Abstract B079: Human 3D ovarian cancer models reveal malignant cell intrinsic and extrinsic factors that influence CAR-T cell activity
Authors: Joy Joash D. , Malacrida Beatrice , Laforets Florian , Kotantaki Panoraia , Maniati Eleni , Manchanda Ranjit , Hopkins Sarah , Garrobo-Calleja Ianire , Annibaldi Alessandro , Gautrot Julien , Balkwill Frances R. 
Published: March 2024

Abstract
Chimeric antigen receptor (CAR)-T cell therapies have a curative effect in some hematologic cancers, but limited efficacy in solid tumors. Using human 3-dimentional (3D) in vitro cell models of increasing complexity, we investigated barriers to CAR-T cell activity in high grade serous ovarian cancer (HGSOC), a disease with poor immunotherapy response rates. We initially identified mucin-1 (MUC1) and TnMUC1 as target antigens in human HGSOC omental metastasis and HGSOC cell lines, OvCAR3 and G164. We then developed second generation CAR-T cells against these targets and tested them in monolayer, spheroids, collagen gels and microfluidic models. In monolayer and spheroid cultures, OvCAR3 cells were sensitive, but G164 cells were resistant to CAR-T cell cytotoxicity. Treating G164 cells with birinapant, an antagonist of cellular inhibitor of apoptosis protein, rendered them sensitive to CAR-T cell killing. Furthermore, treating OvCAR3 cells with anti-tumor necrosis factor alpha (TNFa) delayed CAR-T cell activity. We concluded that malignant cell-intrinsic antigen independent resistance to CAR-T cell killing may involve defective death receptor signaling especially involving TNFa pathway. Surprisingly, when we co-cultured G164 cells as spheroids with primary omental fibroblasts from ovarian cancer patients, CAR-T cells were activated and cytotoxic. C-C motif chemokine ligand 2 (CCL2) produced by fibroblasts activated CCR2/4 positive CAR-T cells to induce cytotoxicity in an antigen-dependent manner. We then investigated CAR-T cell activity in collagen gels seeded with co-cultures of cancer cell and fibroblasts. In OvCAR3 gels, CAR-T cells penetrated the gels and were cytotoxic against the cancer cells. However, in G164 gels, a dense extracellular matrix (ECM) produced mainly by fibroblasts, abrogated CAR-T cell migration and cytotoxicity. Inhibiting transforming growth factor beta (TGFb) signaling pathway reduced ECM content and density in G164 gels, thus promoting CAR-T cell migration and cytotoxicity. To recapitulate systemic delivery of CAR-T cells, we developed vascularized microfluidic ovarian cancer models. CAR-T cells successfully penetrated and killed malignant cells in co-culture collagen gels when delivered through vascularized microfluidic devices. These models further confirmed that CAR-T cell activity is influenced by the death receptor signaling in malignant cells. Complex 3D human cell models may provide an efficient way of screening multiple cytotoxic human immune cell constructs while also revealing novel mechanisms of resistance, thus accelerating pre-clinical research into immunotherapies in solid tumors.
Citation Format: Joash D. Joy, Beatrice Malacrida, Florian Laforets, Panoraia Kotantaki, Eleni Maniati, Ranjit Manchanda, Sarah Hopkins, Ianire Garrobo-Calleja, Alessandro Annibaldi, Julien Gautrot, Frances R. Balkwill. Human 3D ovarian cancer models reveal malignant cell intrinsic and extrinsic factors that influence CAR-T cell activity [abstract]. In: Proceedings of the AACR Special Conference on Ovarian Cancer; 2023 Oct 5-7; Boston, Massachusetts. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_2):Abstract nr B079.