PO.TB04.03 · 肿瘤生物学
利用高通量球体微阵列平台模拟NSCLC中肿瘤微环境介导的耐药
Modeling tumor microenvironment-mediated resistance in NSCLC using a high-throughput spheroid microarray platform
作者与单位 Authors & Affiliations
摘要 Abstract
中文摘要
EGFR和KRAS突变型非小细胞肺癌（NSCLC）中的治疗耐药仍是一项重大临床挑战，通常由肿瘤微环境（TME）内的动态相互作用所驱动。尽管第三代EGFR抑制剂改善了预后，但代偿性基质信号可能削弱疗效。传统模型无法再现旁分泌介导的耐药机制。为研究旁分泌介导的耐药机制，我们开发了一种高通量球体微阵列平台，以探究成纤维细胞和内皮细胞来源的因子如何影响不同NSCLC基因型的药物敏感性。该平台用于生成携带KRAS G12C或EGFR突变的NSCLC细胞系（H358、H1975、HCC827）的均一3D球体。使用通过软光刻制作的PDMS印章，在标准24孔板内塑造琼脂糖微孔。将球体培养于经WI-38成纤维细胞、HUVEC内皮细胞或两者共同条件化的培养基中，以模拟基质信号。用奥希替尼（osimertinib）或阿达格拉西布（adagrasib）处理72小时后，采用钙黄绿素AM（Calcein AM）、碘化丙啶（Propidium Iodide）和Hoechst染色评估球体活力，随后进行荧光成像和定量分析。在各条件间比较药物反应以评估TME介导的耐药，结果显示H1975球体在HUVEC条件化培养基中的活力比对照高约25%。同样，暴露于WI-38条件化培养基的H358球体对阿达格拉西布的反应减弱，与既往发现一致。成纤维细胞和内皮细胞联合条件化进一步降低了药物疗效，提示存在相加或协同效应。各条件下球体活力保持较高水平（>83%），并维持均一性（变异系数<22%），支持该平台在机制研究中的稳健性。这些发现凸显了可溶性基质因子在调节治疗反应中的作用，并强调了该平台的潜力。未来与患者来源类器官及多重信号分析的整合，可能揭示可用于克服耐药的可操作靶点，并指导EGFR和KRAS突变型NSCLC的联合治疗策略。
查看英文原文 English abstract
Therapeutic resistance in EGFR- and KRAS-mutant non-small cell lung cancer (NSCLC) remains a major clinical challenge, often driven by dynamic interactions within the tumor microenvironment (TME). While third-generation EGFR inhibitors have improved outcomes, compensatory stromal signaling can undermine efficacy. Conventional models fail to capture paracrine-mediated resistance mechanisms. To investigate paracrine-mediated resistance mechanisms, a high-throughput spheroid microarray platform was developed to investigate how fibroblast- and endothelial-derived factors influence drug sensitivity across NSCLC genotypes. The platform was used to generate uniform 3D spheroids of NSCLC cell lines (H358, H1975, HCC827) harboring KRAS G12C or EGFR mutations. Agarose microwells were molded within standard 24-well plates using PDMS stamps fabricated by soft lithography. Spheroids were cultured in media conditioned by WI-38 fibroblasts, HUVEC endothelial cells, or both to simulate stromal signaling. Following a 72-hour treatment with osimertinib or adagrasib, spheroid viability was assessed using Calcein AM, Propidium Iodide, and Hoechst staining, followed by fluorescent imaging and quantitative analysis. Drug response was compared across conditions to evaluate TME-mediated resistance, with H1975 spheroids showing approximately 25% higher viability in HUVEC-conditioned media compared to control. Similarly, H358 spheroids exposed to WI-38-conditioned media showed attenuated response to adagrasib, consistent with prior findings. Combined fibroblast and endothelial conditioning further diminished drug efficacy, suggesting additive or synergistic effects. Spheroid viability remained high (>83%) across conditions, and uniformity was maintained (<22% coefficient of variance), supporting the platform's robustness for mechanistic studies. These findings highlight the role of soluble stromal factors in modulating therapeutic response and underscore the potential of this platform. Future integration with patient-derived organoids and multiplexed signaling analyses may uncover actionable targets to overcome resistance and guide combination strategies in EGFR- and KRAS-mutant NSCLC.
利益披露 Disclosure
A. Rahnama, None..
M. Arrighi, None.