Mature tumoroids recapitulate clinically relevant drug response through extended 3D culture in PDAC

Abstract

BackgroundDrug responses in pancreatic ductal adenocarcinoma (PDAC) vary sharply across in vitro culture formats, but most 2D-3D comparisons conflate microenvironmental cues with time-dependent cellular adaptation. As a result, conventional assays frequently overestimate drug efficacy and poorly reflect clinical pharmacology. Main findingsWe profiled MiaPaCa-2, PANC-1, and CFPAC-1 grown in an extracellular-matrix (ECM) hydrogel for 1-12 days, defining extended 3D cultures ([&ge;]10 days) as mature tumoroids, and quantified 72 h drug responses to a multi-class oncology panel using growth-rate (GR) metrics to normalize for proliferation across formats and durations. Prolonged 3D pre-culture induced broad tolerance, with typical 10-100x reductions in sensitivity to standards of care (5-fluorouracil, SN38, oxaliplatin, gemcitabine, paclitaxel), following a reproducible susceptibility hierarchy (MiaPaCa-2 > PANC-1 > CFPAC-1) after GR correction. In mature tumoroids, GR values closely approximated clinically observed plasma exposures (e.g., within <4x for 5-FU and <0.5x for gemcitabine), whereas 2D and short-term organoid assays markedly underestimated resistance, often by >100x, thereby overstating drug activity. Notably, CFPAC-1 exhibited increased sensitivity to SN38 and trametinib under mature-organoid conditions, demonstrating that microenvironmental conditioning can invert responses for selected mechanisms. Transcriptomic profiling revealed coordinated up-regulation of multiple ABC transporters with extended 3D residence, tracking resistance phenotypes across lines and implicating transporter-linked tolerance programs. SignificanceTogether, these data identify time-in-3D and the emergence of mature tumoroids as dominant, previously under-controlled determinants of PDAC pharmacology that both induce tolerance and unmask context-dependent vulnerabilities. We propose incorporating both short-term and mature-tumoroid screening arms into preclinical workflows, reporting pre-culture duration alongside GR-normalized effect sizes, and leveraging transporter-informed biomarkers to guide regimen prioritization and sequencing. This framework enhances physiological relevance, reproducibility, and translational fidelity in PDAC drug discovery.