Supplementary_Information_PDAC_Patternoids_Raw_data

Paper Title: “3D Patterned Organoid Model System for Characterization of Invasive Phenotypes and Drug Sensitivity in PDAC”

Authors & Affiliations:

\author[1,2,3]{Sophie C. Kurzbach}\author[1,2]{Violetta Carvajal-Heckele}\author[2,3,4,5,6]{Maximilian Reichert}\author[1,2,3]{Andreas R. Bausch}

\affil[1]{Technical University of Munich, TUM School of Natural Sciences, Department of Bioscience, Chair for Cellular Biophysics E27, 85748 Garching, Germany.}\affil[2]{Technical University of Munich, Center for Functional Protein Assemblies (CPA), 85748 Garching, Germany.}\affil[3]{Technical University of Munich, Center for Organoid Systems and Tissue Engineering (COS), 85748 Garching, Germany.}\affil[4]{Technical University of Munich, School of Medicine, Klinikum rechts der Isar, Medical Clinic and Polyclinic II, 81675 Munich, Germany.}\affil[5]{German Cancer Research Center (DKFZ), German Cancer Consortium (DKTK), Partner site Munich, 69120 Heidelberg, Germany.}\affil[6]{Technical University of Munich, Klinikum rechts der Isar, Medical Clinic and Polyclinic II, Translational Pancreatic Cancer Research Center, 81675 Munich, Germany.}

Abstract:

Pancreatic ductal adenocarcinoma (PDAC) is a highly invasive and heterogeneous malignancy, posing challenges for reproducible modeling and functional phenotypic analysis. To address these limitations, we developed a standardized 3D patternoid platform using collagen-based microcavity arrays to enhance organoid formation consistency and quantify subtype-specific invasion mechanisms. We utilized murine primary PDAC cells stratified by epithelial-mesenchymal transition (EMT) into three subtypes: epithelial (\textit{E-9591}), hybrid EMT (\textit{Mlow-8028}), and mesenchymal (\textit{M-16992}). The platform’s sensitivity was verified by a strong correlation between EMT scores and invasive phenotypes, as well as responses to physiological concentrations of the protease inhibitor Batimastat. Key invasion parameters—including invasive area, maximum invasion distance, and branching complexity—were measured under both genomic and drug-induced conditions. The platform demonstrated high inter-organoid reproducibility, with precise control over initial cell numbers ensuring batch-to-batch comparability. Invasion dynamics analysis revealed that epithelial cells (\textit{E-9591}) primarily relied on spatial constraints within the microcavity to invade, while hybrid EMT cells (\textit{Mlow-8028}) exhibited a mixed invasion mechanism. Batimastat drug sensitivity assays further distinguished invasion dependencies, confirming that \textit{M-16992} cells predominantly relied on protease activity, whereas \textit{Mlow-8028} exhibited both spatial constraint-driven and protease-dependent invasion. This scalable and adaptable 3D patternoid platform enables high-throughput analysis of invasive behaviors and therapeutic responses, offering significant potential for preclinical cancer research and personalized medicine.

Dataset Description:

Raw Data: Original invasion dynamics measurements, fluorescence imaging data.

Data Structure: Folders of raw data for each corresponding paper figure as .zip file.

Methodology: PDAC organoids were generated using a 3D patterned culture system that provides spatial control over cell seeding and growth. Briefly, PDAC cells were seeded onto micropatterned substrates and maintained in a defined, growth factor–supplemented medium under standard incubator conditions (37 °C, 5% CO₂). Data acquisition was performed via whole sample overview scans 2h upon cell seeding and after 3 days of cultivation, time-lapse confocal microscopy, capturing high-resolution z-stacks at fixed intervals to monitor organoid formation, invasive behavior, and morphological changes over several days or at the end time point. Raw imaging data were processed through a custom analysis pipeline that included processing, segmentation using threshold-based fiji macros, and quantitative measurements of invasion metrics using provided fiji macros. All experimental conditions—including drug concentrations, imaging parameters, and data processing protocols—were rigorously standardized across replicates to ensure reproducibility and robust statistical analysis.

Access and Licensing:

A dedicated reviewer access link has been provided with the DAS to facilitate data verification during the peer review process.

Keywords and Related Information:“PDAC,” “organoid model,” “invasive phenotype,” “drug sensitivity,” and “3D culture.”