Application of NLRC5 in targeted screening of medicine for improving immunotherapy esophageal squamous cell carcinoma

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to application of NLRC5 in targeted screening of medicines for improving immunotherapy of esophageal squamous carcinoma. NLRC5 expression is inhibited or regulated, and immunotherapy of esophageal squamous cell carcinoma is improved. The NLRC5 is highly methylated, and the expression of the NLRC5 in the ESCC is reduced. The NLRC5 is over-expressed, immune cell infiltration is promoted, meanwhile, immune escape is promoted by intensifying T cell depletion, immune checkpoint expression is up-regulated, and an immunosuppression state is created, so that anti-tumor immune response is potentially weakened. The invention discloses a polyhedral function of the NLRC5 in immune regulation and control from a single cell level, and particularly discloses close association of the NLRC5 with an immune checkpoint pathway and T cell depletion. A theoretical basis is provided for developing a therapy for targeting NLRC5, and a new way is possibly opened up for enhancing anti-tumor immune response. Researches show that NLRC5 is an important regulatory factor of immune response in ESCC, and plays a dual role in immune activation and immunosuppression.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to the application of NLRC5 in targeted screening of drugs to improve immunotherapy for esophageal squamous cell carcinoma. Background Technology

[0002] Esophageal squamous cell carcinoma (ESCC) is one of the deadliest malignant tumors worldwide, currently ranking seventh in incidence and sixth in cancer-related mortality. Despite advancements in treatment, its 5-year survival rate remains below 30%, highlighting the serious challenge. The poor prognosis of ESCC can be attributed to multiple factors, including difficulties in early detection, limited treatment options, insufficient understanding of its molecular mechanisms, and a lack of effective biomarkers. Although ESCC patients typically receive comprehensive treatment including surgical resection, chemotherapy, and radiotherapy, the overall prognosis remains poor, particularly for advanced-stage patients. Therefore, to improve early diagnosis, monitor tumor progression, and prevent metastasis in ESCC, it is crucial to develop novel and effective treatment strategies and identify reliable biomarkers that can predict prognosis. In recent years, immunotherapy, especially immune checkpoint inhibitors and chimeric antigen receptor (CAR) T-cell therapy, has achieved significant breakthroughs in the treatment of hematologic malignancies, becoming one of the most important advances in cancer treatment.

[0003] Despite the progress made by CAR-T cell therapy in hematological malignancies, its application in solid tumors remains challenging. The complex tumor microenvironment can induce T cell exhaustion and hinder the ability of CAR-T cells to infiltrate and clear solid tumors. Therefore, exploring the mechanisms of T cell immunosuppression in ESCC and identifying potential intervention targets are crucial to overcoming T cell exhaustion. Such research is urgently needed to improve the efficacy of immunotherapy for ESCC.

[0004] NLRC5, as an inflammasome sensor, exerts immunomodulatory effects by regulating major histocompatibility complex class I molecules and promoting widespread apoptosis of immune cells. Its role in tumor immunity is dual: evidence suggests it enhances the immune response, while also exhibiting immunosuppressive effects. However, the specific relationship between NLRC5 and ESCC immune regulation remains unclear. Summary of the Invention

[0005] To address this issue, this invention provides the application of NLRC5 in targeted screening of drugs to improve immunotherapy for esophageal squamous cell carcinoma (ESCC). This invention utilizes a comprehensive analysis combining the TCGA public dataset, published CancerCell cohort data (HRA003107), single-cell RNA sequencing data, and GEO databases (GSE53625 and GSE52826). Results show that high NLRC5 expression is associated with poor prognosis in ESCC patients. Further investigation explored the association between NLRC5 expression and tumor DNA methylation, employed various immune cell algorithms to study the relationship between NLRC5 and T cell infiltration, and performed correlation analysis between NLRC5 and immune checkpoints, while also observing its expression characteristics in single-cell data. The study found that high NLRC5 expression leads to poor ESCC outcomes by promoting T cell exhaustion and widespread immune cell apoptosis.

[0006] This invention is achieved through the following technical solution: the application of NLRC5 in targeted screening of drugs to improve immunotherapy for esophageal squamous cell carcinoma (ESCC), inhibiting or regulating NLRC5 expression to improve immunotherapy for ESCC. Furthermore, NLRC5 hypermethylation leads to downregulation of NLRC5 expression in ESCC.

[0007] Furthermore, NLRC5 overexpression promotes immune cell infiltration, while simultaneously exacerbating T cell exhaustion to promote immune escape, upregulating immune checkpoint expression, and creating an immunosuppressive state, thereby potentially weakening the anti-tumor immune response. Even further, NLRC5 overexpression upregulates the PD-1 / LAG3 pathway, participating in the immune exhaustion mechanism, and thus promoting tumor immune escape.

