肝癌是全球癌症相关死亡率的一个重要原因。尽管有可用的靶向疗法，但对索拉非尼和乐伐替尼等酪氨酸激酶抑制剂（TKI）的耐药性构成了巨大的挑战。癌症相关成纤维细胞 (CAF) 居住的肿瘤微环境 (TME) 深刻地影响着这种抵抗力。为了揭示这些机制，我们制造了复制肝脏结构的 3D 微流控芯片来探究 TKI 耐药的复杂机制。该芯片设计反映了肝小叶的六边形结构，将肝癌细胞置于核心，周围环绕着成纤维细胞，并经过严格的评估确认了生物相容性和一致的细胞生长。在确定 2D 共培养中索拉非尼和仑伐替尼的 IC50 值后，Transwell 设置揭示了共培养细胞中耐药性的发展。在 3D 微流控芯片中，活/死检测强调了药物暴露下活力的提高，强调了成纤维细胞驱动的耐药性。该研究确定 AHSG 和 CLEC3B 是共培养中耐药性的潜在介质，在共培养培养基中显着上调。功能测试证实了它们的作用，因为引入重组 AHSG 和 CLEC3B 在 2D 和 3D 场景中增强了肝癌细胞对索拉非尼和乐伐替尼的耐药性。总之，通过使用微流控技术复制复杂的 TME，本研究揭示了 AHSG 和 CLEC3B 的作用以及提高肝癌治疗效果的可能方法。

Liver cancer is a significant global contributor to cancer-related mortality. Despite available targeted therapies, resistance to tyrosine kinase inhibitors (TKIs) like sorafenib and lenvatinib poses a formidable challenge. The tumor microenvironment (TME), inhabited by cancer-associated fibroblasts (CAFs), profoundly influences this resistance. To uncover the mechanisms, a 3D microfluidic chip replicating liver architecture was fabricated to probe the intricate mechanisms of TKI resistance. The chip design mirrors the hexagonal structure of liver lobules, situating liver cancer cells at the core, encircled by fibroblasts, with rigorous assessments confirming biocompatibility and consistent cell growth. After determining the IC50 values of sorafenib and lenvatinib in 2D co-culture, a transwell setup revealed drug resistance development in co-cultured cells. Within the 3D microfluidic chip, live/dead assays highlighted elevated viability under drug exposure, emphasizing fibroblast-driven drug resistance. The study identifies AHSG and CLEC3B as potential mediators of drug resistance in co-culture, significantly upregulated in the co-cultured medium. Functional tests confirmed their roles, as introducing recombinant AHSG and CLEC3B enhanced liver cancer cell resistance to sorafenib and lenvatinib in both 2D and 3D scenarios. In conclusion, by replicating the complex TME using microfluidic technology, this study sheds light on the roles of AHSG and CLEC3B as well as possible approaches for improving the effectiveness of liver cancer treatment.