Abstract 3350: Immuno-reactive cancer organoid models to examine immune checkpoint blockade efficacy
Authors: Shelkey Ethan N. W. , Soto-Pantoja David , Lu Yong , Soker Shay 
Published: September 2020

Abstract
Introduction: As the number of available immunotherapies for solid tumors increase, their prevalence in the clinic continues to rise as well. While the results are promising, and immunotherapies have benefits over traditional chemotherapeutics, a sizable percentage of patients are non-responders to all types of immunotherapy. These differences in sensitivity can be either innate or acquired. Yet, there has been limited number of 3D in vitro models to assess tumor immune-reactivity. These models allow directed study of the mechanisms of immunotherapy resistance in solid tumors. Our goal was to create organoids containing cancer cells from a variety of origins paired with cytotoxic T-cells of various origins to model immunotherapy efficacy. We pursued a system in which we could validate cancer cell killing due to immune cell action in an easily modifiable environment. Our choice of immune checkpoint inhibitor antibodies were a paired course of PD-1 and CTLA-4, which are used extensively in the clinic for several cancer types, or anti-CD47. The immune reactive tumor organoid system could be more generally applied to answer questions about immunotherapy resistance.
Methods: We created extracellular matrix (ECM)-like structures using collagen/hyaluronic acid-based hydrogels into which primary tumor cells or tumor cell lines were mixed, along with T-cells. The cancer cells incorporated were either MC-38 OVA cells for a positive T-cell-mediated cytotoxicity control, freshly-isolated CT-26 colon adenocarcinoma cells, 4T1 breast cancer cells, or cultured CT-26 cells. The T-cells were either derived from murine lymph nodes or were isolated from the murine tumor infiltrating mononuclear cell population. Organoids were treated with therapeutic equivalent doses. Additional factors such as microbial metabolites were evaluated for their effects on immunotherapy efficacy. We performed viability assays, flow cytometry, RT-qPCR, and immunohistochemistry (IHC) staining to characterize results.
Results and Discussion: We have been able to successfully show that the immune checkpoint inhibitor regimens stimulated internally localizing T-cells, inducing T-cell-mediated tumor cell killing our organoid model. Checkpoint inhibitor treated samples resulted in proportionally greater loss of viability with increased significance when compared to positive controls. The results were corroborated by IHC, showing increased numbers of CD-4+ T-cells and cytotoxic proteins such as granzyme B in the stimulated samples.
Conclusion: We have created an ex-vivo tumor immune-reactive tumor organoid model for studying immunotherapy. This will allow us to modulate facets of the tumor in the organoid system including cancer type, tumor cell mutations, biochemical signals, and physical properties of the microenvironment. We can then observe the impacts of these changes on immunotherapy efficacy to determine what factors could potentially be contributing to differences in patient sensitivity.
Citation Format: Ethan N. W. Shelkey, David Soto-Pantoja, Yong Lu, Shay Soker. Immuno-reactive cancer organoid models to examine immune checkpoint blockade efficacy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 3350.