[0008] This invention reveals the multifaceted functions of NLRC5 in immune regulation at the single-cell level, particularly its close association with immune checkpoint pathways and T cell exhaustion. These findings provide a theoretical basis for developing NLRC5-targeted therapies and may open new avenues for enhancing anti-tumor immune responses. Studies have shown that NLRC5 is an important regulator of the immune response in ESCC, playing a dual role in both immune activation and immunosuppression. While high NLRC5 expression is associated with increased T cell infiltration, it also promotes T cell exhaustion, leading to the formation of immunosuppressive tumor cells (TMEs). Therefore, NLRC5 may be a promising therapeutic target for ESCC. Further research is needed to elucidate the exact mechanisms by which NLRC5 affects immune cell behavior and to evaluate whether strategies targeting NLRC5 can enhance the efficacy of ESCC immunotherapy. This invention emphasizes the crucial role of NLRC5 in the immune regulation and prognosis of ESCC and suggests its significant research and application prospects in the context of cancer immunotherapy, especially given the severe limitations imposed by the immunosuppressive mechanisms in ESCC. Attached Figure Description

[0009] Figure 1The figures show the expression of NLRC5 in various cancers. In the figures: (A) is a box plot based on the TCGA database for pan-cancer analysis, showing the comparison of NLRC5 expression levels (log2(TPM+1)) between tumor and normal tissues. The boxes represent the interquartile range, the horizontal line inside the box represents the median, the dashed line extends to 1.5 times the interquartile range, and discrete points represent outliers; (B) is a paired scatter plot of NLRC5 expression between paired tumors and adjacent normal tissues in the TCGA database. The lines show the expression differences between paired samples from the same patient, and the statistical significance markers are the same as above; (CE) are unpaired t-test box plots of NLRC5 mRNA expression in tumors and normal tissues in three independent cohorts: (C) is the TCGA cohort; (D) is the CancerCell cohort (HRA003107); (E) is the GSE53625 cohort. Box plot parameters were set as before; (F) is a bar chart of NLRC5 expression in ESCC cell lines from the CCLE database; (GH) is the qPCR validation result of relative NLRC5 mRNA expression in ESCC cell lines compared to normal esophageal epithelial cells (NE2(G) and NE3(H)); asterisks in the figure indicate statistical significance between groups: p<0.05, p<0.01, p<0.001; Figure 2 The prognostic significance of NLRC5 expression in different cohorts of esophageal squamous cell carcinoma (ESCC) is shown in the figure. In the figure: (AB) are Kaplan-Meier survival curves for overall survival in ESCC patients with high / low NLRC5 expression; (A) is the TCGA cohort (grouped according to median NLRC5 expression); (B) is the CancerCell cohort (grouped using log-rank test). p-values ​​were calculated using the log-rank test. (CD) are univariate Cox regression analyses of overall survival; (C) is the TCGA cohort; (D) is the CancerCell cohort. The hazard ratios and 95% confidence intervals for each variable, including NLRC5 expression grouping, are shown, with p-values ​​indicating variable significance. (EF) are multivariate Cox regression analyses of overall survival; (E) is the TCGA cohort; (F) is the CancerCell cohort (adjusted for other clinical variables). The hazard ratios, 95% confidence intervals, and p-values ​​for each factor are shown, highlighting the independent prognostic value of NLRC5 expression. Figure 3The relationship between NLRC5 methylation levels and mRNA expression in each cohort is shown. In the figure: (A) Box plot of average NLRC5 methylation levels in tumor and normal tissues in the TCGA cohort (analyzed using an unpaired t-test); (B) Lollipop plot of the correlation between NLRC5 methylation and expression. Red dots represent transcription start sites, and green dots represent sites with a correlation coefficient greater than 0.3; (C) Spearman correlation scatter plot of NLRC5 mRNA expression and overall gene average methylation level; (D) Spearman correlation scatter plot of NLRC5 mRNA expression and average methylation level in the TSS region; (E) Spearman correlation scatter plot of NLRC5 mRNA expression and average gene-wide average methylation level; (F) Box plot of average NLRC5 methylation levels in tumor and normal tissues in the CancerCell cohort (analyzed using an unpaired t-test); (G) Box plot of average NLRC5 methylation levels in tumor tissue, adjacent normal tissue, and normal muscle tissue in the GSE52826 cohort (analyzed using an unpaired t-test). Figure 4 Functional enrichment analysis of NLRC5-related genes in the TCGA and CancerCell cohorts; Figure: (AD) shows the results of the TCGA cohort analysis: (A) GO biological process enrichment analysis showed significant enrichment of immune-related processes such as T cell activation, viral defense response, and adaptive immune response; (B) GO cell component analysis showed enrichment of cell surface and various membrane-related components; (C) GO molecular function analysis showed functional enrichment of protein binding, receptor binding, and MHC class II protein complex binding; (D) KEGG pathway analysis showed significant enrichment of pathways such as allogeneic transplant rejection, cell adhesion molecules, type I diabetes, and antigen processing and presentation; (EH) shows the results of the CancerCell cohort analysis: (E) GO biological process analysis showed significant enrichment of pathways such as immune response, T cell receptor signaling pathway, and γ-interferon production; (F) GO cell component analysis was similar to that of the TCGA cohort, showing enrichment of various membrane-related components; (G) GO molecular function analysis showed enrichment of receptor binding, MHC... Significant enrichment was observed in functions such as class II receptor activity and T-cell receptor binding; (H)KEGG pathway analysis showed significant enrichment in pathways related to type 1 diabetes, allogeneic transplant rejection, and antigen processing and presentation; In the figure: bubble size represents the number of genes contained in the enriched pathway, color depth indicates the significance of enrichment, expressed as -log 10 (P-value) indicates that the deeper the blue color, the more significant the enrichment. Figure 5This study compares immune infiltration between the TCGA and CancerCell cohorts for