Cancer drug screening in patient‐derived cells holds great promise for personalized oncology and drug discovery but lacks standardization. Whether cells are cultured as conventional monolayer or advanced, matrix‐dependent organoid cultures influences drug effects and thereby drug selection and clinical success. To precisely compare drug profiles in differently cultured primary cells, we developed DeathPro, an automated microscopy‐based assay to resolve drug‐induced cell death and proliferation inhibition. Using DeathPro, we screened cells from ovarian cancer patients in monolayer or organoid culture with clinically relevant drugs. Drug‐induced growth arrest and efficacy of cytostatic drugs differed between the two culture systems. Interestingly, drug effects in organoids were more diverse and had lower therapeutic potential. Genomic analysis revealed novel links between drug sensitivity and DNA repair deficiency in organoids that were undetectable in monolayers. Thus, our results highlight the dependency of cytostatic drugs and pharmacogenomic associations on culture systems, and guide culture selection for drug tests.

Pancreatic ductal adenocarcinoma (PDAC) is projected to be the second leading cause of cancer mortality by 2030. Bulk transcriptomic analyses have distinguished ‘classical’ from ‘basal-like’ tumors with more aggressive clinical behavior. We derive PDAC organoids from 18 primary tumors and two matched liver metastases, and show that ‘classical’ and ‘basal-like’ cells coexist in individual organoids. By single-cell transcriptome analysis of PDAC organoids and primary PDAC, we identify distinct tumor cell states shared across patients, including a cycling progenitor cell state and a differentiated secretory state. Cell states are connected by a differentiation hierarchy, with ‘classical’ cells concentrated at the endpoint. In an imaging-based drug screen, expression of ‘classical’ subtype genes correlates with better drug response. Our results thus uncover a functional hierarchy of PDAC cell states linked to transcriptional tumor subtypes, and support the use of PDAC organoids as a clinically relevant model for in vitro studies of tumor heterogeneity.

Background Cancer-associated fibroblasts (CAFs) are considered to play a fundamental role in pancreatic ductal adenocarcinoma (PDAC) progression and chemoresistance. Patient-derived organoids have demonstrated great potential as tumor avatars for drug response prediction in PDAC, yet they disregard the influence of stromal components on chemosensitivity. Methods We established direct three-dimensional (3D) co-cultures of primary PDAC organoids and patient-matched CAFs to investigate the effect of the fibroblastic compartment on sensitivity to gemcitabine, 5-fluorouracil and paclitaxel treatments using an image-based drug assay. Single-cell RNA sequencing was performed for three organoid/CAF pairs in mono- and co-culture to uncover transcriptional changes induced by tumor-stroma interaction. Results Upon co-culture with CAFs, we observed increased proliferation and reduced chemotherapy-induced cell death of PDAC organoids. Single-cell RNA sequencing data evidenced induction of a pro-inflammatory phenotype in CAFs in co-cultures. Organoids showed increased expression of genes associated with epithelial-to-mesenchymal transition (EMT) in co-cultures and several potential receptor-ligand interactions related to EMT were identified, supporting a key role of CAF-driven induction of EMT in PDAC chemoresistance. Conclusions Our results demonstrate the potential of personalized PDAC co-cultures models not only for drug response profiling but also for unraveling the molecular mechanisms involved in the chemoresistance-supporting role of the tumor stroma.