Paired functional screening reveals GMI‑modulated drug response behavior in heterogeneous tumor models

Abstract

Tumor drug sensitivity is often evaluated under static experimental conditions despite the dynamic biological environments that influence therapeutic response. Here, we established a paired ex vivo functional screening framework to evaluate modulation-dependent drug-response behavior in heterogeneous tumor-derived systems. Using three-dimensional spheroids generated from companion-animal tumors, we performed matched high-throughput screening of an oncology-focused compound library under baseline phosphate-buffered saline (PBS) and biologically modulated conditions using GMI (Ganoderma microsporum immunomodulatory protein). Drug-response landscapes varied substantially across tumors and paired conditions, demonstrating that biological modulation does not uniformly enhance or suppress therapeutic sensitivity. Instead, delayed GMI exposure following compound treatment was associated with bidirectional viability shifts, revealing structured patterns of sensitization-associated and attenuation-associated responses across compounds and cases. Pathway-level aggregation identified recurrent modulation-associated response patterns across drug classes linked to DNA replication and repair, cell-cycle regulation, receptor tyrosine kinase signaling, developmental signaling, and metabolic stress pathways. Unsupervised clustering further resolved three modulation-associated response groups corresponding to sensitized, mixed, and attenuation-associated response states. These modulation-associated response patterns were strongly influenced by tumor context while retaining substantial case-specific variability across species and tumor types. Together, these findings demonstrate that delayed biological perturbation can alter therapeutic-response behavior in a strongly case-dependent manner and establish paired ex vivo functional screening as a strategy for resolving context-dependent modulation patterns in heterogeneous tumor systems.