Abstract A071: Pancreas organoid immune co-culture system identifies immunomodulators in pancreas adenocarcinoma
Authors: Ebben Johnathan D , Turicek David , Hossan Md Shahadat , Stram Austin , Lin Ethan , Hess Nicholas , Mayhew Zachary , Kinney Melissa A , Burpee Devin , Patel Anikait , Capitini Christian M , Kratz Jeremy D 
Published: December 2023

Background Metastatic pancreas adenocarcinoma (PDAC) is highly lethal and minimally responsive to immunotherapy. Gamma delta T cells (gDTs) are a unique T cell subset found within the PDAC microenvironment. Animal models have shown that gDTs promote oncogenic progression and ablating gDT delays cancer progression and improves survival. Understanding the activity of immune effector cells against cancer organoids provides a scalable platform to understand the impact of activation and small molecule modulation to redirect gDT killing in PDAC.

Methods Patient-derived organoids (PDOs) were generated from fresh tissue sampling and exapanded using a defined, serum-free media. Upon passaging, media was collected from PDAC PDOs. Allogeneic gDTs isolated from healthy donors were cultured in PDAC PDO conditioned media from 3 separate PDOs, in 3 separate biological replicates, and then subjected to bulk RNAseq. Allogeneic gDTs were isolated and cultured with PDAC PDOs at ratio of 5:1 (gDTs:PDAC PDOs) for 48 hours. After 48 hours, staining was performed for induction of apoptosis (cleaved caspase-3/7FITC). Images were acquired by high content imaging (Cytation 5, BioTek) and quantified for response across organoid populations. Based on RNAseq nominated candidates, low density lipoprotein (LDL) was added (10 ug/mL) as well as a small molecule inverse agonist of the Liver X Receptor (LXR) beta transcription factor (SR9243) to assess the ability of these factors to modulate gDT mediated PDAC killing.

Results Exposure of gDTs to PDAC PDO secreted factors results in differential regulation of cholesterol handling, including upregulation of cholesterol efflux, and downregulation of cholesterol uptake and biosynthesis related genes in conditioned T cell populations. gDTs cause marked apoptosis in PDAC PDOs, including 40% staining of % organoid area versus negligible staining in control populations of 1.9% organoid area (effect size = 9.5, p&lt;0.001). This response was directly compared to standard chemotherapy at the time of clinical resistance to FOLFIRINOX which conferred only 2% staining of mean organoid area (effect size = 0.11, NS). Given RNAseq data demonstrating cholesterol handling changes after exposure to PDAC secreted factors, we investigated LDL supplementation in our model. Supplementation of LDL significantly improves gDT mediated killing, with induction of apoptosis seen in 52% of the PDAC PDO area (p=0.003). This effect is mimicked by the small molecule SR9243 (an LXR inverse agonist), with 56% of PDAC PDO demonstrating apoptotic induction at 48 hours.

Conclusions A PDAC PDO model system can be used to investigate gDT mediated PDAC killing when applied in low-volume medium throughput screening applications.  Modulation of LXR activity and microenvironment LDL results in substantially increased immune-mediated killing of PDAC. This screening platform identifies potentially clinically actionable modulators of immune response to PDAC, and suggests potential future immunologically relevant targets that may improve the efficacy of immunotherapy.

Citation Format: Johnathan D Ebben, David Turicek, Md Shahadat Hossan, Austin Stram, Ethan Lin, Nicholas Hess, Zachary Mayhew, Melissa A Kinney, Devin Burpee, Anikait Patel, Christian M Capitini, Jeremy D Kratz. Pancreas organoid immune co-culture system identifies immunomodulators in pancreas adenocarcinoma [abstract]. In: Proceedings of the AACR-NCI-EORTC Virtual International Conference on Molecular Targets and Cancer Therapeutics; 2023 Oct 11-15; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2023;22(12 Suppl):Abstract nr A071.