Background Tertiary lymphoid structures (TLS) are non-encapsulated immune cell aggregates that form at sites of chronic inflammation, including in and around tumors. Recent studies have shown that the presence of TLS in human tumors is an indicator of positive clinical outcome. However, due to dysregulated angiogenesis, many tumors have a poorly-organized and leaky vasculature that impedes entry of immune effector cells into tumors and consequently, TLS formation. It has been shown in pre-clinical studies that low doses of antiangiogenic agents normalize the tumor vasculature, leading us to hypothesize that treating tumors with low-doses (well below drug MTD) of vascular normalizing (VN) therapies will improve immune cell infiltration and TLS formation within the tumor microenvironment (TME).

Methods To test this hypothesis, melanoma-bearing mice were treated intratumorally with VN agents. Five days post-treatment, tumors were digested into single cell suspensions and RNA was isolated and used for RT-PCR. Transcript levels of TLS-promoting factors (CCL19, CCL21, CXCL13) and markers of vascular normalization (HIF1A, GLUT1) and inflammation/immune cell infiltration (CXCL10, VCAM1, CD8A) were measured and compared to PBS treated controls. Changes in tumor vasculature were evaluated using immunofluorescence microscopy (IFM) of tumor sections stained with CD31, PNAd, and PDGFRβ. Fluorescently-labeled lectin was injected into the mice to observe tumor perfusion. TLS formation was evaluated in tumor sections using IFM, with TLS being defined as PNAd+ vessels surrounded by clusters of CD45+ cells.

Results We observed that the VN agents dasatinib, STING agonist, bevacizumab, and agonist anti-TNFR1 antibody each induced global changes in the TME that are consistent with enhanced immune cell infiltration and TLS formation. These changes include increases in expression of CCL19, CCL21, and VCAM1. Dasatinib and STING agonists were shown to promote VN, as demonstrated by improved lectin perfusion into the tumor and increased pericyte coverage of blood vessels on-treatment.

Conclusions VN agents induce global changes in immune cell infiltration and TLS-promoting factors in the TME. In vivo, these agents induce VN in the TME and promote TLS formation. This knowledge can be used to develop optimal combination immunotherapy designs in the cancer setting.