high / low NLRC5 expression groups. In the figures: (AD) is a box plot of tumor purity, matrix score, immune score, and ESTIMATE composite score based on NLRC5 expression levels (low vs. high) in the TCGA cohort. Unpaired t-tests were used to assess statistical differences between groups; (EH) is a box plot of the corresponding immune scores in the CancerCell cohort. The consistency between the two cohorts further validates the reliability of NLRC5 as an immune regulator. (IJ) CIBERSORT analysis shows the relative proportions of various immune cells in the low / high NLRC5 expression groups in the TCGA and CancerCell cohorts. The analysis shows significant differences in immune cell infiltration, particularly in T cells and macrophage subsets, where high NLRC5 expression is associated with increased immune cell infiltration. (KL) Single-sample gene set enrichment analysis shows the differential expression of immune-related gene sets between the different NLRC5 expression groups in the two cohorts. Notably, high NLRC5 expression is associated with enhanced activation of multiple immune pathways, suggesting its role in the regulation of the tumor immune microenvironment. ( p<0.05, p<0.01, p<0.001); Figure 6 This section presents analyses of immune cell infiltration and immune checkpoints in the TCGA and CancerCell cohorts, as well as TIDE analysis of the NLRC5 high / low expression groups. In the figure: (AB) shows the correlation between NLRC5 expression and immune cell infiltration in the TCGA cohort (A) and CancerCell cohort (B) using CIBERSORT analysis. Memory CD4 + T cells and macrophages, among other immune cells, showed a significant correlation, highlighting the immunomodulatory potential of NLRC5; (CD) is a radar plot showing the correlation between NLRC5 expression and key immune checkpoint molecules in the TCGA cohort (C) and CancerCell cohort (D). High NLRC5 expression was associated with the upregulation of multiple immunosuppressive checkpoint molecules, suggesting its potential involvement in immune escape; (EF) is a box plot further illustrating the distribution of different immune cell types in the NLRC5 high / low expression groups of the TCGA cohort (E) and CancerCell cohort (F). p<0.05, p<0.01, (p<0.001); (GH) is a violin plot showing the TIDE analysis results of the TCGA cohort (G) and the CancerCell cohort (H). High NLRC5 expression was associated with higher TIDE scores, suggesting that tumors with high NLRC5 expression may have an immune escape mechanism; Figure 7 Identification of differentially expressed genes in the TCGA and CancerCell cohorts and their correlation with T cell subtypes: focusing on the T cell exhaustion pathway; Figure: (A) Venn diagram showing the intersection of differentially expressed genes in the NLRC5 high / low expression groups in the TCGA and CancerCell cohorts; (B) Protein interaction network diagram of the intersection DEGs, highlighting the key gene interactions driven by NLRC5 expression, in which multiple immune-related genes show high connectivity; (CD) Gene set enrichment analysis of the TCGA cohort (C) and CancerCell cohort (D), showing that the T cell exhaustion pathway is significantly upregulated in the NLRC5 high expression group. Key exhaustion markers and immune checkpoint pathways are significantly enriched, suggesting that NLRC5 is involved in the regulation of T cell exhaustion; (EM) Correlation analysis of NLRC5 expression and various T cell subtypes based on GEPIA data. Notably, both (G) exhausted T cells and (I) settled T cells showed a significant positive correlation with NLRC5 expression, with the exhausted T cell subtype exhibiting the highest correlation, suggesting that NLRC5 may be involved in promoting or maintaining T cell exhaustion. Other T cell subtypes also showed moderate to high correlations, confirming the broad-based influence of NLRC5 on T cell immune regulation. Figure 8 Single-cell RNA sequencing analysis of NLRC5 subtypes in different tissues and immune cells (with a focus on CD8) + Expression and correlation of NLRC5 in T cells; Figure: (A) is a dot plot showing the expression distribution of NLRC5 RNA in different tissue types, including peripheral blood mononuclear cells, lymph nodes (normal and metastatic lesions), and esophageal tissue. Notably, NLRC5 is highly expressed in PBMCs, suggesting its important role in circulating immune cells; (B) is a visualization of T cell subtypes using single-cell data UMAP, clearly marking CD8. + (C) shows the spatial distribution characteristics of immune subsets such as T cells and Tregs; (D) is a UMAP projection map of NLRC5 expression levels in T cell subtypes, showing its enrichment in specific immune cell populations, suggesting its immunomodulatory function; (EH) is a dot plot quantifying the expression level of NLRC5 in various immune cell subtypes, especially showing high expression in T cells and some myeloid cells, further confirming its participation in the tumor immune microenvironment; (EH) is CD8. + Correlation analysis of NLRC5 with immune checkpoint molecules within T cell subsets. NLRC5 showed significant positive correlations with PDCD1, ENTPD1, ​​LAG3, and HAVCR2, with the strongest correlation observed with PDCD1. These findings suggest that NLRC5 may participate in CD8 activation by regulating the immune checkpoint pathway. + T cell depletion process; Figure 9This paper presents the correlation network and pathway enrichment analysis of NLRC5 and PANoptosis-related genes in three cohorts. In the figure: (AC) shows the correlation network of NLRC5 and PANoptosis-related genes in the TCGA cohort (A), CancerCell cohort (B), and GSE53625 cohort (C); the thickness of the lines indicates the correlation strength, and the color intensity represents the direction and significance of the correlation; (DF) shows the gene set enrichment analysis of the high / low NLRC5 expression groups in the TCGA cohort, displaying the enrichment of the (D) apoptosis signaling pathway, (E) necroptosis signaling pathway, and (F) pyroptosis pathway. Each pathway is labeled with a normalized enrichment score and a false discovery rate (q-value); (GI) shows the GSEA analysis in the CancerCell cohort, displaying the enrichment of apoptosis (G), necroptosis (H), and pyroptosis (I) pathways; (JL) shows the GSEA analysis in the GSE53625 cohort, exhibiting a similar enrichment pattern of apoptosis (J), necroptosis (K), and pyroptosis (L) pathways to the cohorts mentioned above. Each queue and path is labeled with its corresponding NES and FDR values. Detailed Implementation

