Comparative characterization of OncoPro and Wnt-Based media reveals distinct phenotypic and pharmacologic states in patient-derived tumor organoids

Abstract

BackgroundPatient-derived tumor organoids (PDTOs) are strongly influenced by culture medium. We compared OncoPro (OP) Tumoroid Culture medium with conventional Wnt/R-spondin/noggin (Wnt) medium. This recently developed OP medium offers a standardized, serum-free alternative to Wnt-based formulations. MethodsWe compared OP and Wnt media across 36 PDTO lines from various malignancies (colorectal, pancreatic, breast, lung, gastric, gastroesophageal junction, biliary head- and neck and uknown primary), assessing establishment success. Selected PDTO models were subjected to downstream characterization, including morphological assessment and bulk transcriptomic profiling with comparison to public single-cell RNA sequencing reference datasets (n=11), whole-exome sequencing (WES) (n=9), and pharmacological response profiling to a 33-drug panel (n=3). ResultsAdaptation from Wnt medium to OP succeeded in 83.3% (15/18), whereas de novo establishment favored Wnt (33.3% vs 11.1%). Key oncogenic driver alterations were retained across matched organoid cultures, supporting preservation of tumor-relevant genomic features. Transcriptomic profiling confirmed preserved tumor-identity across media, while revealing different epithelial state programs: Wnt upregulated proliferation/stemness-associated genes (e.g. LGR5) and OP enriched adhesion-associated genes and inflammatory/TGF-{beta} programs. In scRNA databases OP signatures preferentially mapped to malignant epithelial compartments in pancreatic cancer, whereas Wnt signatures were linked to non-malignant epithelium. Similarly, in colon cancer OP signature mapped predominantly to the malignant epithelial compartments. Drug (n=33) screening in pancreatic- and colorectal cancer PDTOs (n=3) demonstrated consistent medium-dependent shifts: Wnt-grown PDTOs were globally more sensitive in the screened subset, particularly to MAPK-axis inhibitors and apoptosis-sensitizers, while OP-grown PDTOs exhibited relative resistance. ConclusionsCulture medium composition is a key determinant of PDTO phenotype, transcriptome and drug sensitivity. Wnt medium was associated with drug-sensitive states, whereas OP medium was associated with adhesion- and inflammatory-related programs, relative resistance in the screened subset of 33 drugs and closer alignment with malignant epithelial programs in the analyzed pancreatic / colorectal cancer single-cell atlases.