Abstract A073: Multiplexed therapeutic sensitivity of patient derived pancreatic cancer organoids after neoadjuvant chemotherapy by high content imaging
Authors: Hossan Md Shahadat , Lin Ethan S. , Riedl Ellie E. , Stram Austin , Warner Jamie M. , Kratz Jeremy D. 
Published: November 2022

Background: Therapeutic development in pancreatic ductal adenocarcinoma (PDAC) requires accurate models for assessing response. Patient-derived cancer organoids (PCOs) have been shown to faithfully recapitulate genomic features of advanced PDAC. Mechanisms of subclonal resistance or intratumoral heterogeneity cannot be characterized using well-level viability assays. Methods: A high-content imaging platform was adapted in low volume, 96-well format for using Cytation 5 (Biotek). PDAC PCOs were plated from organoid suspension in 1:1 reduced growth factor Cultrex plating 10uL per well. After 24h, physiologic chemotherapy was applied to match pharmacologic exposure of FOLFIRINOX (FFX) and gemcitabine/nab-paclitaxel (g/n-pac). Initial response was assessed by normalized change in PCO area at 72h. Subsequent viability staining was performed for single organoid level response for apoptosis (caspase-3/7-FITC) and necrosis (TO-PRO-3). Analyses were automated in Gen5.12 (Biotek) with selection for elements using circularity (&gt;0.4) and alignment by location within &lt;88 mm. Response was assessed using descriptive statistics and effect size (Glass’s Delta) normalized to media control. Results: PCOs were generated from patient-derived pancreatic tumor without prior treatment. Low volume plating facilitated imaging with reduced Z stack (300 mm) with an average of 14 PCOs evaluable per well. Increasing markers of apoptosis by caspase-3/7 staining stratified serial decrease in mean organoid growth by quartile [+285, +175, +42%, +28]. Increasing markers of necrosis using TO-PRO-3 staining stratified serial decrease in mean organoid growth by quartile [+259, +191, +54%, +26]. Normalized change in area was reduced with g/npac +11.9% (GD=1.43) and FFX +20.4% (GD=1.38) versus control +281%. Subclonal populations existed with normalized change in area &gt;50% which represented 17.8% of g/npac and 18.5% of FFX treated PCOs. Individual organoids with mean area staining positive for apoptosis was increased in g/npac +67.3% (GD=2.96) and FFX +65.1 (GD=2.84) versus background control +12.8%. Individual organoids with mean with area staining positive for necrosis was increased in g/npac +47.3% (GD=1.88) as compared to both FFX +34.9 (GD=1.20, p&lt;0.002) and background control staining of +11.1% (p&lt;0.001). Conclusions: High content imaging in PDAC PCOs provide scalable model for response assessment. This method can track subclonal populations with residual growth after treatment with chemotherapy. Here, we provide proof of concept in patient-derived tissue for clinically relevant response assessment with increased induced necrosis with g/npac in comparison to FFX. Ongoing investigations are generating sensitivity thresholds to define subclonal populations with residual growth and viability for application in clinical prediction.

Citation Format: Md Shahadat Hossan, Ethan S. Lin, Ellie E. Riedl, Austin Stram, Jamie M. Warner, Jeremy D. Kratz. Multiplexed therapeutic sensitivity of patient derived pancreatic cancer organoids after neoadjuvant chemotherapy by high content imaging [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A073.