[0010] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. Obviously, the described embodiments are some embodiments of the present invention, but not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0011] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains, and all materials publicly cited herein and cited by them are incorporated herein by reference.

[0012] Equivalent technologies of the specific embodiments described herein that are readily apparent to those skilled in the art through routine experimentation are included in this application.

[0013] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all standard laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from regular biochemical reagent stores.

[0014] I. Materials and Experimental Methods: 1. Clinical Samples: This invention includes three ESCC batch sequencing cohorts. These include transcriptome and methylation data from 155 ESCC tissue samples and paired adjacent normal tissues (HRA003107, PMID: 36584672). Additionally, GSE53625 transcriptome data from 179 paired ESCC samples were obtained from the GEO database. Furthermore, TCGA pan-cancer datasets and ESCC-specific data were integrated, and GSE52826 methylation data from 12 samples in the GEO database were also used.

[0015] 2. Cell Culture: All cell lines were preserved in the laboratory. Immortalized esophageal epithelial cells NE2 and NE3 were cultured in GIBCOdKSFM medium supplemented with EDGS and 1% penicillin-dip antibiotics; ESCC cell lines were cultured in RPMI-1640 medium containing 10% fetal bovine serum and 1% penicillin-dip antibiotics at 37°C and 5% CO2. All cells were identified by short tandem repeats and were negative for mycoplasma.

[0016] 3. Functional enrichment analysis: Pearson correlation coefficients between genes in each cohort and NLRC5 were calculated. Genes with the strongest correlations or genes characteristic of specific cell clusters were selected and submitted to the DAVID database for annotation analysis. Using official gene symbols as identifiers, Homo sapiens was selected as the species. Gene Ontology and KEGG pathway enrichment results were obtained, and the top 5 significant entries were displayed in ascending order of p-value.

[0017] 4. Methylation Analysis: Methylation and gene expression data were obtained from the CancerCell, TCGA, and GSE52826 cohorts, and gene annotation was performed using the bioomaRt package in RStudio. Unpaired tests were used to compare the mean methylation levels between cancer and adjacent normal tissues, and the Spearman assay was used to assess the correlation between methylation and gene expression.

[0018] 5. Immune Infiltration Analysis: The estimate R package was used to calculate the matrix score, immune score, overall score, and tumor purity of ESCC samples from three cohorts. The CIBERSORT deconvolution algorithm was used to quantify the proportion of 22 immune cell types based on expression profiles, with the sum of cell scores for each sample limited to 1. The ssGSEA method of the GSVA package was used to assess the infiltration degree of 28 immune cell types.

[0019] 6. xCell analysis: The xCell R package was used to analyze the transcriptome data of the three cohorts, calculate the relative abundance of 64 cell types, compare the differences in cell scores between cancer and normal tissues, and reveal the compositional characteristics of immune / stromal cells in the tumor microenvironment.

