Therapeutic vascular normalization to promote tumor-associated tertiary lymphoid structures

Abstract

Abstract Tertiary lymphoid structures (TLS) are non-encapsulated immune cell aggregates that form at sites of chronic inflammation. Recent studies have shown that the presence of TLS in human tumors indicates positive clinical outcome. However, many tumors have poorly organized and leaky vasculature that impedes entry of immune effector cells into tumors and consequently TLS formation. Recently, studies have shown that low doses of antiangiogenic agents normalize tumor vasculature, leading us to hypothesize that treating tumors with low doses of vascular normalizing (VN) therapies will improve immune cell infiltration and TLS formation within the tumor microenvironment (TME). To test this hypothesis, tumor-bearing mice were treated intratumorally with VN agents. RNA was isolated from digested tumors and transcript levels of TLS-promoting factors and markers of inflammation/immune cell infiltration were measured and compared to PBS treated controls. Changes in tumor vasculature were evaluated using immunofluorescence microscopy. Additionally, primary cultures of murine T cells and DCs and the BPR melanoma cell line were treated in vitro with VN agents. We observed that the VN agents dasatinib, STING agonist, bevacizumab, and agonist anti-TNFR1 antibody each induced global changes in the TME that are potentially supportive of immune cell infiltration and TLS formation. In vitro, dasatinib induced DCs and BPR melanoma cells to express higher levels of co-stimulatory receptors, MHC class I and II, and TLS-promoting chemokines. Treatment with a STING agonist was able to enhance T cell activation, while treatment with dasatinib inhibited T cell activation.