Application of ITPRIPL1 as a biomarker for gastric cancer diagnosis or prognosis

Abstract

This invention discloses the application of ITPRIPL1 as a biomarker for the diagnosis or prognosis of gastric cancer. Database analysis revealed high expression of ITPRIPL1 in gastric cancer patients. Immunohistochemical staining of samples from clinically diagnosed gastric cancer patients further validated its high expression in gastric cancer tumor cells, with higher ITPRIPL1 expression levels observed in lower-differentiated tumor tissues. This invention provides a novel biomarker for the diagnosis and prognosis of gastric cancer, offering new ideas and directions for immunotherapy of gastric cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology and relates to the application of ITPRIPL1 as a biomarker for the diagnosis or prognosis of gastric cancer. Background Technology

[0002] Gastric cancer (GC) is the fifth most common cancer worldwide and the fourth leading cause of cancer death. Because gastric cancer is almost asymptomatic in its early stages, most patients are diagnosed at an advanced stage, resulting in a poor prognosis. Therefore, early diagnosis remains a challenging and crucial area of ​​research. Currently, gastric cancer diagnosis primarily relies on endoscopic targeted biopsy, but its early sensitivity is low and it cannot effectively assess the patient's condition. Therefore, there is an urgent need to find new and effective diagnostic biomarkers. Furthermore, gastric cancer exhibits greater heterogeneity than other cancer types, being the third most common heterogeneous cancer worldwide with the highest mortality rate. Systemic treatment options for gastric cancer are limited. While immune checkpoint blockade (ICB) has brought profound changes to cancer treatment, a large number of patients still do not respond well to existing immunotherapies. Therefore, finding new and effective immunotherapeutic targets is a key research issue.

[0003] In recent years, the role of inositol 1,4,5-trisphosphate receptor-interacting protein-like 1 (ITPRIPL1) in tumor immune escape has attracted much attention. ITPRIPL1 is a single transmembrane protein that functions as a natural inhibitory ligand of CD3ε, its expression suppressing T cells in the tumor microenvironment and promoting tumor growth. The binding of the ITPRIPL1 extracellular domain to CD3ε on T cells significantly reduces calcium ion concentration and ZAP70 phosphorylation, thereby hindering the initial activation of T cells. Treatment with neutralizing antibodies inhibits tumor growth and promotes T cell transformation in mouse models of various types of solid tumors. Antibodies targeting canine ITPRIPL1 have shown significant therapeutic effects on naturally occurring tumors in pets. These findings highlight the role of ITPRIPL1 (or CD3L1, CD3ε ligand 1) in inhibiting T cell activation at a crucial "signal-1" stage. Therefore, ITPRIPL1 holds promise as a therapeutic target for various tumor types. However, ITPRIPL1 has not been systematically and clearly studied in gastric cancer. Summary of the Invention

[0004] The purpose of this invention is to address the above-mentioned problems by providing an application of ITPRIPL1 as a biomarker for the diagnosis or prognosis of gastric cancer.

[0005] To achieve its objective, the present invention employs the following technical solution:

[0006] The first aspect of the present invention provides the use of ITPRIPL1 as a biomarker in the preparation of diagnostic or prognostic agents for gastric cancer.

[0007] Compared with normal controls, the expression level of ITPRIPL1 in gastric cancer patients was significantly upregulated.

[0008] High expression of ITPRIPL1 in gastric cancer patients indicates a poor prognosis.

[0009] A second aspect of the invention provides the use of a reagent for detecting the expression level of the biomarker ITPRIPL1 in a sample in the preparation of gastric cancer diagnostic or prognostic products.

[0010] In the above-mentioned application technology solution, the sample includes tissues and cells.

[0011] In the above application technical solution, the tissue is gastric cancer tissue, and the cells are gastric cancer cells.

[0012] In the above application technology solutions, the product is a chip, test strip, or reagent kit.

[0013] In the above-mentioned application technology solutions, the product also includes negative controls, positive controls, or antibodies.

[0014] In the above application technology solution, the product includes reagents required for qPCR, immunohistochemistry or single-cell sequencing methods for ITPRIPL1 detection.