[0020] 7. TIDE analysis: Based on the TIDE framework, gene expression characteristics were integrated to assess the degree of T cell dysfunction and rejection in three cohorts of ESCC patients and to predict tumor immune escape mechanisms.

[0021] 8. Gene set enrichment analysis: Gene sets related to T cell exhaustion, apoptosis, necroptosis and pyroptosis were obtained from the MSigDB database. The TCGA, CancerCell and GSE53625 cohorts were divided into high / low expression groups based on the median expression of NLRC5. Pathway enrichment analysis was performed using GSEA software.

[0022] 9. scImpute Analysis and Correlation Calculation: scImpute was used to process single-cell RNA-seq data to correct for missed gene detections. TF tissues and CD8 were screened in this cohort. + T cell subset data were used, with missing values ​​imputed using default parameters. The correlation between NLRC5 and immune checkpoint genes was calculated using the Pearson correlation method, and a scatter plot with linear regression fitting was plotted, with correlation coefficients and p-values ​​labeled.

[0023] II. Experimental Results 1. High expression of NLRC5 in pan-cancer and esophageal squamous cell carcinoma: To assess the expression of NLRC5 in various cancers, we analyzed pan-cancer datasets using the Xiantao Academic Platform. The results showed that in most cancer types, NLRC5 expression in tumor tissues was significantly higher than in normal tissues (…). Figure 1 AB). In all three ESCC cohorts, NLRC5 expression levels in tumor tissues were significantly higher than in adjacent normal tissues (AB). Figure 1 C), this finding was further validated in two independent datasets ( Figure 1 DE).

[0024] To investigate NLRC5 expression in ESCC cell lines, we searched the CCLE database but found no normal esophageal epithelial control cells. Therefore, we used qPCR to detect NLRC5 expression in laboratory-cultured cell lines and found that most ESCC cell lines showed higher NLRC5 expression than NE2 and NE3 cells. However, the expression levels in COLO680N, TE5, and TE9 cell lines were similar to those in NE2 and lower than those in NE3 cells. Figure 1 FH).

[0025] 2. High NLRC5 expression predicts poor prognosis in esophageal squamous cell carcinoma: To assess the prognostic value of NLRC5 in ESCC, we conducted survival analyses in the TCGA and CancerCell cohorts. Kaplan-Meier curves showed that patients with high NLRC5 expression had significantly shorter overall survival in both cohorts (TCGA cohort: p = 0.016; CancerCell cohort: p = 0.012). Figure 2 AB).

[0026] Univariate Cox regression analysis confirmed that NLRC5 was a risk factor for poor prognosis (TCGA cohort: HR = 3.103, 95% CI = 1.216–7.915, p = 0.018; CancerCell cohort: HR = 2.613, 95% CI = 1.202–5.678, p = 0.015). Figure 2 CD). However, other variables such as age and N stage show inconsistent correlations.

[0027] Multivariate Cox analysis further validated that NLRC5 was an independent prognostic factor (TCGA cohort: HR = 3.391, 95% CI = 1.161–9.907, p = 0.026; CancerCell cohort: HR = 2.392, 95% CI = 1.077–5.314, p = 0.032). Figure 2 EF). N stage also remained significantly correlated. These findings suggest that NLRC5 holds promise as an effective prognostic biomarker for overall survival in ESCC patients across various clinical contexts.

[0028] 3. NLRC5 methylation level is associated with its downregulation in esophageal squamous cell carcinoma: To explore the epigenetic regulatory mechanism of NLRC5 in ESCC, we analyzed the methylation and expression data of the TCGA, CancerCell, and GSE52826 cohorts.

[0029] Compared with normal esophageal tissue, the NLRC5 methylation level in tumor tissue showed an overall increasing trend (TCGA cohort: no statistical significance, P>0.05; CancerCell and GSE52826 cohorts: P<0.05). Figure 3 (A, FG). In the GSE52826 cohort, methylation levels progressively increased from normal tissue, adjacent normal tissue to tumor tissue, suggesting that it may be involved in the progression of ESCC.

[0030] Spearman correlation analysis showed that NLRC5 methylation levels were significantly negatively correlated with mRNA expression across all cohorts. Figure 3 CE). Notably, methylation modifications near the transcription start site showed the strongest negative correlation, a feature visually represented by a lollipop diagram. Figure 3 (B, D). These findings suggest that hypermethylation is a key mechanism leading to the downregulation of NLRC5 expression in ESCC.

[0031] 4. Functional enrichment analysis of NLRC5-related genes in esophageal squamous cell carcinoma: To explore the biological function of NLRC5 in ESCC, we screened NLRC5-related genes in the TCGA, CancerCell, and GSE53625 cohorts using Pearson correlation analysis (|R|>0.5, P<0.05) and performed GO and KEGG enrichment analyses.

