Abstract 42: The importance of intact tumor immune microenvironment in unpropagated 3D fresh patient tumoroids in immuno-oncology drug testing for immunogenic tumor cell death and T-cell activation
Authors: Agrawal Vijayendra , Mediavilla-Varela Melanie , Page Melba Marie , Kreahling Jenny , Altiok Soner 
Published: August 2019

Background: The tumor stroma contains important components of the tumor microenvironment including fibroblasts, immune cells, and the extracellular matrix. While none of these cells are malignant, the dynamic interplay of these cells drive alterations in cellular functions. While informative, conventional 2D dissociated tumor models do not maintain the stromal-stoichiometry of the tumor microenvironment to accurately assess ex-vivo tumor cell viability and T-cell activation after drug treatment. In this study we performed a comparative assessment of tumor immune microenvironment, and therapy mediated changes in tumor cell viability and T-cell activation in live 3D tumoroids versus matched 2D dissociated cells of the same patients’ fresh tumors side-by-side.

Methods: All human tumor samples were obtained with patients’ consent and relevant IRB approval. Un-propagated live 3D tumoroids measuring 100-150 micron in size were prepared from fresh patient tumor samples. In parallel, the same patient tumor sample was enzymatically digested into 2D tumor dissociations to obtain single cell populations for direct comparison to live 3D tumoroids. Cytokine analysis was performed to determine immunomodulatory cytokine profile. To better understand the role of tumor extracellular matrix on immuno-oncology drug response, tumor models were treated with pembrolizumab (Keytruda) and atezolizumab (Tecentriq) (10ug/ml) for 48 hours and multiplex cytokine release assay, flow cytometry and high content confocal image analysis were performed.

Results: Comparative studies revealed that removal of extracellular matrix by digestive enzymes significantly affects tumor immune microenvironment as assessed by multiplex cytokine release assays. Flow cytometry analysis demonstrated differential T-cell and NK cell activation upon immuno-oncology drug treatment between the unpropagated 3D-tumoroid and dissociated cell suspension models. High-content imaging studies combined with flow death analysis indicated that tumor extracellular matrix plays an important role in tumor cell killing by immuno-oncology drugs.

Summary: Our data shows the importance of maintaining the stoichiometry and cell-to-cell interactions of the tumor microenvironment. Using a powerful combinatorial approach including high-content image analysis, flow cytometry, and cytokine analysis we are able to accurately assess therapy-mediated tumor cell death in a quantitative manner. Furthermore, these data demonstrate that intact tumor extracellular matrix within unpropagated 3D tumoroids is critical for a more accurate assessment of therapy-mediated changes in tumor immune microenvironment.

Citation Format: Vijayendra Agrawal, Melanie Mediavilla-Varela, Melba Marie Page, Jenny Kreahling, Soner Altiok. The importance of intact tumor immune microenvironment in unpropagated 3D fresh patient tumoroids in immuno-oncology drug testing for immunogenic tumor cell death and T-cell activation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 42.