SLC9A9 links tumor immune infiltration to therapeutic response in colorectal cancer with emphasis on mismatch repair proficient subtype

Abstract

Background: Immunotherapy has revolutionized colorectal cancer (CRC) treatment, however, predictive biomarkers for mismatch repair proficient (pMMR) tumors remain scarce. The involvement of the SLC9A9 (solute carrier family 9 member A9) gene in the tumor immune microenvironment is poorly understood.

Methods: We analyzed publicly available gene expression datasets to assess SLC9A9 expression levels in CRC patients. The relationships between SLC9A9 expression and lymphocyte infiltration, specifically CD8 + and CD4 + T cells, were examined using correlation and regression analyses.

Results: Expression of SLC9A9 was notably higher in normal CRC tissues compared to the adjacent tumor tissues, particularly among pMMR CRC patients. Moreover, a significant association was found between SLC9A9 expression and lymphocyte infiltration in these pMMR patients. Interestingly, pMMR patients exhibiting high SLC9A9 expression showed enhanced sensitivity to immunotherapy compared to their counterparts with low SLC9A9 expression.

Conclusions: Our study highlights the prognostic value of SLC9A9 in pMMR CRC and its correlation with lymphocyte infiltration, particularly CD8 + and CD4 + T cells. These results provide a foundation for further investigation into the mechanistic link between SLC9A9 expression and tumor immunity, potentially facilitating the creation of innovative therapeutic approaches for pMMR CRC patients. Collectively, SLC9A9 may serve as a novel immune checkpoint to assess the efficacy of immunotherapy and predict therapeutic outcomes in pMMR CRC patients.

Supplementary Information: The online version contains supplementary material available at 10.1186/s12935-025-04139-5.

Keywords: Biomarker; Colorectal cancer; Immune checkpoint; Immunotherapy; SLC9A9.

Fig. 1

A strong correlation between high SLC9A9 expression and increased lymphocyte infiltration in CRC patients. (A) In the TCGA database, a comprehensive analysis of the correlation between all genes and CD8 + T cell infiltration in pMMR colorectal cancer (n = 249) was performed using TIMER and Xcell algorithms. Among the top 3,500 genes, SLC9A9 emerged as a highly ranked candidate. Several other members of the SLC gene family were also present within these 3,500 genes, highlighting their potential significance in the context of CD8 + T cell infiltration in pMMR colorectal cancer. (B) Correlation between SLC family gene expression and CD8⁺ T cell infiltration in pMMR CRC patients from the TCGA cohort (n = 249), based on TIMER analysis. Notably, among the SLC gene family members analyzed, only SLC9A9 demonstrated a robust and significant positive correlation with CD8⁺ T cell infiltration, indicating its potential immune-related regulatory function. (C-F) Expression of SLC9A9 from the TCGA (C, n = 434; D, n = 280) and GSE39582 (E, n = 585; F, n = 459) database. D, F correspond to pMMR CRC samples, and show consistent downregulation of SLC9A9 in tumors compared to normal tissues. (G) Correlation between SLC9A9 expression and CD8⁺ T cell infiltration across multiple cancer types using TCGA pan-cancer data (TIMER analysis). Significant correlations were observed in several cancer histotypes, including CRC, LUAD, LIHC, and KIRC, among others. Abbreviations: BLCA (Bladder urothelial carcinoma), BRCA (Breast invasive carcinoma), CESC (Cervical squamous cell carcinoma and endocervical adenocarcinoma), CHOL (Cholangiocarcinoma), COAD (Colon adenocarcinoma), DLBC (Diffuse large B-cell lymphoma), ESCA (Esophageal carcinoma), GBM (Glioblastoma multiforme), HNSC (Head and neck squamous cell carcinoma), KICH (Kidney chromophobe), KIRC (Kidney renal clear cell carcinoma), KIRP (Kidney renal papillary cell carcinoma), LAML (Acute myeloid leukemia), LGG (Lower grade glioma), LIHC (Liver hepatocellular carcinoma), LUAD (Lung adenocarcinoma), LUSC (Lung squamous cell carcinoma), MESO (Mesothelioma), OV (Ovarian serous cystadenocarcinoma), PAAD (Pancreatic adenocarcinoma), PCPG (Pheochromocytoma and paraganglioma), PRAD (Prostate adenocarcinoma), READ (Rectum adenocarcinoma), SARC (Sarcoma), SKCM (Skin cutaneous melanoma), STAD (Stomach adenocarcinoma), TGCT (Testicular germ cell tumors), THCA (Thyroid carcinoma), THYM (Thymoma), UCEC (Uterine corpus endometrial carcinoma), UCS (Uterine carcinosarcoma), UVM (Uveal melanoma). (H) Correlation between immune check points and SLC9A9 expression in pMMR patients by Spearman’s correlation coefficient. n=249. (I, J) Unsupervised hierarchical clustering of colorectal tumors using ssGSEA scores for immune signatures identifies increasing levels of immune infiltrates. I from TCGA database, n=367, J from GSE39582 database, n=519

