BACKGROUND Metastasis and recurrence, wherein circulating tumour cells (CTCs) play an important role, are the leading causes of death in colorectal cancer (CRC). Metastasis-initiating CTCs manage to maintain intravascular survival under anoikis, immune attack, and importantly shear stress; however, the underlying mechanisms remain poorly understood. METHODS In view of the scarcity of CTCs in the bloodstream, suspended colorectal cancer cells were flowed into the cyclic laminar shear stress (LSS) according to previous studies. Then, we detected these suspended cells with a CK8+/CD45-/DAPI+ phenotype and named them mimic circulating tumour cells (m-CTCs) for subsequent CTCs related researches. Quantitative polymerase chain reaction, western blotting, and immunofluorescence were utilised to analyse gene expression change of m-CTCs sensitive to LSS stimulation. Additionally, we examined atonal bHLH transcription factor 8 (ATOH8) expressions in CTCs among 156 CRC patients and mice by fluorescence in situ hybridisation and flow cytometry. The pro-metabolic and pro-survival functions of ATOH8 were determined by glycolysis assay, live/dead cell vitality assay, anoikis assay, and immunohistochemistry. Further, the concrete up-and-down mechanisms of m-CTC survival promotion by ATOH8 were explored. RESULTS The m-CTCs actively responded to LSS by triggering the expression of ATOH8, a fluid mechanosensor, with executive roles in intravascular survival and metabolism plasticity. Specifically, ATOH8 was upregulated via activation of VEGFR2/AKT signalling pathway mediated by LSS induced VEGF release. ATOH8 then transcriptionally activated HK2-mediated glycolysis, thus promoting the intravascular survival of colorectal cancer cells in the circulation. CONCLUSIONS This study elucidates a novel mechanism that an LSS triggered VEGF-VEGFR2-AKT-ATOH8 signal axis mediates m-CTCs survival, thus providing a potential target for the prevention and treatment of hematogenous metastasis in CRC.

中文翻译：

---

剪切应力通过自分泌VEGF激活ATOH8，从而促进循环中结直肠癌细胞的糖酵解依赖性生存。

背景技术其中循环肿瘤细胞（CTC）起重要作用的转移和复发是结直肠癌（CRC）死亡的主要原因。转移引发的四氯化碳设法维持在血管紧张，免疫攻击和重要的切应力作用下的血管内存活；但是，其基本机制仍然知之甚少。方法根据先前研究的结果，鉴于血液中四氯化碳的缺乏，悬浮的结直肠癌细胞流入了循环层流剪切应力（LSS）。然后，我们检测到这些具有CK8 + / CD45- / DAPI +表型的悬浮细胞，并将其命名为模拟循环肿瘤细胞（m-CTC），以用于后续的CTC相关研究。定量聚合酶链反应，蛋白质印迹，用免疫荧光法和免疫荧光法分析对LSS刺激敏感的m-CTC的基因表达变化。此外，我们通过荧光原位杂交和流式细胞术检查了156例CRC患者和小鼠CTC中的非离子bHLH转录因子8（ATOH8）表达。通过糖酵解测定，活/死细胞活力测定，无细胞测定和免疫组化测定ATOH8的代谢和存活功能。此外，还探索了ATOH8促进m-CTC存活的具体起伏机制。结果m-CTC通过触发ATOH8的表达对LSS产生积极反应，ATOH8是一种流体机械传感器，在血管内存活和代谢可塑性中起重要作用。具体而言，通过由LSS诱导的VEGF释放介导的VEGFR2 / AKT信号通路的激活来上调ATOH8。然后ATOH8转录激活HK2介导的糖酵解，从而促进循环中结直肠癌细胞的血管内存活。结论这项研究阐明了一种新的机制，即LSS触发VEGF-VEGFR2-AKT-ATOH8信号轴介导m-CTC的存活，从而为预防和治疗CRC的血源性转移提供了潜在的靶标。