[0032] In the TCGA cohort, the relevant genes are mainly enriched in biological processes such as T cell activation, adaptive immune response, and antiviral defense (GO-BP), located in cellular components such as the endoplasmic reticulum membrane and plasma membrane (GO-CC), and involved in molecular functions such as MHC class II molecules and peptide antigen binding. Figure 4 AC. KEGG pathway analysis showed significant enrichment in pathways such as antigen presentation, graft-versus-host disease, and cell adhesion. Figure 4 D).

[0033] CancerCell and the GSE53625 cohort showed similar enrichment patterns: GO analysis included items such as interferon-gamma regulation and T cell receptor signaling pathways, and cellular components remained concentrated in membrane-associated regions. Figure 4 EG). KEGG analysis further confirmed its association with antigen processing and immune signaling pathways. Figure 4 H; Appendix Figure 2 This suggests that NLRC5 is closely related to the immune microenvironment of ESCC through antigen presentation and T cell-mediated immune regulation.

[0034] 5. NLRC5 Regulation of Immune Infiltration Characteristics in Esophageal Squamous Cell Carcinoma: Based on the significant enrichment of NLRC5-related genes in immune pathways, we used ESTIMATE, CIBERSORT, ssGSEA, and xCell algorithms to explore their association with the tumor microenvironment.

[0035] In the TCGA and CancerCell cohorts, ESTIMATE analysis showed that high NLRC5 expression was closely associated with lower tumor purity, higher immune score, matrix score, and overall score. Figure 5 AH) indicates enhanced infiltration of immune cells and stromal cells. The CIBERSORT algorithm further reveals that CD8+ expression was higher in the NLRC5-high expression group in both cohorts. + The proportion of T cells was significantly increased ( Figure 5 IJ) indicates that it may promote T cell infiltration.

[0036] ssGSEA analysis confirmed that CD8 activation was present in the NLRC5 high expression group. + T cells, CD4 + T cell subsets, NK cells, macrophages, and helper T cells were all enriched. Figure 5 KL), reflecting widespread immune activation. These findings were consistently validated in the GSE53625 cohort and xCell analysis (see appendix). Figure 3-4 ).

[0037] Notably, although high NLRC5 expression is associated with enhanced immune infiltration, it is also associated with poor prognosis. This paradoxical phenomenon suggests that NLRC5 may simultaneously participate in the process of immune dysfunction or T cell depletion, acting similarly to a potential immune checkpoint regulator. This invention reveals for the first time that NLRC5 has a dual regulatory function of immune activation and inhibition in ESCC.

[0038] 6. Analysis of Immune Infiltration Characteristics and Functional Impairment Indicators: The correlation between NLRC5 expression and immune cell infiltration in the TCGA and CancerCell cohorts was evaluated using the CIBERSORT algorithm. Figure 6 AB). Analysis showed that in tumor tissues with high NLRC5 expression, CD8 + T cells, activated memory CD4 + T cells and NK cells increased significantly. At the same time, the infiltration levels of macrophage subtypes such as M0, M1 and M2 were also higher, which further confirms the important role of NLRC5 in regulating the immune microenvironment.

[0039] Figure 6 The radar plot of CD showed the correlation between NLRC5 expression and immune checkpoint markers. Notably, PD1, CTLA4, TIM3, and LAG3 showed higher expression levels in the NLRC5 high-expression group. Figure 6 These markers are typically associated with T cell exhaustion. This suggests that while high NLRC5 expression may promote immune cell infiltration, it may also potentially weaken anti-tumor immune responses by creating an immunosuppressive state through upregulation of immune checkpoint expression.

[0040] Finally, we assessed tumor immune escape using TIDE analysis. Figure 6 Although the total TIDE score between the high and low NLRC5 expression groups did not reach statistical significance, the dysfunction score of the high expression group was significantly higher, indicating more severe T cell dysfunction. This result supports the hypothesis that NLRC5 overexpression may promote immune escape by exacerbating T cell exhaustion, and its mechanism of action is similar to that of PD1 immunosuppressive effects.

[0041] These results indicate that while NLRC5 enhances immune infiltration, it paradoxically induces an immunosuppressive state, impairs T cell function, and ultimately leads to a poor prognosis in ESCC patients. This dual role of NLRC5—both promoting immune responses and suppressing immune function— warrants further investigation into its therapeutic significance.

[0042] 7. Identification of Differentially Expressed Genes in TCGA and CancerCell Cohorts and Correlation Analysis with T Cell Subtypes: This invention analyzed differentially expressed genes between the NLRC5 high / low expression groups in the TCGA and CancerCell cohorts. Venn diagrams showed significant overlap in DEGs between the two cohorts, indicating that NLRC5 has a common mechanism of action across different datasets. Protein-protein interaction networks constructed based on shared DEGs highlighted the close association of multiple immune-related genes, with immune checkpoint molecules such as PD-1, CTLA-4, and TIM-3 being particularly prominent, suggesting that NLRC5 may regulate immune-related pathways in the tumor microenvironment.

