Abstract LB-B06: A novel ex vivo 3D tumor organoid model of fresh patient tumors (3D-ACT) to assess efficacy of cellular therapy in immuno-oncology
Authors: Agrawal Vijayendra , Pabon Mibel , Page Melba Marie , Kreahling Jenny , Altiok Soner 
Published: March 2020

Abstract
Introduction: The spatial organization and dynamic interplay of the complex cell-to-cell interactions in patient tumors play an important role in cellular phenotypes that can result in permanent alterations in cellular functions and response to immuno-oncology (IO) treatments. Therefore, to assess the therapeutic efficacy of IO treatments, including cellular therapeutics, it is imperative to develop models that preserve the stromal-stoichiometry of the tumor microenvironment. Here we describe a patient-derived high content confocal imaging approach using an unpropagated tumor organoid model with intact tumor stroma for quantitative assessment of infiltration and target tumor cell killing activity of ex vivo expanded autologous tumor-infiltrating lymphocytes (TILs) in non-small cell lung cancer. Materials and Methods: All human tumor samples were obtained with patient consent and relevant IRB approval. For the ex vivo assays 3D tumoroids measuring 150 micron in size were prepared and cryopreserved during the process of ex vivo propagation of autologous TILs. Ex vivo propagated TILs were characterized using multiparameter flow analysis, fluorescently labeled and exposed to fresh tumor organoids. Computational confocal image analysis was used to quantify TILs infiltration into the tumor organoids and target tumor cell killing. Multiplex cytokine assays and flow cytometry analysis were performed to assess activation of TILs upon exposure to tumor organoids. Results: We successfully prepared matched autologous TILs and unpropagated 3D tumor organoids from 75% of NSCLC patient tumors. The characteristics of tumor immune microenvironment and tumor cell viability was evaluated in previously cryopreserved organoids using a custom image analysis algorithm that was developed for the collection of data in a structurally relevant environment on quantification of marker-specific cell number, cell viability and apoptosis in addition to structural and functional analysis of cells in intact 3D tumor organoids. High content confocal imaging analysis demonstrated that CD3/CD28 pre-activated TILs with increased activation phenotypes and enhanced pro-inflammatory cytokine release had marked infiltration into the 3D organoids compared to untreated TILs and PBMCs. The data was correlated with quantitative tumor cell killing assessment for each tumor organoid. Conclusion: These results demonstrate that our 3D-ACT model using ex vivo expanded TILs and 3D tumor organoids is an effective tool for the therapeutic assessment of autologous TILs and indicate that it can also be used to assess efficacy of other cellular therapy applications in Immuno-oncology. Furthermore, implementation of this platform in the clinical studies may also allow determining the most effective combinatorial cellular therapy strategies for individual patients.
Citation Format: Vijayendra Agrawal, Mibel Pabon, Melba Marie Page, Jenny Kreahling, Soner Altiok. A novel ex vivo 3D tumor organoid model of fresh patient tumors (3D-ACT) to assess efficacy of cellular therapy in immuno-oncology [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference on Molecular Targets and Cancer Therapeutics; 2019 Oct 26-30; Boston, MA. Philadelphia (PA): AACR; Mol Cancer Ther 2019;18(12 Suppl):Abstract nr LB-B06. doi:10.1158/1535-7163.TARG-19-LB-B06