets in DC cells that express different functions. Statistics of the cell numbers at different stages of the disease revealed that the proportion of monoDC cells increases with progression of GCPM and that its antigen presenting ability decreases significantly. The probes for detecting various genes provided in panel can evaluate monoDC cells by means of the expression degree, and further evaluate the risk and prognosis of peritoneal metastasis of gastric cancer patients based on the monoDC cells, so as to provide a practical and reliable basis for the decision of clinical treatment schemes. While targeted therapeutic strategies against subpopulations of monoDC cells may be helpful in the prevention and treatment of gastric cancer peritoneal metastasis.
The monoDC cells have higher correlation with monocytes, so the present disclosure provides the above scheme, based on the characteristics of monoDC cells highly expressing monocyte lineage related genes such as CD14, MAFB and S100A9, detection probes for the above genes are used to evaluate monoDC cells.
The present disclosure constructs the characteristic gene panel provided by the preferred scheme described above based on differential gene expression analysis of monoDC cells and other DC cells during peritoneal transfer, and selection of the above-described most representative 9 high-expression differential genes in combination with a COX proportional risk model. The model is verified by a TCGA gastric cancer database, and the fact that the gene panel is really related to prognosis of gastric cancer peritoneal metastasis is verified, namely, the higher the gene panel is expressed, the worse the prognosis of gastric cancer patients.
Use of a detection panel provided according to a second aspect of the present disclosure for the preparation of a reagent or composition, wherein the reagent or composition is for:
for assessing the risk of a gastric cancer patient for developing peritoneal metastasis;
is used for prognosis evaluation of patients with gastric cancer peritoneal metastasis.
According to a third aspect of the present disclosure there is provided a use for constructing a risk prediction model of gastric cancer peritoneal metastasis, wherein such a risk prediction model is programmed and inputted into a computer or a gene detector in the form of a mathematical software package, and the risk prediction model is capable of outputting a risk level value of gastric cancer peritoneal metastasis, the risk level value being positively correlated with the expression level of each gene in a detection panel.
Preferably, such a risk prediction model is configured to: based on the detection of the expression level of each gene in panel, dendritic cells having monocyte characteristics in the sample are evaluated, and based on the result of the evaluation, a risk level value of peritoneal metastasis of gastric cancer is output.
The sample is malignant ascites sample or abdominal cavity flushing fluid sample.
According to a fourth aspect of the present disclosure there is provided a kit for peritoneal transfer evaluation of a gastric cancer patient, wherein the kit comprises reagents capable of separately detecting the extent of expression of genes selected from the group consisting of:
SPP1、CD14、S100A9、MAFB、APOE、FN1、CTSL、RNASE1、FCGR3A。
the selection of reagents in the fourth aspect of the present disclosure is based on the same principle as the selection of genes for detecting panel in the first aspect of the present disclosure, and will not be repeated here.
Preferably, the kit is used for detecting malignant ascites samples or peritoneal irrigation fluid samples.
Preferably, the kit is used for evaluating dendritic cells having monocyte characteristics in a sample based on the expression level of the genes.
And, further preferably, the above-mentioned kit further includes a case body, and at least one of the following reagents:
pepsin, tissue fixative, prehybridization, oligonucleotide probe hybridization, blocking, biotinylated murine anti-digoxin, SABC-POD, biotinylated peroxidase, DEPC, 3% citric acid, 2 XSSC, 0.5 XSSC, 0.2 XSSC, PBS for in situ hybridization.
The risk and prognosis of the occurrence of peritoneal metastasis of gastric cancer patients can be evaluated by screening the monosdc cells in malignant ascites or peritoneal irrigation solution and detecting the expression level of gene panel by the above kit.
