Deciphering the Tumor Microenvironment in KRAS-Mutated Metastatic Colorectal Cancer Using Single-Cell RNA Sequencing

Single-cell RNA sequencing (scRNA-seq) technology has enabled in-depth analysis of intercellular heterogeneity across various diseases, and recent studies have utilized this technology to provide cell type–specific insights into pathological changes in colorectal cancer (CRC). CRC, a highly prevalent and lethal malignancy worldwide, results from a combination of genetic, epigenetic, and environmental factors. Although immunotherapy has significantly improved CRC outcomes, intolerance remains a major issue for many patients. Among CRC cases, microsatellite instability-high (MSI-H) tumors, characterized by ۳۰% or more mutations in microsatellites due to somatic or germline mutations in DNA mismatch repair machinery, are particularly common in endometrial, stomach, and colon cancers. Given that CRC is the second most lethal and third most common malignancy globally, effective treatment options remain limited. Methods: Raw single-cell sequencing data from a patient with metastatic colorectal cancer treated with polyclonal tumor-infiltrating lymphocytes (TILs) targeting the KRAS(G۱۲D) hotspot mutation, identified by the code GSE۱۳۶۳۹۴, was obtained from the NCBI database. The Scanpy toolbox was then used for data processing. During the initial phase, data from low-quality cells, such as dead, dividing, and stressed cells, were excluded. Next, the scVI package was employed for the normalization of the data, which is designed for comprehensive analysis of single-cell omics data. Batch effects were removed, and dimensionality reduction was performed with the PCA technique. Finally, cell clustering was carried out based on the expression of specific gene markers, and the Bonferroni correction was applied in statistical analyses to calculate the adjusted P-value. Results: Enrichment of biological processes related to translation, ribosomal biogenesis, and mRNA processing suggests a highly active tumor microenvironment. At the molecular function level, RNA binding and ubiquitin-protein transferase activity point to regulatory mechanisms controlling gene expression and protein turnover in response to therapy. Pathway analysis underscores the importance of MYC signaling, G۲-M checkpoint, and p۵۳ pathway activation, reflecting the tumor's response to immune-mediated DNA damage and stress. Furthermore, the enrichment of apoptosis and angiogenesis pathways emphasizes the therapeutic intent of TILs to induce tumor cell death and modulate the tumor's vascular supply. Lastly, the KEGG pathways associated with ribosome biogenesis and TNF signaling suggest a high level of metabolic activity and inflammation, which is crucial for the tumor's response to immune intervention. This data highlights the complex interplay between tumor cells and the immune system under TIL therapy, offering potential insights into the mechanisms driving treatment responses in KRAS-mutated metastatic CRC. Conclusion We filtered the adjusted p-values to retain those below ۰.۰۵, highlighting significant results. Pathway analysis reveals the importance of MYC signaling, G۲-M checkpoint, and p۵۳ activation, reflecting the tumor’s response to immune-mediated DNA damage. The enrichment of apoptosis and angiogenesis pathways underscores TILs' role in inducing tumor cell death and modifying the tumor’s blood supply. KEGG pathways related to ribosome biogenesis and TNF signaling indicate high metabolic activity and inflammation, essential for the tumor's response to immune interventions. These findings enhance the reliability of our conclusions and offer valuable insights into treatment responses in KRAS-mutated metastatic CRC, guiding future research directions.

کلیدواژه ها:

Single-Cell RNA-seq ، Immune Checkpoints ، Immunotherapy