[0043] Gene set enrichment analysis showed that the NLRC5 high-expression group in both cohorts was significantly enriched in T cell exhaustion-related pathways. In particular, immune checkpoint-related genes such as PD-1, CTLA-4, and TIM-3 were significantly enriched, suggesting that NLRC5 may participate in tumor immunosuppression by regulating T cell exhaustion.

[0044] Correlation analysis of NLRC5 expression with T cell subtypes using the GEPIA database revealed a strong positive correlation between exhausted T cells and NLRC5 expression. Furthermore, subtypes such as resident T cells, effector Treg cells, central memory T cells, and effector memory T cells also showed moderate to high correlations. These findings collectively confirm the broad influence of NLRC5 on T cell immune regulation, particularly its potential role in promoting T cell exhaustion.

[0045] 8. Single-cell transcriptome analysis of NLRC5 in different tissue types and immune subtypes: Using single-cell RNA sequencing technology, we systematically elucidated the expression profile of NLRC5 in different human tissues and immune cell subtypes, highlighting its role in regulating immune responses (especially CD8+ in the tumor microenvironment). + The key role of T cells in T cell function. Figure 8 As shown in AD, NLRC5 exhibits a differential expression pattern: significantly high expression in peripheral blood mononuclear cells ( ). Figure 8 A), suggesting its involvement in the systemic immune surveillance process.

[0046] UMAP map identification by integrating T cell subtypes ( Figure 8 B) and NLRC5 expression profile ( Figure 8 C), NLRC5 was found in CD8 + NLRC5 is specifically enriched in T cells, suggesting its potential involvement in T cell function regulation and exhaustion processes. Dot plot analysis further confirms the high expression of NLRC5 in T cells and some myeloid cells. Figure 8(D) suggests that this molecule may influence tumor immunogenicity by shaping interactions between immune cells.

[0047] Correlation analysis showed that NLRC5 was significantly associated with key immune checkpoint molecules such as PDCD1, ENTPD1, ​​LAG3, and HAVCR2, especially in CD8. + In T cell subsets, NLRC5 showed a strong positive correlation with PDCD1 and a significant positive correlation with LAG3. These results suggest that NLRC5 may participate in immune exhaustion mechanisms by regulating pathways such as PD-1 / LAG3, thereby promoting tumor immune escape.

[0048] This study reveals the multifaceted functions of NLRC5 in immune regulation at the single-cell level, particularly its close association with immune checkpoint pathways and T cell exhaustion. These findings provide a theoretical basis for developing therapies targeting NLRC5 and may open new avenues for enhancing anti-tumor immune responses.

[0049] 9. NLRC5 promotes pan-apoptosis of immune cells: In addition to inducing functional exhaustion, tumor-infiltrating immune cells may also undergo various forms of programmed cell death. Given the role of NLRC5 in immune regulation, we further investigated whether it participates in the totipotency apoptosis of immune cells, thus constituting an additional tumor-associated immunosuppressive mechanism.

[0050] Previous studies have suggested that NAD + A deficiency may lead to increased NLRC5 expression, subsequently triggering immune cell death. To test this hypothesis, we focused on analyzing key genes associated with pluripotency apoptosis. The results showed that in three independent cohorts, NLRC5 was significantly positively correlated with several important regulatory factors, especially AIM2 and GSDME (…). Figure 9 AC).

[0051] Panapoptosis integrates three major cell death pathways: necrosis, apoptosis, and pyroptosis. To further explore the role of NLRC5, we conducted gene set enrichment analysis (GSEA) using cohort data from groups with different NLRC5 expression levels. The results showed that in the TCGA cohort, the high NLRC5 expression group had apoptosis (…). Figure 9 D) Necrosis ( Figure 9 E) and pyrolysis ( Figure 9 The F) pathway was significantly upregulated. This finding was subsequently validated in the CancerCell cohort. Figure 9 GI). Furthermore, in the GSE53625 cohort, we detected [apoptosis-related factor]. Figure 9 J) and pyrolysis ( Figure 9 L)-related pathways were significantly enriched, although necrosis pathways ( Figure 9K) did not reach statistical significance, but still showed an upward trend.

[0052] The above results were validated in both the TCGA and GSE53625 independent cohorts, confirming that NLRC5 plays a key role in promoting apoptosis of immune cell pluripotency.

[0053] In recent years, immunotherapy has revolutionized cancer treatment, but its efficacy varies significantly across different malignant tumors. In China, esophageal squamous cell carcinoma (ESCC), which accounts for the majority of esophageal cancers, still faces significant challenges in treatment, primarily due to its highly immunosuppressive tumor microenvironment (TME). This TME is characterized by low levels of immune cell infiltration, typically resembling an "immunely cold" tumor, leading to resistance to immune checkpoint inhibitors and CAR-T therapies in ESCC. A deeper understanding of the molecular mechanisms driving immune escape in ESCC is crucial for overcoming treatment bottlenecks and improving patient prognosis.