Drawings
The above and other features, advantages and aspects of embodiments of the present disclosure will become more apparent by reference to the following detailed description when taken in conjunction with the accompanying drawings. In the drawings, wherein like or similar reference numerals denote like or similar elements, in which:
FIG. 1 is a schematic flow chart of differential gene expression analysis of monoDC cells versus other DC cells in accordance with the present disclosure;
FIG. 2 is a single cell transcriptome sequencing UMAP cluster map of sample cells referred to in the present disclosure;
FIG. 3 is a UMAP cluster map of DC cells referred to in the present disclosure;
FIG. 4 is a violin graph of gene expression levels characteristic of various subpopulations of DC cells contemplated in the present disclosure;
FIG. 5 shows a correlation analysis heat map between DC cell subpopulations and monocytes;
FIG. 6 is a GSEA enrichment analysis of monoDC cells and other DC cell subsets referred to in the present disclosure;
FIG. 7 shows the cell proportion and functional changes of the monoDC cells involved in the present disclosure in GCPM progression;
wherein a is the proportion of cells in the different groups of monoDC cells in GCPM progression, and b is the expression level of antigen presenting function in the different groups of monoDC cells in GCPM progression;
FIG. 8 is a differentiation trace and gene heat map of various subpopulations of DC cells contemplated in the present disclosure;
wherein a is a differentiation track based on cell types, b is a differentiation track with a timing sequence, c is a gene heat map, and dynamic changes of gene expression level and related channels along with the timing sequence differentiation track are displayed;
FIG. 9 is the immunofluorescent staining results of peritoneal wash samples and malignant ascites samples referred to in this disclosure, DC cell marker CD1c (red), monoDC cell marker CD163 (yellow) and nuclear marker DAPI (blue);
FIG. 10 is a flow analysis of MonoDC cells in a peritoneal irrigation fluid sample and malignant ascites sample as referred to in this disclosure;
wherein a is a flow analysis gate strategy of the monoDC cells in the sample, and b is the proportion level of the monoDC cells in the abdominal cavity flushing fluid sample and malignant ascites sample;
FIG. 11 shows MonoDC cells and CD8 in the peritoneal irrigation solution sample and malignant ascites sample according to the present disclosure + Results of flow analysis after T co-cultivation;
wherein a is the detection of CD8 by flow analysis + Proliferative CD8 after co-culture of T and MonoDC cells + Proportional level of T cells, b is detection of CD8 by flow analysis + IFN-gamma after co-culture of T and MonoDC cells + CD8 + Proportional level of T cells;
FIG. 12 shows the correlation between the characteristic gene panel of the monoDC cells and gastric cancer prognosis in TCGA gastric cancer database.
Detailed Description
For purposes of clarity, embodiments and advantages of the disclosed embodiments, the embodiments in this disclosure will be described more clearly and fully below with reference to the accompanying drawings in which embodiments of the disclosure are shown, it being apparent that the embodiments described are some, but not all, of the embodiments of the disclosure. All other embodiments, which can be made by one of ordinary skill in the art based on the embodiments in this disclosure without inventive faculty, are intended to be within the scope of this disclosure.
Various reagents, software, instruments, or devices are commercially available as contemplated by the present disclosure.
Reagents, instruments, software and equipment: CD45 Monoclonal Antibody (HI 30), eFluor TM 506；CD3 Monoclonal Antibody(OKT3)，FITC；CD56(NCAM)Monoclonal Antibody(TULY56)，FITC；CD19 Monoclonal Antibody(HIB19)，FITC；APC anti-human CD1c；CD163 Monoclonal Antibody (eBioGHI/61 (GHI/61)), PE; hu CD14 BV421 MPhiP9; BV510 Mouse Anti-Human CD3 (HIT 3 a); CD8a Monoclonal Antibody (RPA-T8), perCP-cyanine5.5; APC Mouse Anti-Human IFN-gamma; CFSE;7-AAD activity staining solution; BD FACSAria III flow cytometer; BD Celesta flow cytometer; chromium (Chromium) TM Next GEM Single Cell 3'Reagent Kit v3.1 kit and chromo mium TM A Next GEM Chip; illumina Novaseq6000 sequencer; cell Ranger (4.0.0); r (4.0.5); rstudio (1.4.1106); doubletFinder (2.0.1); semat (3.2.3); harmony (1.0); single (1.0.1); biocManager (1.30.12); clusteriprofiler (4.4.4); monocle2 (2.4.0); pheatmap (1.0.12); ggplot2 (3.3.3); graphPad Prism (8.0.1); loupe Cell Browser (5.0.0).
Aiming at the current situation that the related pathophysiological mechanism of gastric cancer peritoneal metastasis is still not clear in the prior art, one of the technical ideas of the present disclosure is to analyze the complex molecular mechanism of gastric cancer peritoneal metastasis and search for a key therapeutic target.