[0015] Preferably, the product includes an anti-ITPRIPL1 antibody and reagents required for immunohistochemical staining.

[0016] The beneficial effects of this invention are as follows: Differential analysis of the TCGA-STAD database revealed that the expression of the target molecule ITPRIPL1 in gastric cancer tissues is higher than that in adjacent normal tissues, and the level is even higher in cancer tissues with higher pathological grades (poorly differentiated / undifferentiated). Univariate Cox regression and survival analysis clarified that high expression of ITPRIPL1 in gastric cancer is associated with poor patient prognosis. GSEA enrichment analysis showed that ITPRIPL1 mainly activates gastric cancer EMT and promotes gastric cancer cell invasion through pathways such as cell adhesion molecules and cytokine receptor interactions. Single-cell sequencing analysis revealed that ITPRIPL1 is highly expressed on T cells and inhibits T cell function, promoting immune escape of tumor cells. Immunohistochemical staining of samples from clinically diagnosed gastric cancer patients further verified the high expression of this target in gastric cancer tumor cells, and the lower the degree of tumor differentiation, the higher the expression level of ITPRIPL1. This invention provides a new diagnostic and prognostic biomarker for gastric cancer and offers new ideas and directions for immunotherapy of gastric cancer. Attached Figure Description

[0017] Figure 1 The study revealed the association between ITPRIPL1 gene characteristics and disease: A. Subcellular localization of CD3L1, the protein encoded by the ITPRIPL1 gene; B. Differential expression of ITPRIPL1 in pan-cancer studies using the TIMER database.

[0018] Figure 2 The study revealed high expression of ITPRIPL1 in gastric cancer, suggesting a poor prognosis: A. Differences in gene expression between gastric cancer tissues and normal gastric tissues in the TCGA database; B. Differences in gene expression between 27 pairs of gastric cancer tissues and adjacent normal tissues in the TCGA database; C. Relationship between ITPRIPL1 expression and T stage, N stage, M stage, and tissue differentiation degree in gastric cancer patients; G. Analysis of ITPRIPL1 expression and overall survival (OS) prognosis using STAD data from the TCGA database; H. Prognostic analysis of ITPRIPL1 expression and progression-free survival (PFS) using STAD data from the TCGA database.

[0019] Figure 3 The study presents the single-gene associations of ITPRIPL1 with gastric cancer and its enrichment in gastric cancer: A. Heatmaps of the top 10 coding genes for ITPRIPL1 at the mRNA level in the TCGA database (positive and negative correlations); B. Predicted protein-protein interaction networks of ITPRIPL1 from the STRING database.

[0020] Figure 4 The study shows the single-gene association between the ITPRIPL1 gene and gastric cancer, as well as its enrichment analysis in gastric cancer: A. KEGG enrichment analysis of the top 100 genes positively associated with ITPRIPL1; B. Gene set enrichment analysis (GSEA) of differentially expressed ITPRIPL1 genes.

[0021] Figure 5 The expression of ITPRIPL1 was shown to be associated with macrophages and CD8+ in GC. + T cell infiltration is closely related to: A. TIMER immune infiltration analysis; B. EPIC immune infiltration analysis; C. XCELL immune infiltration analysis.

[0022] Figure 6 The results of single-cell analysis of the GSE184198 dataset are shown: A. Cell cluster annotation diagram; B. Violin plot of ITPRIPL1 expression in each cell population.

[0023] Figure 7The results of immunohistochemical staining were shown to confirm: A. ITPRIPL1 was significantly upregulated in tumors compared with adjacent normal tissues; B. The difference in ITPRIPL1 expression in solid tumor tissues and stroma suggests that ITPRIPL1 is significantly upregulated during carcinogenesis and is negatively correlated with the degree of tumor differentiation. Detailed Implementation

[0024] The present invention will be further described below with reference to embodiments, but these embodiments are not intended to limit the scope of the invention.

