Modeling tumor microenvironment-mediated resistance in NSCLC using a high-throughput spheroid microarray platform

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.