Abstract PR04: Intravital imaging of cytolytic activity reveals how poor tumor vascularization and hypoxia limit tumor rejection

Abstract

Abstract Background: Immunotherapies that harness cytotoxic T lymphocytes (CTLs) to eradicate solid tumors, melanoma in particular, have re-emerged as potent clinical options in recent years. Improving the dispersion and cytotoxicity of intratumoral CTLs may further benefit patients. One of the obstacles to successful treatment is thought to be poor vascularization of tumors, which 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 melanoma tumors expressing ovalbumin (OVA) 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, adoptively transferred OT-I CTLs, specific for OVA, preferentially infiltrated B16-OVA tumors through peripheral ICAM1+ vessels. CTLs remained initially at their entry areas, crawling vigorously only within 50µm of flowing blood vessels. These dense CTLs swarms efficiently cleared perivascular areas of tumor cells but left avascular areas intact. In contrast, Foxp3+ regulatory T cells could venture farther away from blood vessels. Target killing was antigen-specific, contact-dependent and perforin-mediated, and required multiple CTLs to engage one target cell to be effective. Several findings converge to suggest that perivascular CTL confinement is oxygen-dependent: occlusion of intratumoral blood vessels resulted in instantaneous and reversible 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 the pro-angiogenic factor bFGF were more easily rejected after CTL injection, displayed delayed growth in untreated mice but were not affected in immunodeficient mice. Although such normalized tumors attracted normal numbers of CTLs, these cells migrated more freely within the tumors. Conclusions: We propose that CTLs depend on oxygen for intratumoral locomotion and cytolysis. Incomplete vascularization promotes patchy infiltration and tumor escape; tumor immunotherapy may thus benefit from an extensive and functional vasculature. Citation Format: Guy Shakhar, Yoav Manaster, Tali Feferman. Intravital imaging of cytolytic activity reveals how poor tumor vascularization and hypoxia limit tumor rejection. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Angiogenesis and Vascular Normalization: Bench to Bedside to Biomarkers; Mar 5-8, 2015; Orlando, FL. Philadelphia (PA): AACR; Mol Cancer Ther 2015;14(12 Suppl):Abstract nr PR04.