Radiation induced regulation of endothelial adhesion receptors

Abstract

The synergy of modern immunotherapeutics and radiation has proven to be a promising strategy to combat cancer. However, fundamental questions regarding the underlying mechanisms of these therapies are unanswered. While clinical studies have demonstrated that radiation can potentially boost immune cell infiltration and tumor immunogenicity, the clinicians’ aim to use radiation to improve immune cell infiltration and turn so called ‘cold’ tumors with little infiltration into ‘hot’ tumors lacks mechanistic research data until today. We hypothesize that alterations of the tumor immune cell composition result from modified endothelial adhesion receptor expression mediating immune cell recruitment from the blood. Thus, in order to investigate the tumor vascular endothelium, we quantified infiltrating immune cells and examined the regulation of endothelial adhesion ligands following different fractionations of radiation. We applied two different radiation doses: 1x10Gy (a dose thought to improve immunogenicity), and 1x2Gy (conventional/normofractionation), analyzed immune cell infiltration via flow cytometry, and investigated the expression of endothelial molecules like ICAM‐1, ICAM‐2, VCAM‐1, E‐selectin, and P‐selectin using real time PCR and histology‐based approaches in the tumor and healthy vasculature. We analyzed transplantable lung tumors of Kras;Trp53 mutant mice 1h, 4h, 8h, 24h, 3d, 5d, and 8d after radiation. Importantly, we observed a generalized decrease of tissue immune cells within the first 24h with recovering populations at day 3 after radiation in all groups. Furthermore, we found that the expression of some endothelial adhesion receptors changes in a dose‐dependent manner, but others are not affected. For instance, ICAM‐2 remains unchanged in both groups, but tumor vasculature shows a rapid upregulation of E‐selectin within the first hour after radiation, followed by a downregulation and a second increase after 2–3 days. The lower fractions (2Gy) elicit an augmented upregulation of VCAM‐1 after 24h peaking at day 3. This is of particular interest as VCAM‐1 is vital to T cell recruitment and currently most research indicates that high doses are needed to improve tumor immunogenicity. However, we observed a more robust repopulation of both CD4 and CD8 T cells following 10Gy – a finding that might be due to a marked downregulation of ICAM‐1 following 2Gy doses that was not observed following 10Gy. An improved understanding of the endothelial adhesion receptor regulation depending on radiation fractionation is of therapeutic relevance: novel immune checkpoint inhibition strategies require efficient T cell recruitment to the tumor to be effective and so do, for instance, isolated and modified T cell (so called CAR‐T cell) therapies: Our results are the first to indicate that endothelial ligand regulation differs depending on radiation dose.Support or Funding InformationDFG (HE 6810/3‐1 to J.M.H., HE 6897/2‐1 to G.H.S.) and the CMMC (CAP‐13 to J.M.H. and CAP‐16 to G.H.S. as well as B02 to G.H.S. and J.M.H.)