Extracellular and immune matrix signature of interstitial fluid as mediator of pathological characteristics of colorectal cancer.

e15653 Background: The interstitial fluid (ISF) in tumor microenvironment harbors abundant proteins and metabolites critically influencing tumor progression, yet its comprehensive molecular profile in colorectal cancer (CRC) remains uncharacterized. We aim to elucidate the proteomic and metabolomic characteristics of colorectal cancer interstitial fluid and investigate their correlation with clinical pathological features. Methods: We collected tumor tissue and matched normal adjacent tissues (NAT) samples from 47 treatment-naïve CRC surgery patients. ISF was extracted through low-speed centrifugation. Multi-omics analysis involving 4D-DIA proteomics and targeted metabolomics (93 metabolites) was performed on all samples (47 tissue samples and their paired 47 interstitial fluid samples, encompassing tumor/NAT). Bioinformatic analysis subsequently comprised differential expression, consensus clustering, and pathway enrichment. Results: We quantified 8,642 proteins in ISF, with significant enrichment for extracellular matrix (ECM) and immune-related proteins compared to tissue lysates. Tumor ISF exhibited upregulated ECM organization but downregulated complement and adaptive immune pathways compared to NAT ISF. Consensus clustering based on 754 immune- and ECM-related proteins within ISF defined three subtypes: High-Cellular immunity/Low-ECM (HCLE, n = 18), High-inflammation/High-ECM (HiHE, n = 22), and Low-immune/Low-ECM (LiLE, n = 7). The HiHE subtype was characterized by concurrent unstable ECM and inflammatory signaling, significantly associated with low differentiation (p = 0.012) and KRAS mutation (p = 0.037). 6 proteins (COL1A1, COL1A2, COL3A1, THBS2, CCDC80, VIM) were significantly overexpressed in the HiHE subtype (fold change > 2, p < 0.05) and were associated with poorer overall survival and disease-free survival in colorectal cancer. Metabolomics revealed elevated proline and hydroxyproline in tumor ISF, particularly in the HiHE subtype. Furthermore, hydroxylation ratios of major collagens (COL1A1, COL3A1) were significantly reduced in HiHE tumor ISF, indicating ECM instability despite high collagen content. Conclusions: This study establishes the first multi-omics of tumor ISF in CRC, revealing that tumor ISF serves as a dynamic reservoir of ECM remodeling proteins, complement, and antibodies. The subtyping based on ISF could provide a unique signature that tissue subtyping could not present. A subtype called HiHE, characterized by concurrent ECM hyperactivation and inflammatory signaling, is correlated with pathological features such as low differentiation.