Fig. 2

SLC9A9 is highly expressed in lymphocyte. (A) t-SNE plot showing clustering of eight cell lineages in CRC single-cell RNA-seq data (GSE146771), including B cells, CD4⁺ T cells, CD8⁺ T cells, innate lymphoid cells (ILCs), myeloid cells, epithelial cells, fibroblasts, and stromal cells (left), and the expression of SLC9A9 across these cell types (right). (B-E) Expression of SLC9A9 (TPM) in cell type subpopulations from GSE146771-cohort (B), GSE132465-cohort (C), GSE144735-cohort (D) and GSE132257-cohort (E). (E) GSE132257 lacks T cell subset-level metadata; therefore, only total T cell expression is shown. Notably, none of the three datasets (GSE132465, GSE144735, GSE132257) provided UMAP coordinates data according to the provided cell types, except annotated UMAP plots with GSE146771

Fig. 3

SLC9A9 is highly correlated with immune infiltration in CRC tissues. (A) Correlation between SLC9A9 expression and immune-related genes in the TCGA pMMR-CRC cohort. (B) Representative IHC images of SLC9A9, CD4 + T cell and CD8 + T cell expression in CRC tissues from the patients. Red dashed lines indicate tumor regions. (C) Expression of SLC9A9. (D, E) Correlation between lymphocyte infiltration and SLC9A9 in CRC patients by Spearman’s correlation coefficient, n = 60

Fig. 4

High SLC9A9 expression is benefit from immunotherapy. (A) Radiological and pathological response to FOLFOX plus sintilimab in a patient with stage T4N2M0 (Case 1) and FOLFOX plus pembrolizumab in a patient with stage T4N2M0 (Case 2). Radiographic imaging shows the tumor in rectum (Ⅰ, a) at initial diagnoses. A notable tumor regression could be seen in primary tumor from Case 1 (Ⅱ). But there was no response in Case 2 after ICI (b). Primary tumor was observed using colonoscopy (Ⅲ, c) at initial diagnosis and after ICI treatment (d). H&E staining shows primary tumor at initial diagnosis (Ⅳ, e) and pathological response after ICI treatment (V, f). Fibrosis and an infiltration with viable density of many lymphocytes (V, arrowheads) can be found, which cannot be found in case 2 (f). IHC staining showed SLC9A9, CD4 + T cells and CD8 + T cells expression with pretreatment tumor samples (Ⅵ, Ⅷ, Ⅹ for Case 1; g, i, k for Case 2) and posttreatment tumor tissues (Ⅶ, Ⅸ, Ⅺ for Case 1; h, j, l for Case 2). Gross and histological changes in CRC patients following immune checkpoint inhibitor treatment. “Marked reduction in tumor burden” refers to visible tumor shrinkage in resected specimens. “Extensive necrotic tissue” refers to large necrotic areas within the tumor bed, confirmed by pathological assessment. These features were more frequently observed in patients who responded to treatment (B- D) Quantification of the score for CD4 + T cells, CD8 + T cells and SL9A9 staining before treatment in CRC tissue from immunotherapy sensitive versus immunotherapy resistance assessed by IHC assay, n = 8