[0025] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0026] The experimental research methods of this invention include: analyzing the molecular characteristics and disease relevance of ITPRIPL1 using public databases; using the TCGA-STAD dataset for prognostic analysis, single-gene correlation, differential expression, functional enrichment, and immune infiltration analysis; constructing an ITPRIPL1 protein-protein interaction network using the STRING database; and performing single-cell analysis using the GEO database GSE184198 dataset. The expression of ITPRIPL1 in clinically diagnosed gastric cancer patients was assessed by immunohistochemical staining of paired samples.

[0027] The experimental results of this invention show that ITPRIPL1 expression in gastric cancer tissues is higher than that in adjacent normal tissues, and the level is even higher in cancer tissues with higher pathological grades (poorly differentiated / undifferentiated). Furthermore, high expression of ITPRIPL1 in gastric cancer is associated with poor patient prognosis. ITPRIPL1 also contributes to CD8+ expression in gastric cancer patients. + It is highly expressed in T cells and mainly activates gastric cancer EMT and promotes gastric cancer cell invasion through pathways such as cell adhesion molecules and cytokine receptor interactions. Immunohistochemical staining of paired samples from clinically diagnosed gastric cancer patients further confirmed the high expression of ITPRIPL1 in gastric cancer tumor cells and its positive correlation with the pathological grade of gastric cancer.

[0028] The specific research process is as follows:

[0029] Example 1

[0030] 1. Materials and Methods

[0031] 1.1 ITPRIPL1 gene information

[0032] The GeneCards database (https: / / www.genecards.org / ) is used to visualize the subcellular localization of human chromosomes and ITPRIPL1.

[0033] 1.2 Data Collection and Preprocessing

[0034] The expression differential of the ITPRIPL1 gene in pan-cancer tissues was displayed using the TIMER database (http: / / timer.cistrome.org / ). To further analyze ITPRIPL1 expression in gastric cancer, RNA expression and clinical data of gastric adenocarcinoma (STAD) from the Cancer Genome Atlas were downloaded from the UCSC-Xena database (https: / / xenabrowser.net / datapages / ). Transcriptome data downloaded from TCGA were in single files per sample; these individual files were merged into a matrix file. The expression level of ITPRIPL1 lncRNA was extracted from the Symbol matrix, and differential box plots of the target gene were constructed using the ggpubr R package. Paired box plots were also extracted from adjacent normal tissues and tumor tissues.

[0035] 1.3 Differential Expression Analysis

[0036] Differential expression of ITPRIPL1 at the mRNA level was analyzed using R (version 4.2.3) under different disease states (tumor or normal) and different cancer stages (including TNM staging and histological grade), and presented in the form of violin plots.

[0037] 1.4 Survival Analysis

[0038] Survival analysis (Kaplan–Meier analysis) based on log-rank P-value was performed using the survminer R package to analyze the relationship between ITPRIPL1 gene expression and overall survival (OS) and progression-free survival (PFS) in patients with TCGA-STAD data.

[0039] 1.5 Co-expression analysis

[0040] Correlation analysis of ITPRIPL1 with other mRNAs in gastric cancer tissues was performed using TCGA-STAD data to identify genes co-expressed with ITPRIPL1, with the selection criteria being (|Cor|>0.3 & P<0.001). The top 10 genes positively and negatively correlated with ITPRIPL1 were visualized using the ComplexHeatmap R package and presented as heatmaps. A protein-protein interaction network for ITPRIPL1 was constructed using the STRING database (http: / / string-db.org).

[0041] 1.6 Enrichment Functional Analysis

[0042] The top 100 genes positively correlated with ITPRIPL1 and the top 100 genes negatively correlated with ITPRIPL1 were selected for KEGG analysis, and the results are represented by a bubble chart. Based on the median expression level of ITPRIPL1, the samples were divided into high-expression and low-expression groups. GSEA enrichment was performed on the data from both groups using the clusterProfiler R package to analyze the differences in functional phenotypes and signaling pathways between the high-expression and low-expression groups of ITPRIPL1.

[0043] 1.7 Independent prognostic factor analysis

[0044] The expression level of ITPRIPL1 and clinical traits were analyzed using the survival R package in a multivariate Cox analysis to obtain the results of independent prognostic analysis.

