PRESERVATION OF DRUG SENSITIVITY STRATIFICATION IN MINIATURIZED TUMOR ORGANOID CULTURES

Pancreatic ductal adenocarcinoma (PDAC), is the most lethal adult malignancy and the 3rd-leading cause of cancer-related death [1].High tumor heterogeneity poses challenges in selecting the optimal treatment for each patient.Patient-derived organoids (PDO), three-dimensional self-organizing models that mimic tumor architecture, show great promise for patient-specific pharmacotyping.Drug sensitivity testing on PDAC PDOs has shown promising correlation to clinical patient response [1][2].Treatments predicted to be effective delayed cancer progression 3 times longer than treatments predicted to be resistant [3].Cell expansion time is currently a major barrier to clinical adoption, with a median of 53 days of expansion needed to screen 5 drugs [3].This can potentially be shortened by reducing organoid culture and assay volumes, but miniaturization has been constrained by limitations in pipetting small volumes of Matrigel.Droplet microfluidics offers a promising solution with various solid tumor PDOs [4], but has not yet been reported for use with pancreatic cancer organoids.Given the rapid progression of PDAC, expediting the path to effective treatment is paramount.Here, we report a 100-fold reduction in culture volume, with PDAC PDOs cultured in 500 nL beads of Matrigel generated with microfluidic droplets.Comparing the drug sensitivity of these miniaturized cultures with standard 50 L Matrigel domes, we address the notable absence of rigorous quantitative comparisons between miniaturized and standard PDO culture systemsOrganoids were successfully grown in 500 nL beads with a mouse PDAC line as well as cells derived from a human PDAC patient.Drug dose responses were quantified with the CellTiter-Glo 3D assay.In the mouse studies, a drug-resistant cell line served as the positive control.These experiments were used to establish a reproducible drug screening protocol.In the human studies, 5 commonly administered chemotherapeutic drugs were stratified based on efficacy, as quantified by the dose response area under the curve (AUC).The response of the miniaturized and standard culture models correlated well.After 4 days of treatment, human PDAC PDOs were found to be sensitive to gemcitabine, paclitaxel, and irinotecan and resistant to oxaliplatin or 5-fluorouracil when screened over a wide range of 19 concentrations.Unpaired t-tests confirm statistical differences in drug sensitivity between the sensitive and resistant drugs, with no significant differences found between multiple runs with the same drug or between drugs in the same sensitivity grouping.Organoid miniaturization by droplet microfluidics therefore holds considerable promise for accelerating the development of targeted PDAC therapies.By assaying PDOs in 3D, rather than expanding biopsy material in 3D and subsequently assaying in 2D as commonly practiced, our approach preserves heterogeneity and enhances patient-specific accuracy without skewing toward the basal tumor phenotype.We now plan to test our protocol on fresh clinical biopsies to establish the reduction in cell expansion time achieved by miniaturization and the consequent reduction in sample-to-assay time in a clinical setting.