Exosomal KRAS Mutation Promotes Promotion of Colorectal Cancer by the Formation of Tumor-Associated Neutrophil Extracellular Traps

Background: Colorectal cancer (CRC) remains one of the leading causes of cancer-related death. The current study aimed to elucidate the mechanism by which exosomes carrying KRAS mutant contribute to neutrophil recruitment as well as the formation of the neutrophil extracellular traps (NETs) in CRC. Methods: APC-WT and APC-KRASG12D mouse models were initially established. The peripheral blood, spleen, bone marrow (BM) and mesenteric lymph nodes (mLN) were isolated to detect neutrophil content. Next, exosomes isolated from APC-WT and APC-KRASG12D mice were injected into APC-WT and APC-KRASG12D mice. The neutrophil ratio, NETs formation and IL-8 protein content in colon tissue were subsequently quantified. DKs-8 (wild type) and DKO-1 (KRAS mutant) cells were employed for in vitro experimentation. Then, DKs-8 cells were cultured with exosome-treated PMA stimulated neutrophil-forming NETs culture medium, with cell viability, invasion, migration, and adhesion evaluated. Results: Compared with APC-WT mice, the number of polyps and neutrophils in the peripheral blood, spleen and mLNs increased in APC-KRASG12D mice, with increased NETs formation, IL-8 expression, and exosomes. IL-8 upregulation, neutrophil recruitment and NETs formation were observed in mice injected with exosomes derived from APC-KRASG12D. The in vitro investigation results revealed that more NETs were formed by DKO-1-Exosomes, which were inhibited by DNAse. In addition, DKs-8 and DKO-1 cells-derived exosomes can adhere to NETs under static conditions in vitro. Exosomal KRAS mutants were noted to exert a stimulatory effect on the production of IL-8 while promoting NETs formation as well as CRC promotion. Conclusion: The results obtained provide evidence suggesting that exosomes may transfer mutant KRAS to recipient cells and trigger an increase in IL-8 production, promote neutrophil recruitment and form NETs, ultimately leading to the promotion of CRC. Funding Statement: This work was supported by the National Natural Science Foundation of China (81873975, 81802084, 81974314, 81902984), the Excellent Academic Leader Training Program of Shanghai Health System (2018BR31), the Medical Guidace Science and Technology Support Project of Shanghai (19411964800), the Natural Science Foundation of Shanghai (19ZR1448800), the Clinical Research and Cultivation Project of Shanghai Tongji Hospital [ITJ(ZD)1803, ITJ(ZD)1905, ITJ(QN)1905]. Declaration of Interests: The authors declare that they have no competing interests. Ethics Approval Statement: The study protocol was approved by the Ethics Committee of Tongji Hospital of Tongji University. All subjects have written informed consent. All animal use and experiments were performed in strict accordance with the procedures approved by the National Cancer Institute Animal Care and Use Committee (ACUC).