[0045] 1.8 Immune infiltration analysis

[0046] Algorithms such as TIMER, EPIC, and XCELL were used to assess the correlation between genes and different types of immune cells in gastric cancer. Timer2 (http: / / timer.cistrome.org / ) was used to analyze the correlation between ITPRIPL1 expression and the expression of common immune cell types, including macrophages and CD8+. + T cells were used to investigate the effect of ITPRIPL1 expression on immune cell infiltration.

[0047] 1.9 Single-cell data analysis

[0048] Download single-cell data (GEO id: GSE184198) from the GEO database (https: / / www.ncbi.nlm.nih.gov / geo / ). Perform cell clustering analysis on the single-cell data using the Seurat R package to identify the cluster number corresponding to each cell. Annotate the cells using the SingleR R package. Using a reference dataset with known cell types (such as the Human Primary Cell Atlas) as a control, compare the gene expression characteristics of each cluster with the reference dataset to achieve cell type annotation and determine the specific cell type corresponding to each cluster. For the target gene ITPRIPL1, extract its expression data across all cells and use a heatmap visualization method to display the expression distribution: the gene is used as the x-axis, and the cell name as the y-axis; the color represents the mean expression of the gene in the cell type, with darker blue indicating a higher expression level of the target gene in that cell.

[0049] 1.10 Immunohistochemical staining

[0050] Ten pairs of fixed, newly diagnosed gastric cancer radical resection tissue blocks and paired adjacent normal tissues were coated with paraffin, sliced ​​into thin sections, and fixed onto glass slides. The tissue samples were obtained from the Department of Pathology, Yubei District People's Hospital, Chongqing, with ethical review approval document: Yubei Medical Ethics Approval (2025C08). First, the slides were dried in a 65℃ oven for 1 hour. Then, they were dewaxed three times in clean xylene, 10 minutes each time. After dewaxing, the slides were hydrated with a series of ethanol solutions of different concentrations (100% ethanol for 5 minutes, 95% ethanol for 5 minutes, 80% ethanol for 5 minutes, 75% ethanol for 5 minutes, ddH2O for 5 minutes). Next, the slides were immersed in clean PBS and washed three times on a shaker at 80 rpm for 5 minutes each time. The hydrated slides were then immersed in sodium citrate antigen retrieval solution and microwaved on high for 5 minutes, then on low for 5 minutes, and allowed to cool naturally afterward. Endogenous peroxidase was used to incubate the sections at room temperature for 10 min to reduce non-specific antibody binding. Anti-ITPRIPL1 antibody (Beijing Solarbio Science & Technology Co., Ltd.) was incubated with the sections at an appropriate dilution (according to the antibody instructions) overnight at 4°C. After three washes with PBS, an appropriate amount of reaction enhancement solution was added, and the sections were incubated at 37°C for 20 min. After incubation, the sections were washed three times with PBS, and the biotin-conjugated secondary antibody was incubated with the sections at room temperature for 30 min, followed by three more washes with PBS. Finally, DAB was added for staining, and hematoxylin and sulfadiazine were added to stain the cell nuclei. The sections were scanned using the iViewer digital slide management system.

[0051] 1.11 Statistical Analysis

[0052] All experiments were performed at least three times. All results are expressed as mean ± standard deviation (SD). Statistical analysis and graph generation were performed using GraphPad Prism 9 and R 4.2.3. Two-tailed Student's t-tests were used to analyze differences between groups, with * / P considered statistically significant.

[0053] 2. Experimental Results and Analysis

[0054] 2.1 Study on the correlation between ITPRIPL1 gene characteristics and tumor disease

[0055] ITPRIPL1 is located on the long arm of human chromosome 2, at 2q11.2, and consists of multiple exons. Subcellular localization results indicate that this gene encodes the protein CD3L1, a single-pass transmembrane protein primarily located on the outer side of the cell membrane. Figure 1A). To further explore the correlation between ITPRIPL1 gene expression and tumor diseases, we analyzed the expression profiles of 33 tumors in the TCGA database. The results showed that ITPRIPL1 expression was significantly higher in various tumors, including glioblastoma, esophageal cancer, primary hepatocellular carcinoma, and gastric cancer, than in normal tissues. Figure 1 B).