[0054] NLRC5, also known as MHC class I transcription activator (CITA), plays a central role in regulating the MHC class I antigen presentation pathway. A series of studies have shown that NLRC5 deficiency can lead to impaired expression of MHC class I genes, thereby affecting CD8 expression. + Insufficient T cell activation promotes tumor immune escape. Meanwhile, NLRC5 gene expression levels are closely related to the survival rate of patients with various cancers, with high expression often indicating longer survival. However, there are also contradictory reports indicating that high NLRC5 expression is associated with poor prognosis. These discrepancies highlight the complex regulatory mechanisms of NLRC5 in tumors. It is noteworthy that although NLRC5 has been extensively studied in other tumors, its role in ESCC has been rarely explored; only one report suggests its potential oncogenic function, but its immunomodulatory effects have not been thoroughly investigated.

[0055] In this study, we explored the prognostic and immunological significance of NLRC5 in ESCC. Based on data from multiple large-scale clinical cohorts, we confirmed that NLRC5 expression was higher in tumor tissues than in normal tissues. Survival analysis and Cox regression showed that NLRC5 overexpression was significantly associated with poor prognosis and was an independent risk factor for ESCC.

[0056] Gene silencing caused by promoter hypermethylation is a common feature in cancer. Previous studies have suggested that the NLRC5 promoter methylation level is closely related to its gene expression²³. Our analysis shows that the NLRC5 promoter methylation level in tumor tissues is lower than that in normal tissues, and methylation is negatively correlated with expression, suggesting that epigenetic regulation may lead to abnormal expression of NLRC5 in ESCC.

[0057] Functional enrichment analysis revealed that NLRC5 is closely related to immune regulation. Using various immune cell infiltration algorithms, we observed a paradox: high NLRC5 expression is associated with both elevated immune scores and CD8+. + Increased T-cell infiltration was associated with poor patient prognosis. This intriguing phenomenon suggests that NLRC5 may be similar to immune checkpoint molecules such as PD-1, participating in T-cell exhaustion and promoting immune escape. Correlation analysis showed that NLRC5 expression was positively correlated with multiple immune checkpoints, and TIDE analysis also indicated that patients with high NLRC5 expression had higher functional impairment scores. Furthermore, differential gene PPI network analysis between the NLRC5 high and low expression groups placed immunosuppressive molecules such as PD-1 at the center. Further GSEA results showed that the T-cell exhaustion pathway was significantly upregulated in tumors with high NLRC5 expression. Single-cell RNA sequencing revealed that NLRC5 expression levels in PBMCs were higher than in other tissues, mainly concentrated in T cells, especially CD8 cells. + T cell subsets. Within this subset, NLRC5 expression was strongly correlated with exhaustion markers such as PDCD1 and LAG3.

[0058] Furthermore, recent research has found that NAD + Deficiency can lead to increased NLRC5 expression, thereby promoting apoptosis of immune cells. Tumor cells often undergo NAD due to metabolic abnormalities (preferring glycolysis, i.e., the Warburg effect). + Deficiency weakens oxidative phosphorylation, a major NAD+ pathway. + Source: NAD + Exhaustion disrupts key cellular functions, promoting tumor progression and treatment resistance. Our analysis showed that NLRC5 was significantly positively correlated with pluripotency apoptosis, necrotizing apoptosis, and genes related to apoptosis and pyroptosis, a result that was also validated by GSEA.

[0059] In summary, our research indicates that NLRC5 is a crucial regulator of the immune response in ESCC, playing a dual role in both immune activation and immunosuppression. While high NLRC5 expression is associated with increased T cell infiltration, it also promotes T cell exhaustion, leading to the formation of an immunosuppressive TME. Therefore, NLRC5 may be a promising therapeutic target for ESCC. Further research is needed to elucidate the exact mechanisms by which NLRC5 influences immune cell behavior and to assess whether strategies targeting NLRC5 can enhance the efficacy of ESCC immunotherapy.

[0060] In summary, this invention highlights the crucial role of NLRC5 in the immune regulation and prognosis of ESCC, and suggests its significant research and application potential in cancer immunotherapy, especially given the severely limited efficacy of ESCC immunosuppressive mechanisms.

[0061] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of NLRC5 in targeted screening of drugs for improving immunotherapy of esophageal squamous cell carcinoma, characterized in that: Inhibition or regulation of NLRC5 expression can improve the immunotherapy of esophageal squamous cell carcinoma.

2. Use according to claim 1, characterized in that: NLRC5 is highly methylated, and NLRC5 expression is down-regulated in ESCC.

3. Use according to claim 1, characterized in that: NLRC5 overexpression promotes immune cell infiltration, and promotes immune escape by exacerbating T cell exhaustion, up-regulates immune checkpoint expression, and creates an immunosuppressive state, thereby potentially weakening the anti-tumor immune response.

4. Use according to claim 1, characterized in that: NLRC5 overexpression up-regulates the PD-1 / LAG3 pathway and participates in the immune exhaustion mechanism, thereby promoting tumor immune escape.

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