Notable influences of estrogen and sex-specific microenvironment in colorectal cancer revealed by single-cell...

Background: Colorectal cancer (CRC), the third most common malignancy worldwide, exhibits notable sex-specific prognostic differences, yet the underlying biological mechanisms remain poorly understood. Methods: In this study, we conducted single-cell sequencing on 32 CRC samples, followed by pathway enrichment analysis, cell-cell interaction analysis, and transcription factor analysis. The co-expression of GZMB and the transcription factor EOMES in CD8+ T cells was detected using multiplex immunohistochemistry. Western blot and TUNEL assays were employed to validate estrogen-induced apoptosis in CRC cell lines. Results: After quality control, we obtained a total of 167,437 cells across 9 cell types from all samples. Specifically, our analysis revealed sex-based variations in cellular composition, functionality, and intercellular interactions within CRC. Notably, female CRC samples exhibited significant positive correlation between estrogen signaling pathway activation and apoptotic activity, with validation through Western blot and TUNEL assays confirming estrogen-mediated apoptosis induction in CRC cell lines. The immune response was notably enhanced in female CRC, with CD8+ T cells showing increased expression of the EOMES gene regulatory network, thereby boosting T cell immunity. Moreover, B cells of female CRC demonstrated improved capabilities in antigen-presenting and MHC-I interactions with T cells. Additionally, Macro_CCL4 cells engaged in sex-specific TNF-TNFRSF1B crosstalk with CD8+ T cells, potentially leading to enhanced antitumor immunity in females. Conversely, CAF_MMP11 cells exhibiting a myofibroblastic CAF phenotype interacted with malignant epithelial cells via signaling pathways such as THBS, MK, and FN1, likely promoting CRC progression. Conclusions: Our research highlights the distinct immunological and hormonal responses in CRC by sex, which may explain the observed prognostic disparities. These findings may offer additional further biological insights for targeted therapies in CRC.