Gastric cancer peritoneal metastasis involves a variety of cell types and molecular signals as a multi-stage, complex cellular biological behavioral process. The current mechanistic study of gastric cancer peritoneal metastasis is widely accepted by the "Seed-Soil" theory ("Seed and Soil" Hypothesis), gastric cancer peritoneal microenvironment as a tumor microenvironment with high complexity and heterogeneity (Tumor Microenvironment, TME), acting as a "Soil" during its peritoneal metastasis. TME includes various types of cells, extracellular matrix and signaling molecules, can provide conditions required for tumor cell growth, and plays a great role and function in proliferation, invasion and metastasis of tumor cells. Thus, the inventors of the present disclosure considered that a deeper study of TME is a trend and a shortcut for understanding the mechanism of tumorigenesis and development more deeply.
Dendritic Cells (DCs) are the most powerful professional antigen presenting cells in vivo, playing a pivotal role in the innate and adaptive immune responses of the body. In early immunization of tumors, DC cells are able to initiate an immune response by T cells through efficient processing and cross-presentation of tumor antigens; upregulation of costimulatory molecules such as CD80/CD86, CD40, CD137L, OX40L, ICOSL, etc., promotes and maintains T cell activation and survival; secretion of chemokines and cytokines affects tumor progression to promote tumor immunity. The characteristics make DC cells have unique functions in inducing and maintaining anti-tumor immunity, and become important regulators for initiating tumor antigen specific immune response in the tumor immune response process. However, as the tumor progresses, factors secreted by tumor cells and their regulated tumor microenvironment will functionally remodel the DC cells, resulting in dysfunction of the DC cells and limited development and maturation of the DC cells, resulting in reduced antigen presentation. The remodeled DC cells cooperate with tumor cells to form a tumor microenvironment that is prone to immunosuppression and tumorigenesis, resulting in the development of immune evasion and tumor progression.
Therefore, the technical idea of the present disclosure is further that: the phenotype and function of DC cells are precisely described from the molecular layer to precisely know the development process of gastric cancer peritoneal metastasis. In other words, the related DC cells and molecular markers thereof which are used for referencing the peritoneal transfer process of the gastric cancer in the abdominal microenvironment are found, so that the early diagnosis of the peritoneal transfer of the gastric cancer is facilitated, the clinical decision is guided, and the method has important significance for prognosis evaluation of patients with the peritoneal transfer of the gastric cancer.
Based on the technical thought, the single-cell transcriptome sequencing technology is used as a core, high-throughput sequencing data are obtained by using peritoneal flushing fluid and malignant ascites clinical samples in different disease stages, and DC cells in a peritoneal microenvironment are subjected to cell grouping by using an unsupervised clustering method and visualized by UMAP (Uniform Manifold Approximation and Projection). DC cell subsets (defined herein as monoDC cells) with monocyte properties associated with gastric cancer peritoneal metastasis were found using differential gene expression analysis, correlation analysis, cell function scoring, gene enrichment analysis, and timing analysis to find the diversity, complexity, heterogeneity, and dynamic changes of DC cell types and functions within the peritoneal microenvironment during peritoneal metastasis. The analysis shows that the proportion of the MonoDC cells is increased in the peritoneal metastasis process of the gastric cancer, and the peritoneal microenvironment is remodeled into an immunosuppressive cancer-promoting microenvironment by reducing the antigen presentation function, so that the peritoneal metastasis of the gastric cancer is facilitated.
In one embodiment of the disclosure, genes related to the monoDC cells, SPP1, CD14, S100A9, MAFB, APOE, FN1, CTSL, RNASE1, FCGR3A, are selected, and based on some or all of these genes, a characteristic gene panel of the monoDC cells is constructed.
In the preferred embodiment of the above examples, 4, 5, 6 and more can be selected therefrom, constituting the characteristic gene panel of the monoDC cell.
In the preferred embodiment of the above examples, the most representative 9 highly expressed differential genes including SPP1, CD14, S100A9, MAFB, APOE, FN1, CTSL, RNASE1 and FCGR3A were selected by differential gene expression analysis of the monoDC cells from other DC cells during peritoneal transfer in combination with a COX scale risk model, and the characteristic gene panel of the monoDC cells was constructed. The model is verified by a TCGA gastric cancer database, and the fact that the gene panel is really related to gastric cancer prognosis is verified, namely, the higher the expression of the gene panel is, the worse the prognosis of gastric cancer patients is. While targeted therapeutic strategies against subpopulations of monoDC cells may be helpful in the prevention and treatment of gastric cancer peritoneal metastasis.