[0056] 2.2 ITPRIPL1 is highly expressed in gastric cancer tissues and is significantly associated with poor prognosis in patients.

[0057] Based on the pan-cancer differential expression results of ITPRIPL1, the differential expression of ITPRIPL1 gene in gastric cancer tissues and normal tissues was analyzed using the TCGA database. The results showed that ITPRIPL1 was significantly and highly expressed in TCGA-STAD. Figure 2 A); In 27 pairs of gastric cancer and paired adjacent normal tissues, ITPRIPL1 also showed a trend of high expression specific to tumor tissue ( Figure 2 B). Clinicopathological correlation analysis showed that ITPRIPL1 expression level was significantly positively correlated with T stage and pathological grade of gastric cancer (B). Figure 2 To evaluate the predictive value of ITPRIPL1 for prognosis and disease progression in gastric cancer patients, Kaplan-Meier survival analysis and log-rank test were performed using the survivor R package to analyze the association between ITPRIPL1 expression and overall survival (OS) and progression-free survival (PFS). Results showed that high ITPRIPL1 expression was significantly associated with shorter OS and PFS in gastric cancer patients, suggesting that high expression predicts a poor prognosis (Figure 2G-H). Further univariate and multivariate Cox regression analyses using the survivor R package showed that ITPRIPL1 expression levels were independent of traditional clinicopathological factors such as age, sex, and TNM stage, and could serve as an independent prognostic risk factor for gastric cancer patients (Tables 1 and 2).

[0058] Table 1

[0059]

[0060] Table 2

[0061]

[0062] 2.3 Gene association and functional enrichment analysis of ITPRIPL1 in gastric cancer

[0063] To further elucidate the regulatory mechanism and potential pathways of ITPRIPL1 in gastric cancer, co-expression correlation analysis was performed on ITPRIPL1 and whole-genome mRNA expression levels in gastric cancer based on the TCGA-STAD dataset. The top 10 genes showing significant positive and negative correlations with ITPRIPL1 were screened and identified. Figure 3 A). Simultaneously, an ITPRIPL1 protein-protein interaction (PPI) network was constructed using the STRING database (http: / / string-db.org). Core interacting proteins were selected based on interaction scores, in the following order: FAHD2B, FAM178B, WDR54, ANKRD39, TMEM102, ANKRD36, GPR183, CNNM3, ANKRD36C, and CIAO1 (…). Figure 3 B). Further KEGG pathway enrichment analysis was performed on the top 100 genes positively correlated with ITPRIPL1. The results showed that the above gene set was significantly enriched in miRNA cancer-related pathways, NSCLC / HCC tumor-driving pathways, and immune-related pathways such as T cell receptor (TCR) signaling pathway and chemokine signaling pathway. Figure 4 A). Based on the median ITPRIPL1 expression, TCGA-STAD samples were divided into high-expression and low-expression groups, and gene set enrichment analysis (GSEA) was performed using the clusterProfiler R package. The results showed that the high-expression group of ITPRIPL1 was significantly enriched in pathways such as cell adhesion molecules, cytokine-receptor interactions, hematopoietic cell lineages, and primary immunodeficiency. Figure 4 B). The above results suggest that high expression of ITPRIPL1 is involved in regulating multiple tumor-related pathways in gastric cancer, especially closely related to the activation of epithelial-mesenchymal transition (EMT) and abnormal regulation of immune signaling pathways.

[0064] 2.4 Correlation between ITPRIPL1 expression and immune cell infiltration in gastric cancer tissues

[0065] To clarify the association between ITPRIPL1 expression and immune cell infiltration in gastric cancer tissues, this study used three algorithms—TIMER, EPIC, and XCELL—to systematically analyze the correlation between ITPRIPL1 expression levels and the degree of infiltration by common immune cells. The TIMER algorithm analysis showed that ITPRIPL1 expression levels were associated with macrophages and CD8+. + The degree of T cell infiltration was significantly positively correlated with ( Figure 5 A); EPIC algorithm ( Figure 5 B) and XCELL algorithm ( Figure 5The analysis results in C) are consistent with the above conclusions, further verifying that ITPRIPL1 expression is related to macrophages and CD8. + A positive correlation exists between T cell infiltration and other factors.

