Abstract 2688: FXIIIa-mediated transferrin sequestration drives cancer stem cell phenotype in colorectal cancer

Abstract Background: Colorectal cancer (CRC) is the third most common cancer worldwide. Tumors, often described as wounds that do not heal, exhibit fibrin clot deposition, which may create a niche for cancer stem cells (CSCs). Factor XIII (FXIII), a transglutaminase, cross-links fibrin (XLF) and functionalizes XLF clots by incorporating additional protein into the matrix. Our previous studies suggest that FXIII-XLF promotes the CSC phenotype. This study aims to identify and validate CSC-promoting proteins enriched in the XLF matrix. Methods: TCGA and CARIS CODEai databases were analyzed for correlations of FXIII expression on CRC survival in primary and CRC peritoneal metastasis (PM). CRC cell lines were grown in an XLF spheroid model with various levels of activated FXIII (FXIIIa). We performed a comparative LC-MS/MS-based label-free proteomics (LFQ) analysis on high-salt washed XLF clots treated with normal plasma (100% FXIIIa activity, N-XLF) and CSC-promoting FXIIIa (150% FXIIIa activity, CP-XLF) matrices. FXIIIa activity was measured using a transglutaminase assay. Differential protein expression in the matrices was further validated by ELISA. The impact of FXIIIa inhibition was tested using the pan-transglutaminase inhibitor T101, and the role of the candidate protein transferrin (Tf) on spheroid formation was assessed with a Tf-neutralizing antibody (Tf ab). We further validated the effects of iron-loaded holo-transferrin versus apo-transferrin on spheroid numbers. Additionally, the effect of FXIIIa on intracellular iron was assessed using an iron-sensitive dye via flow cytometry. Results: FXIIIA1 RNA expression was 2.7-fold higher in CRC PM compared to primary sites, and higher expression correlated with poor survival in all stages. Further, XLF protein expression in a CRC tumor microarray was present in 39.5% of patients, correlating with notably reduced survival (HR=2.2, p<0.05). Proteomics analysis revealed 21 differentially expressed proteins in CP-XLF compared to N-XLF (FDR <0.05). Tf was upregulated (4.5-fold) in CP-XLF. FXIIIa inhibition with T101 disrupted Tf sequestration in the XLF matrix and the FXIIIa-mediated promotion of spheroid formation. Tf neutralizing antibodies significantly reduced spheroid formation in both N-XLF and CP-XLF conditions. FXIIIa preferentially sequestered iron-loaded holo-transferrin over apo-transferrin. FXIIIa was associated with a 2.2-fold increase in cells with high intracellular iron (p=0.002). Conclusions: FXIII RNA expression is associated with worse overall survival in CRC. Transferrin (Tf) and iron saturation are crucial in promoting the CSC phenotype in the CP-XLF matrix. This finding positions Tf as a potential mechanistic lead in the FXIII-XLF-CSC axis and a target for therapeutic intervention. Citation Format: Savannah Gregg, Rio Boothello, Sareh Bayatpour, Shoja M. Haneefa, Adam Hawkridge, Umesh Desai, Gretchen Hubbard, Andrew Elliott, Jose J. Trevino, Leopoldo Fernandez, Mary Garland-Kledzik, Bhaumik Patel, Adam Khader. FXIIIa-mediated transferrin sequestration drives cancer stem cell phenotype in colorectal cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2688.