Abstract PR-005: CAR T-cell motility response to interstitial fluid flow and tumor microenvironment
Authors: Mendes Gabriela Geraldo , Owens Joseph , Gutova Margarita , Brown Christine , Munson Jennifer M. 
Published: March 2024

Abstract
Background: Glioblastoma (GBM) is the most common and aggressive primary brain tumor in adults with an average survival of 15 months. Standard of care treatments for GBM include surgical resection, radiotherapy and chemotherapy, but do not result in long-term survival. This poor response is due to the invasive nature of GBM, so immunotherapies like chimeric antigen receptor (CAR) T cells are being investigated as a novel treatment. However, solid tumors still present challenges to this therapy, including a hostile tumor microenvironment (TME) and enhanced interstitial fluid flow (IFF). We have shown that IFF can increase invasion of glioma cell lines in vitro, but the impact of IFF along with tumor growth dynamics remains poorly understood for CAR T cells. Therefore, we aim to identify the impact of IFF on T cell migration and distribution in the GBM TME. Methods: Our lab has developed a 3D tissue-engineered model that recapitulates the brain TME. This system consists of tissue culture inserts containing hyaluronan collagen I gel with T cells (labeled with Hoescht), and CellTracker-labeled primary brain-tumor-derived cells (PBTs), astrocytes, and microglia. Flow is then applied to the gel via a pressure head for over 3 hours. Cell viability and transmigration are measured via flow cytometry analysis by collecting gels and flow-through media 3h from flow application. To study CAR T cell behavior and interaction with the TME in our 3D model we used live cell imaging techniques. Results: Different mechanisms have been identified to be involved with IFF-mediated invasion, including the receptor Sphingosine-1-Phosphate Receptor 3 (S1P3R). We have investigated whether T cells have their viability or motility affected by TY52156, a selective S1P3R antagonist, and found that T cell viability is significantly reduced in the presence of the drug when CAR T cells are resuspended in the gels. Additionally, CAR T cell migration through the gels was not affected by TY52156 under static (0.3µm/s) and flow (0.7µm/s) conditions compared to T cell media or DMSO; however, we have seen a positive trend on cell migration under fast flow (3µm/s). We found that in static conditions CAR T cell motility does not significantly change in the presence of astrocytes and microglia but the average cell speed is much higher in the same condition. Conclusion: Further analyses of other parameters like net displacement and directedness are ongoing to better understand the correlation between T cell migration and TME. Because T cell flow-mediated motility was not influenced by blockade of S1PR3, we are going to extend our studies to other mechanisms of IFF-stimulated invasion, like CD44. Immunohistochemistry (IHC) has been used to identify the distribution of CAR T-cells infused via intracerebroventricular (ICV) injection post tumor formation and astrocytes and microglia have been stained to investigate the correlation between T cell distribution and interaction with the brain TME which will be analyzed.
Citation Format: Gabriela Geraldo Mendes, Joseph Owens, Margarita Gutova, Christine Brown, Jennifer M. Munson. CAR T-cell motility response to interstitial fluid flow and tumor microenvironment [abstract]. In: Proceedings of the AACR Special Conference on Brain Cancer; 2023 Oct 19-22; Minneapolis, Minnesota. Philadelphia (PA): AACR; Cancer Res 2024;84(5 Suppl_1):Abstract nr PR-005.