[0066] 2.5 ITPRIPL1 is highly expressed on T cells and inhibits T cell function.

[0067] Single-cell sequencing analysis of GSE184198 cells showed that ITPRIPL1 was highly expressed mainly in T cells, with CD8 as the dominant expression level. + The expression level was most significant in T cells. Figure 6 (AB). This study found a significant positive correlation between ITPRIPL1 expression and the degree of T cell infiltration. This result, seemingly contradicting previous reports that ITPRIPL1 can inhibit T cell activation and mediate tumor immune escape, actually reflects the cell-origin specificity and functional stratification of this molecule in the tumor microenvironment. Previous studies have confirmed that ITPRIPL1 mainly exerts its inhibitory effect through T cell self-expression and binding to CD3ε. The positive correlation between ITPRIPL1 expression and T cell infiltration observed in this study is essentially a correlation at the cellular composition level, rather than at the T cell functional activation level, indicating that higher T cell abundance corresponds to higher levels of ITPRIPL1 transcripts. Based on these results, we hypothesize that ITPRIPL1 may be a characteristic molecule of tumor-infiltrating T cells, playing a depletion / inhibitory checkpoint role within T cells. Its high expression suggests an increased number of T cells in tumor tissue but suppressed function, ultimately promoting tumor cell immune escape.

[0068] 2.6 ITPRIPL1 can be used as a biomarker for gastric cancer diagnosis.

[0069] Preliminary bioinformatics analysis suggests that ITPRIPL1 may be involved in CD8+ in the gastric cancer tumor microenvironment. + To further verify the expression characteristics and clinical significance of ITPRIPL1 in gastric cancer tissues, this study used immunohistochemistry to detect the expression level of ITPRIPL1 protein in paired tumor tissues and adjacent normal tissues of patients who underwent radical gastrectomy. The results showed that ITPRIPL1 positive expression was mainly located in the cytoplasm, and the expression level of ITPRIPL1 protein in gastric cancer tissues was significantly increased compared with adjacent normal tissues. Figure 7 A). Further analysis revealed that the expression level of ITPRIPL1 protein was closely related to the degree of differentiation of gastric cancer tissue; the lower the degree of tumor tissue differentiation, the higher the expression level of ITPRIPL1. Figure 7B). The above immunohistochemical results are consistent with the previous bioinformatics analysis results, confirming that ITPRIPL1 is abnormally highly expressed in gastric cancer tissues, and that its high expression may be closely related to the malignant phenotype of tumors and the regulation of the immune microenvironment.

Claims

1. Application of ITPRIPL1 as a biomarker in the preparation of diagnostic or prognostic agents for gastric cancer.

2. The application according to claim 1, characterized in that: Compared with normal controls, the expression level of ITPRIPL1 was significantly upregulated in patients with gastric cancer.

3. The application according to claim 1, characterized in that: High expression of ITPRIPL1 in gastric cancer patients indicates a poor prognosis.

4. Application of reagents for detecting the expression level of the biomarker ITPRIPL1 in samples in the preparation of gastric cancer diagnostic or prognostic products.

5. The application according to claim 4, characterized in that: The samples include tissues and cells.

6. The application according to claim 5, characterized in that: The tissue is gastric cancer tissue, and the cells are gastric cancer cells.

7. The application according to claim 4, characterized in that: The product is a chip, test strip, or reagent kit.

8. The application according to claim 7, characterized in that: The product also includes negative controls, positive controls, or antibodies.

9. The application according to claim 7, characterized in that: The products include reagents required for qPCR, immunohistochemistry, or single-cell sequencing methods for ITPRIPL1 detection.

10. The application according to claim 9, characterized in that: The product includes anti-ITPRIPL1 antibody and reagents required for immunohistochemical staining.

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