Comprehensive profiling of immune and stromal alterations in colorectal cancer with diabetes mellitus through single-cell transcriptomics and proteomics.

e15635 Background: Colorectal cancer (CRC) and diabetes mellitus have become significant global health challenges, reaching epidemic levels. Evidence increasingly shows that diabetes raises cancer risk and mortality. The complexities of this link are still being explored. Methods: We performed single-cell RNA sequencing on diabetes mellitus-associated colorectal cancer (DM-CRC), non-diabetes mellitus-associated colorectal cancer (NDM-CRC), and paracancerous tissues obtained from 7 patients receiving radical resection. Tissue microarrays derived from 72 CRC patients were utilized for multi-color immunofluorescence analysis. Additionally, data from The Cancer Genome Atlas (TCGA) were employed for further analysis. Proteomic analyses were conducted on tissue samples from 12 paired patients and on serum exosome samples from 4 paired patients, both with and without diabetes. Results: We demonstrate that IFIT2+ neutrophils characterized by active cytokine and NFĸB signaling, are more pronounced in DM-CRC compared to NDM-CRC. CXCL5+ macrophages, during the later stages of development,tend to become M2 phenotype in DM-CRC, potentially worsening survival. CD8+ LAG3+ T cells in DM-CRC are activated and progress towards an exhausted state. Collectively, these alterations in immune cell dynamics within the DM-CRC microenvironment suggest a trend towards immunosuppressive characteristics. Subsequent tissue proteomics analysis identified 76 differentially expressed proteins and the upregulated signaling pathways predominantly involve the negative regulation of immune system processes, response to oxidative stress, fatty acid metabolic processes, collagen-containing extracellular matrix and epithelial-mesenchymal transition (EMT). Furthermore, serum exosome proteomics identified the upregulated exosomal collagens (COL6A1, COL6A3, COL1A1) and vimentin recognized as potential biomarkers for CRC patients with diabetes. The proteomic data imply highly heterogeneous and aggressive stromal changes in DM-CRC. Notably, MMP1+ cancer-associated fibroblasts, associated with decreased progression-free survival in CRC patients, are more prevalent in DM-CRC with elevated glycolytic activity, EMT, hypoxia, and oxidative phosphorylation. These fibroblasts also upregulate collagen-containing extracellular matrix and TGFβ signaling via BHLHE41. Consequently, the interaction among the three specific clusters of immune cells and stromal cells has been obviously intensified in DM-CRC, particularly through the TGFβ1-CVCR1 and TGFβ receptor pathways. Conclusions: This study characterizes an altered tumor microenvironment in DM-CRC, highlighting immune-stroma interactions and identifying protein markers, which may guide personalized treatments for CRC patients with diabetes.