International Journal of Molecular Medical Science, 2024, Vol.14, No.4, 239-251 http://medscipublisher.com/index.php/ijmms 243 4.3 Understanding clonal evolution Clonal evolution is a fundamental aspect of cancer biology, where tumor growth is driven by competing subclones. Single-cell sequencing has provided a powerful tool to study clonal evolution by tracking genetic and transcriptomic changes at the single-cell level. For example, in renal cell carcinoma, scRNA-seq combined with deep sequencing techniques has revealed the presence of subclonal single-nucleotide variants, supporting the clonal evolution model (Gerstung et al., 2012). This approach can be applied to colon cancer to understand how different subclones evolve, compete, and contribute to tumor progression and treatment resistance. 4.4 Mapping the immune landscape The immune landscape of tumors is highly heterogeneous and plays a critical role in cancer progression and response to immunotherapy. Single-cell sequencing has been extensively used to map the immune landscape in various cancers. For instance, in breast cancer, scRNA-seq has profiled diverse immune cell phenotypes, revealing continuous phenotypic expansions specific to the tumor microenvironment (Azizi et al., 2018). In colon cancer, similar approaches can be used to map the immune landscape, identify key immune cell populations, and understand their roles in tumor immunity and immunotherapy response. This detailed mapping can inform the development of more effective immunotherapeutic strategies. 4.4.1 Differences immune microenvironment between left- and right- sided CRC Right-sided CRC, originating from the cecum, ascending colon, and hepatic flexure, and left-sided CRC, originating from the splenic flexure, descending colon, and sigmoid colon, are often grouped as one disease. However, they represent clinically distinct entities with significant differences in prognosis and treatment outcomes (Gallois et al., 2018). Right-sided CRC typically has a worse prognosis than left-sided CRC (Stintzing et al., 2017). Extensive sequencing analyses have described a characteristic branching pattern of cancer evolution that supports the notion that tumor biology is characterized by both intratumor heterogeneity and the preservation of ancestral aberrations within the primary tumor and corresponding metastatic sites. The transcriptomes of 27,927 single human CRC cells from 3 left-sided and 3 right-sided CRC patients were analyzed by Guo et al. using single-cell RNA sequencing. This study revealed that right-sided CRC contains a significant proportion of exhausted CD8+ T cells with a highly migratory nature. In contrast, a cluster of cells from left-sided CRC exhibited states preceding exhaustion and a high ratio of preexhausted to exhausted T cells, which were identified as favorable prognostic markers. Additionally, these researchers identified a potentially novel subpopulation of cancer cells characterized by the expression of Retinol-binding protein 4 (RBP4) and Neurotensin (NTS), which exclusively expanded in left-sided CRC. Regulatory T cells (Tregs) from left-sided CRC showed higher levels of immunotherapy-related genes compared to those from right-sided CRC, suggesting that left-sided CRC may have increased responsiveness to immunotherapy. Furthermore, antibody-dependent cellular phagocytosis (ADCP) and antibody-dependent cellular cytotoxicity (ADCC) induced by M2-like macrophages were found to be more pronounced in left-sided CRC and correlated with a good prognosis in CRC. These findings provide valuable insights into the molecular and cellular differences between left-sided and right-sided CRC, which may help in the development of more targeted and effective treatment strategies for these distinct subtypes of CRC. 4.4.2 Differences in tumor microenvironment components between dMMR and pMMR CRC patients Immunotherapy responses to cancer are highly variable, recent study demonstrated that tumors with deficient mismatch repair (dMMR) display a higher level of anti-tumor immunity when compared to tumors with proficient mismatch repair (pMMR) (Zhu et al., 2023). In order to better understand the potential mechanisms governing these diverse immune responses, Pelka et al. conducted a transcriptional profiling study with single-cell sequencing on 371 223 cells obtained from CRC and adjacent normal tissues of 28 individuals with pMMR and 34 individuals with dMMR (Pelka et al., 2021). Through an extensive analysis of 88 cell subsets and their associated gene expression programs, significant transcriptional and spatial remodeling was observed across the tumors. To identify key interactions between malignant and immune cells, the researchers identified expression programs that exhibited co-variation across tumors from affected individuals and utilized spatial profiling techniques to localize coordinated programs. Additionally, an immune hub enriched in dMMR tumors was discovered within the tumor microenvironment, characterized by the presence of activated T cells and the
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