Abstract B059: Mechanisms of Sustainable T-cell Recruitment in a Perfusion-Enabled 3D Interstitial Network

Abstract All immunotherapy modalities depend on the ability of effector T-cells, including chimeric antigen receptor (CAR) T cells, to efficiently migrate into target tissue. In addition, the persistence of T-cells—survival, migration, and effector function—is integral to an efficient immune response. The dynamic metabolic landscape of T-cells during migration and effector function is poorly understood, and there is a lack of appropriate preclinical models to address this issue. Key factors include 1) the ability for spatial and temporal control of metabolic transport, 2) holistic investigation of T-cell migration in complex yet well-controlled multi-component ecosystems, 3) and physiologically and patient-specific relevance for examining T-cell persistence. We developed an immunology model with long-term (> 2 weeks) 3D ex vivo co-culture of human-derived tumors and autologous immune cells. We define a balance that supports efficient T-cell migration and effector function within well-known heterogeneous tumor microenvironments. Compared to static culture, perfusion culture (with constant flowrate 60 μL/h) notably reduced T cell metabolic stress, increased proliferation, and shortened the doubling time from 2 days to 1.5 days. In perfusion culture, CellROX and MitoSOX staining revealed a significant reduction in oxidative stress in both the cytoplasm (general cellular oxidative stress) and mitochondria (superoxide production) of CD4+ and CD8+ human-derived T cells. Multiplex immunofluorescence cell profiling on patient-derived microtumors revealed heterogeneous distribution of immune cell populations, including CD8+ (Cytotoxic T Lymphocytes) and CD4+ (Helper T Cells) T cells, as well as MDSCs, defined by markers CD11b+/CD33+/HLA-DRlow/CD15+ CD14- for PMN-MDSC). Intriguingly, a case study involving a microsatellite stable (MSS) CRC phenotype harboring POLE mutation and associated ultrahigh TMB demonstrated a response to PD-1 blockade, potentially from the intratumoral immune cell population. Furthermore, we have employed this approach for T cell trafficking and track their chemotaxis along interstitial paths. We assessed T-cell haptokinesis properties based on cell viability, shape, migration direction, and velocity under the influence of three (3) different fluid flow rates: 1-10 μL/h (low), 40-60 μL/h (medium), and 70-100 μL/h (high) for at least three (7) days. Our results suggested that chemotactic axes regulate T cell migration at baseline for both target and wild-type tumors. Furthermore, the increased infiltration and cytotoxic activities of T-cells enhanced the production of chemoattractants, thereby recruiting more T cells from distant site. These results help us understand physiologically relevant 3D T-cell persistence and infiltration patterns influenced by long-term perfusion that have never been studied. For the first time, we showed evidence of the complex microenvironment components, including physical barriers, tissue heterogeneity, immune suppressor cells, and perfusion transport, collectively contribute to overall immune function. Citation Format: Duy T. Nguyen, Laxmi Swetha Karanam, Said J Cifuentes Maury, Zoe Zhou. Mechanisms of Sustainable T-cell Recruitment in a Perfusion-Enabled 3D Interstitial Network [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr B059.