It should be understood that (gene) detection panel is a term used after development of high throughput gene detection and gene sequencing, which means that several probes corresponding to genes are designed onto the same capture chip in the detection to capture the target nucleic acid sequence and used for subsequent gene sequencing. In the detection, not only one locus and one gene are detected, but a plurality of loci and a plurality of genes are detected simultaneously, and the loci and the genes need to be selected and combined according to a standard to form a detection panel. Based on the above, the genetic panel related in the disclosure includes a vector such as a capture chip or the like, and the selection of the vector may be selected by those skilled in the art as appropriate based on the prior art and the actual application scenario, which is not repeated in the disclosure.
In one embodiment of the present disclosure, a kit based on the above-described characteristic gene panel and detection model is provided. The kit can be used for screening malignant ascites or monoDC cells in peritoneal irrigation liquid and detecting the expression level of gene panel so as to evaluate the risk and prognosis of peritoneal metastasis of gastric cancer patients, thereby providing a practical and reliable basis for the decision of clinical treatment schemes.
The experimental procedure for obtaining the representative differential gene to construct the above-described test gene panel is exemplarily described as follows. The method specifically comprises the following steps:
sample collection: samples of malignant ascites including patients with peritoneal metastasis and other peritoneal irrigation fluid samples of patients without peritoneal metastasis are obtained. Sufficient samples were taken for transport to the laboratory for preparation of single cell suspensions for subsequent single cell transcriptome sequencing. The samples are divided into the following groups according to different stages of gastric cancer occurrence and development:
g0: benign uterine fibroid peritoneal irrigation solution is used as a negative control group;
g1: early gastric cancer abdominal cavity flushing liquid;
and G2: gastric cancer abdominal cavity flushing liquid in the progressive stage;
and G3: malignant ascites before treatment of patients suffering from gastric cancer peritoneal metastasis;
and G4: malignant ascites after treatment of patients with peritoneal metastasis from gastric cancer.
Sample processing: all experimental procedures were completed within 2-4 hours after sample collection to ensure cell activity and status. Filtering malignant ascites or abdominal cavity flushing fluid samples according to steps, centrifugally collecting cells, completely splitting red, washing, resuspension and counting cells, adjusting the concentration of single cell suspension to reach the standard of single cell sequencing, forming micro-reaction liquid drops with a ' water-in-oil ' structure through ChromiumTM Next GEM Chip chips according to the specification provided by ChromiumTM Next GEM Single Cell 3'Reagent Kit v3.1 kit of 10x Genomics company, and carrying out barcode marking on single cells. The samples were then reverse transcribed into cDNA by PCR, thus constructing a single cell cDNA sequencing library. Next, PE-150 double-ended sequencing was performed on samples successfully constructed from the library using an Illumina Novaseq6000 sequencer.
Single cell sequencing screening gastric cancer peritoneal transfer related DC cell subsets:
the method specifically comprises the following steps:
(1) Single cell RNA sequencing data processing: as shown in FIG. 1, the original expression matrix for single Cell RNA sequencing was obtained by aligning GRCh38 human reference genomes using Cell Ranger software (version 4.0.0, 10x Genomics,https:// support.10xgenemics.com). The low quality cells were then filtered and the quality control inclusion criteria were as follows:
(a) The UMI number of the cells is more than or equal to 800 or is arranged at the front 99%;
(b) The total gene number of the cell detected is more than 200;
(c) The total number of mitochondrial genes in the cell is less than 20%;
(d) Genes can be detected in at least 3 cells;
(e) The single cells were determined by the "double Finder" R-package algorithm. Then, a normalized gene expression matrix was obtained using the "normazedata" function in the "setup" R package. The sample sequencing data were integrated by the Harmony algorithm for subsequent downstream data analysis.
