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 trap (NET) in CRC. APC-WT and APC-KRASG12D mouse models were initially developed. Peripheral blood, spleen, bone marrow (BM) and mesenteric lymph nodes (mLN) were isolated to detect neutrophil content. Then, APC-WT and APC-KRASG12D mice were injected with exosomes isolated from APC-WT and APC-KRASG12D mice. The ratio of neutrophils, NETs formation and IL-8 protein content were subsequently quantified in colon tissues. 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. Compared with APC-WT mice, the numbers of polyps and neutrophils in the peripheral blood, spleen and mLNs were increased in APC-KRASG12D mice, accompanied with increased NET formation, IL-8 expression and exosomes. Meanwhile, IL-8 upregulation, neutrophil recruitment and NET formation were observed in the mice injected with exosomes derived from APC-KRASG12D. The in vitro investigation results revealed that more NETs were formed in the presence of DKO-1-Exos, which were inhibited by DNAse. In addition, DKs-8- and DKO-1 cells-derived exosomes could adhere to NETs under static conditions in vitro. Exosomal KRAS mutants were noted to exert stimulatory effects on the IL-8 production and NET formation to promote the growth of CRC cells. The results provide evidence suggesting that exosomes may transfer mutant KRAS to recipient cells and trigger increases in IL-8 production, neutrophil recruitment and formation of NETs, eventually leading to the deterioration of CRC.

中文翻译：

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外体KRAS突变通过诱导IL-8表达促进肿瘤相关的中性粒细胞胞外陷阱的形成，并导致结直肠癌恶化。

大肠癌（CRC）仍然是与癌症相关的死亡的主要原因之一。当前的研究旨在阐明携带KRAS突变体的外泌体促成中性粒细胞募集以及在CRC中形成中性粒细胞胞外陷阱（NET）的机制。最初开发了APC-WT和APC-KRASG12D小鼠模型。分离外周血，脾脏，骨髓（BM）和肠系膜淋巴结（mLN）以检测中性粒细胞含量。然后，向APC-WT和APC-KRASG12D小鼠注射从APC-WT和APC-KRASG12D小鼠分离的外泌体。随后在结肠组织中定量中性粒细胞的比率，NETs形成和IL-8蛋白含量。DKs-8（野生型）和DKO-1（KRAS突变体）细胞用于体外实验。然后，用外来体处理的PMA刺激的中性粒细胞形成NETs培养基培养DKs-8细胞，并评估细胞活力，侵袭，迁移和粘附。与APC-WT小鼠相比，APC-KRASG12D小鼠外周血，脾脏和mLNs的息肉和中性粒细胞数量增加，并伴有NET形成，IL-8表达和外泌体增加。同时，在注射来自APC-KRASG12D的外来体的小鼠中观察到IL-8上调，中性粒细胞募集和NET形成。体外研究结果表明，在DKO-1-Exos存在下，更多的NETs被DNAse抑制。另外，DKs-8和DKO-1细胞衍生的外泌体在体外静态条件下也可以粘附到NETs上。已知外泌体KRAS突变体对IL-8产生和NET形成具有刺激作用，以促进CRC细胞的生长。结果提供了证据，表明外来体可能会将突变的KRAS转移至受体细胞并触发IL-8产生，中性粒细胞募集和NETs形成的增加，最终导致CRC恶化。