Abstract B84: Live imaging reveals hypoxia to be a limiting factor in CTL-mediated tumor rejection

Abstract

Abstract Background: One of the obstacles to successful immunotherapy by CTLs is thought to be poor vascularization of tumors that leads to sub-optimal tumor infiltration. In contrast, the effects of disrupted vascularization and the hypoxia it introduces on CTL migration and function within tumors have not been investigated. Methods: To study whether tumor vascularity can shape anti-tumor CTL responses, we combined immunohistochemistry of human melanoma samples with dynamic two-photon imaging within B16-OVA tumors in live mice. Results: In patients, CD8 T cells were twofold denser around peripheral vessels than around deep vessels, and fourfold denser than in avascular areas. This pattern was clearest in poorly-infiltrated tumors. Correspondingly, in mice, OT-I CTLs preferentially infiltrated B16-OVA tumors through peripheral ICAM1+ vessels. CTLs remained initially at their entry areas, crawling vigorously only within 50 µm of flowing blood vessels. Gradually, CTLs formed dense perivascular swarms, producing locally-high CTL-to-target ratios at their advancing front. These swarms advanced through the tumors, leading to patchy elimination of tumor cells. Target killing was antigen-specific, contact-dependent and perforin-mediated, and required several CTLs to engage one target cells to be effective. Several findings suggest that CTL confinement to perivascular areas is oxygen-dependent: occlusion of intratumoral blood vessels resulted in immediate arrest of CTLs. Immunohistology indicated that CTLs avoided hypoxic tumor areas. Hypoxia and mitochondrial uncoupling interfered with CTL migration in vitro and, finally, highly vascular tumors grown in a matrix containing pro-angiogenic factors were more easily rejected after CTL injection, displayed delayed growth in untreated mice but were not affected in immunodeficient mice. Conclusions: We suggest that CTLs depend on oxygen for intratumoral locomotion and cytolysis. Incomplete vascularization promotes patchy infiltration and tumor escape; tumor immunotherapy may thus benefit from normal and extensive tumor angiogenesis Citation Format: Guy Shakhar, Tali Feferman, Yoav Manaster, Anat Hutzler. Live imaging reveals hypoxia to be a limiting factor in CTL-mediated tumor rejection. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B84.