(2) DC cell clustering and grouping: the hypervariable genes of each cell were screened and normalized using the "serat" v 3R package, and then unsupervised dimensionality reduction and cluster analysis were performed on the sequencing data. Specific genes for each cell population were determined based on differentially expressed genes with expression levels |log2 (Fold Change) | >0.50 and FDR <0.01, then cell annotation was performed according to the known marker gene set, while the cell grouping annotation results were visualized using the UMAP map (fig. 2). Further extraction of the DC cell expression matrix, again with a second round of clustering and cell annotation, the DC cells were seen to be divided into 3 sub-populations, defined as DC1, DC2 and monosdc cell populations (fig. 3), demonstrating the presence of heterogeneous sub-populations of cells expressing different functions in the DC cells. Meanwhile, monoDC cells highly expressed monocyte lineage-related genes such as CD14, MAFB, S100A9, and FCGR3A (FIG. 4). To further determine the correlation between each subpopulation of DC cells and monocytes, the present disclosure calculated correlation coefficients for the relevant cell populations based on gene expression levels, indicating higher correlation between monoDC cells and monocytes (fig. 5).
(3) Pathway enrichment analysis (GSEA) and cell function analysis: enrichment analysis (https:// www.gsea-msigdb. Org/GSEA/index. Jsp) of monose cells with other DC cell populations on a predefined gene set on the GSEA website demonstrated that monose cells had poor antigen presenting function (fig. 6). Meanwhile, cell proportion change and cell remodeling of the monoDC cell subset during GCPM progress were explored by single cell analysis. Based on differential expression gene analysis and known marker genes, the present disclosure defines a set of antigen presentation function-related genes. Cell function scoring was performed on each DC subpopulation based on the z-score value of the average expression level of the gene set, and the statistical significance of the difference results between groups was assessed by a two-sided unpaired Wilcoxon test method. The results indicate that the proportion of monoDC cells increases with the progression of GCPM and that its antigen presenting capacity decreases significantly (fig. 7). Thus, monoDC cells may play an important role in remodelling the immunosuppressive abdominal microenvironment.
(4) Cell timing analysis: to further investigate the potential differentiation trajectories between different DC cell types, the present disclosure used the "Monocle2" R package with the original expression matrix as input data, simulated the dynamic changes of the DC cell trajectories, and established a pseudo-time axis (https:// gitub. Com/cole-trapnell-lab/Monocle-release) between different cell populations. Meanwhile, by means of BEAM test in Monocle2, differential expression genes with the gene expression level changing along with the time-series-planned track are calculated, and enrichment analysis is carried out on the genes, so that the results are visualized in the form of a heat map. As shown in fig. 8, the subset of monoDC cells was at the end of the dendritic cell timing trajectory, indicating that DC cells tended to switch to cell types with low antigen presenting function during peritoneal transfer.
Experimental verification of Single cell sequencing results
In addition, clinical samples of GCPM malignant ascites and GCPM-free gastric cancer abdominal cavity flushing fluid are collected for carrying out immunofluorescence experiments and flow cytometry to verify single-cell results. Immunofluorescence experiments are carried out on clinical samples by using CD1c and CD163 antibodies in the embodiment of the disclosure, and the experiments show that the proportion of the monoDC cells with double positive CD1c and CD163 in GCPM ascites is higher than that of peritoneal flushing fluid (figure 9); the proportion of monoDC cells and antigen presenting function were examined by flow cytometry, and as a result, it was confirmed that monoDC cells were significantly increased in GCPM (fig. 10), and their antigen presenting ability to activate cd8+ T cells was decreased (fig. 11), consistent with the single cell analysis results (fig. 7). The results show that in the GCPM process, the increase and the remodelling of the monoDC cell subset play an important role in the formation of the intraperitoneal immunosuppressive microenvironment, are beneficial to the occurrence and the development of gastric cancer peritoneal metastasis, and are hopeful to become therapeutic targets for inhibiting gastric cancer peritoneal metastasis.
