Van Schil, P. | bioRxiv Science

bioRxiv2025

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

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.

cancer biology↗

bioRxiv2021

OrBITS: A High-throughput, time-lapse, and label-free drug screening platform for patient-derived 3D organoids

BackgroundPatient-derived organoids are invaluable for fundamental and translational cancer research and holds great promise for personalized medicine. However, the shortage of available analysis methods, which are often single-time point, severely impede the potential and routine use of organoids for basic research, clinical practise, and pharmaceutical and industrial applications. MethodsHere, we developed a high-throughput compatible and automated live-cell image analysis software that allows for kinetic monitoring of organoids, named Organoid Brightfield Identification-based Therapy Screening (OrBITS), by combining computer vision with a convolutional network machine learning approach. The OrBITS deep learning analysis approach was validated against current standard assays for kinetic imaging and automated analysis of organoids. A drug screen of standard-of-care lung and pancreatic cancer treatments was also performed with the OrBITS platform and compared to the gold standard, CellTiter-Glo 3D assay. Finally, the optimal parameters and drug response metrics were identified to improve patient stratification. ResultsOrBITS allowed for the detection and tracking of organoids in routine extracellular matrix domes, advanced Gri3D(R)-96 well plates, and high-throughput 384-well microplates, solely based on brightfield imaging. The obtained organoid Count, Mean Area, and Total Area had a strong correlation with the nuclear staining, Hoechst, following pairwise comparison over a broad range of sizes. By incorporating a fluorescent cell death marker, intra-well normalization for organoid death could be achieved, which was tested with a 10-point titration of cisplatin and validated against the current gold standard ATP-assay, CellTiter-Glo 3D. Using this approach with OrBITS, screening of chemotherapeutics and targeted therapies revealed further insight into the mechanistic action of the drugs, a feature not achievable with the CellTiter-Glo 3D assay. Finally, we advise the use of the growth rate-based normalised drug response metric to improve accuracy and consistency of organoid drug response quantification. ConclusionsOur findings validate that OrBITS, as a scalable, automated live-cell image analysis software, would facilitate the use of patient-derived organoids for drug development and therapy screening. The developed wet-lab workflow and software also has broad application potential, from providing a launching point for further brightfield-based assay development to be used for fundamental research, to guiding clinical decisions for personalized medicine.

cancer biology↗