Expression profiling of inhibitory immune checkpoints in colorectal cancer stem cells and their association with tumor immunity and immunotherapy biomarkers

Background: Colorectal cancer stem cells (CSCs) represent a rare subpopulation within tumors endowed with self-renewal capabilities and are recognized as key drivers of tumor initiation, metastasis, and therapeutic resistance. These cells also display immunomodulatory properties that enable them to evade immune surveillance. However, the mechanisms underlying their immune evasion, including the role of immune checkpoints (ICPs), remain poorly understood. Therefore, this study aimed to characterize the inhibitory ICP expression landscape in colorectal CSC-enriched models and to evaluate its association with tumor microenvironment and biomarkers related to immune checkpoint inhibitor (ICI) response. Methods: CSC-enriched spheroids, cancer stem-like cells (CSLCs), were generated from four colorectal cancer cell lines (HCT-116, HT-29, SW480, SW620). Differential expression of stemness markers and inhibitory ICPs between spheroid cultures and bulk cancer cells were assessed by real-time PCR, Western blotting, Immunofluorescence, and flow cytometry. Additionally, RNA-seq and clinical data from colorectal adenocarcinoma patients in The Cancer Genome Atlas (TCGA) were retrieved and stratified using the mRNA expression-based stemness index (mRNAsi), a stemness score derived using the one-class logistic regression machine learning algorithm. Correlations between cancer stemness and the tumor immune microenvironment, as well as ICIs-related biomarkers, including ICP expression levels, tumor mutational burden (TMB), and microsatellite instability (MSI), were subsequently analyzed. Results: Spheroid cultures exhibited a significant elevation in the expression of stemness markers (e.g., ALDH, NANOG, and SOX9), confirming the successful enrichment of CSC subpopulations. This upregulation was accompanied by increased expression of multiple inhibitory ICPs (e.g., PD-L1, B7-H3, and CD47) compared with their parental adherent cells (cancer), suggesting a potential role for these ICPs in mediating CSC characteristics. Consistently, patients with high stemness scores displayed reduced immune cell infiltration, increased TMB, higher MSI prevalence, and elevated expression of multiple ICPs, after adjusting for tumor purity, indicating an association between the tumor stemness and factors predictive of ICI responsiveness. Conclusion: The unique immunological profile of colorectal CSLCs identified in this study highlights the role of ICPs in CSC-mediated immune evasion and underscores the potential of CSCs both as targets for checkpoint blockade-based immunotherapies and as biomarkers of response.