Construction of MonoDC cell Gene Panel and TCGA verification
Based on differential expression gene analysis performed on monosed dc cells, 9 representative differential genes, SPP1, CD14, S100A9, MAFB, APOE, FN1, CTSL, RNASE1 and FCGR3A, respectively, were selected as characteristic genes panel for predicting the occurrence and progression of gastric cancer peritoneal metastasis. In order to verify the relation between the gene panel and the prognosis of gastric cancer survival, the median of the average expression level is used as the demarcation value of the high-low expression group, and comparison analysis is carried out in bulk RNA sequencing data of TCGA-STAD (The Cancer Genome Atlas-Stomach Adenocarcinoma). The survival rate of the high expression group is obviously lower than that of the low expression group through a Kaplan-Meier survival curve, and the Log-rank test proves that the difference between the two groups has statistical significance (figure 12), which shows that the characteristic gene set of the monoDC cell subset is obviously related to the poor prognosis of the gastric cancer and can be used as a prediction index of the poor prognosis of the gastric cancer.
In one embodiment of the present disclosure, there is provided a use of the detection panel as in the previous embodiment: for preparing an agent or composition. The prepared reagent or composition is used for one or more purposes selected from the following:
for assessing the risk of a gastric cancer patient for developing peritoneal metastasis;
is used for prognosis evaluation of patients with gastric cancer peritoneal metastasis.
In one embodiment of the present disclosure, there is provided a use of the detection panel as in the previous embodiment: the method is used for constructing a risk prediction model of gastric cancer peritoneal metastasis, and the risk prediction model is input into a computer or a gene detector through programming and in a mathematical software package.
In a preferred embodiment, the risk prediction model is capable of outputting a risk level value for peritoneal metastasis of gastric cancer, which is positively correlated with the expression level of each gene in the detection panel.
According to an embodiment of the present disclosure, the risk prediction model is configured to: based on the detection of the expression degree of each gene in panel, evaluating dendritic cells with monocyte characteristics in a sample, and outputting a risk degree value of gastric cancer peritoneal metastasis based on the evaluation result; the sample is malignant ascites sample or abdominal cavity flushing fluid sample.
It should be understood that modeling, training, programming of the risk prediction model and parameters, weight values and the like applied in each step can be completed by a technician based on the technical ideas of the embodiments of the present disclosure by relying on the prior art, wherein specific modeling, training, programming modes and details are not important in the present application, and are limited in space and not repeated.
In one embodiment of the present disclosure, a computer or gene detector configured with the risk prediction model described above is provided.
In an exemplary embodiment, the computer or the gene assaying instrument has:
the data acquisition module is configured to acquire the expression degree of the following genes in the sample: SPP1, CD14, S100A9, MAFB, APOE, FN1, CTSL, RNASE1, FCGR3A, and the above expression levels are recorded or output to other modules in digitized form. The sample is malignant ascites sample or abdominal cavity flushing fluid sample.
The operation module can execute a risk prediction model, evaluate dendritic cells with monocyte characteristics in a sample based on the expression degree of each gene in the detection panel, and output a risk degree value of gastric cancer peritoneal metastasis based on the evaluation result.
Wherein, the operation module can also output the evaluation data for prognosis evaluation of patients with gastric cancer peritoneal metastasis.
And the storage module is used for storing the data of the expression degree and the risk degree value data generated by the operation module.
The computer or the gene assaying instrument may further have a manual input device such as a keyboard, a camera, etc., and a display device for displaying the evaluation result such as a display screen, etc. But may include necessary buses, transceivers, memory, etc.
In one embodiment of the present disclosure, a computer readable storage medium is provided having stored thereon a computer program which, when executed by a processor, is capable of running the risk prediction model described above.
In one embodiment of the present disclosure, a kit is provided comprising reagents capable of separately detecting the extent of expression of at least a plurality of genes selected from the group consisting of:
SPP1、CD14、S100A9、MAFB、APOE、FN1、CTSL、RNASE1、FCGR3A。
in a preferred embodiment, the above kit may include at least one of the following items, respectively, in addition to the various primers: a carrying means, the space of which is divided into a defined space that can house one or more containers, such as kits, vials, test tubes, and the like, each of which contains a separate component for use in the genetic probes of the present disclosure; the instructions, which can be written on bottles, test tubes and the like, or on a single piece of paper, or outside or inside the container, for example paper with a video APP download window for the operation demonstration, such as a two-dimensional code, can also be in the form of multimedia, such as a CD, a U-disc, a netdisk, etc.
The sample aimed by the kit is a malignant ascites sample or an abdominal cavity flushing fluid sample.
In a preferred embodiment, the kit further comprises at least one of the following reagents:
pepsin, tissue fixative, prehybridization, oligonucleotide probe hybridization, blocking, biotinylated murine anti-digoxin, SABC-POD, biotinylated peroxidase, DEPC, 3% citric acid, 2 XSSC, 0.5 XSSC, 0.2 XSSC, PBS for in situ hybridization.
In one embodiment of the present disclosure, a targeted drug is provided that targets genes based on the aforementioned detection panel.
In one embodiment of the present disclosure, a targeted drug is provided that has a target based on the following genes:
SPP1、CD14、S100A9、MAFB、APOE、FN1、CTSL、RNASE1、FCGR3A。
the above examples in this disclosure, or preferred embodiments thereof, by single cell transcriptome sequencing analysis of gastric cancer ascites or peritoneal irrigation solutions at different stages of development, find that there are a population of monoDC cells with low antigen presenting function in the peritoneal microenvironment, which can remodel the peritoneal microenvironment into immunosuppressive, cancer-promoting microenvironment, which is beneficial for the occurrence and development of gastric cancer peritoneal metastasis. For this finding, the present disclosure picked out the characteristic gene panel of the monoDC cells associated with prognosis, and established a detection kit based on the gene panel. The method can predict the survival prognosis of the gastric cancer patient, screen out high risk groups which are easy to generate peritoneal metastasis in the gastric cancer patient, and have diagnostic significance for early identification of the occurrence of gastric cancer peritoneal metastasis, thereby guiding positive clinical treatment strategies. In addition, the method is also beneficial to providing a new target point for the immunotherapy of the peritoneal metastasis of the gastric cancer, and also provides reference significance for the subsequent study of the application of the peritoneal microenvironment in the clinical diagnosis and treatment of the peritoneal metastasis of the gastric cancer.
Based on the foregoing, it will be appreciated by those skilled in the art that the presently claimed technology and equivalents thereof will be apparent. In addition, the technical scheme disclosed can be appropriately modified and changed according to the need by those skilled in the art, and the modified and improved technical scheme is also within the protection scope of the claims of the present disclosure.
Claims (10)
1. A detection panel for peritoneal metastasis evaluation of gastric cancer patients, characterized in that the detection panel comprises detection probes for the following genes:
SPP1、CD14、S100A9、MAFB、APOE、FN1、CTSL、RNASE1、FCGR3A。
2. use of the test panel according to claim 1 for the preparation of an agent or composition for one or more of the following uses:
for assessing the risk of a gastric cancer patient for developing peritoneal metastasis;
is used for prognosis evaluation of patients with gastric cancer peritoneal metastasis.
3. A kit for peritoneal metastasis evaluation of a gastric cancer patient, comprising reagents capable of detecting the expression levels of the following genes, respectively:
SPP1、CD14、S100A9、MAFB、APOE、FN1、CTSL、RNASE1、FCGR3A。
4. the kit of claim 3, further comprising a cartridge body and at least one of the following reagents:
pepsin, tissue fixative, prehybridization, oligonucleotide probe hybridization, blocking, biotinylated murine anti-digoxin, SABC-POD, biotinylated peroxidase, DEPC, 3% citric acid, 2 XSSC, 0.5 XSSC, 0.2 XSSC, PBS for in situ hybridization.
5. A kit according to claim 3, wherein the kit is used for assessing dendritic cells having monocytic characteristics in a sample based on the extent of expression of the gene.
6. The kit according to claim 5, wherein the sample is a malignant ascites sample or an abdominal cavity wash sample.
7. Use of the test panel according to claim 1 for constructing a risk prediction model of gastric cancer peritoneal metastasis, which is programmed and entered into a computer or genetic tester in the form of a mathematical software package.
8. The use of a test panel according to claim 7, wherein the risk prediction model is capable of outputting a risk level value for peritoneal metastasis of gastric cancer, which risk level value is positively correlated with the expression level of each gene in the test panel.
9. The use of the detection panel of claim 8, wherein the risk prediction model is configured to: based on the expression degree of each gene in the detection panel, evaluating dendritic cells with monocyte characteristics in a sample, and outputting a risk degree value of gastric cancer peritoneal metastasis based on the evaluation result; the sample is malignant ascites sample or abdominal cavity flushing fluid sample.
10. Use of a detection panel according to claim 1 for the preparation of a targeted drug targeting a gene detectable by the detection probe in the detection panel.
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