Session Item

Effective immunotherapy requires a tumour microenvironment (TME) that can enable effective invasion of and reception to immune cells. Abnormal blood vessel architecture, low perfusion, hypoxia and an immunologically “cold” nature may restrict the efficacy of immunotherapy and account for the relatively low proportion of patients in which immunotherapy has high efficacy. This study aimed to determine the changes in TME following low-dose fractionated radiotherapy in order to capitalise on their temporal nature for optimising subsequent immunotherapy.

This work examined subcutaneous flank tumours comprising AB-1 murine mesothelioma cells in BALB/c mice. Tumour irradiations were performed using a PXi XRAD Smart small animal image-guided radiotherapy system when tumours were approximately 20-30 mm2, with mice receiving 5 daily doses of either 0 Gy (sham) or 2 Gy. Animals were euthanised in groups of 4 on either the final day of 5-fraction radiotherapy, the day after or every subsequent 3 days for up to 14 days. Tumours were excised and cut onto slides for immunohistochemistry (IHC) staining of blood vessel markers, hypoxia and the presence/nature of T cells. Samples were also processed for RNA sequencing and network analysis.

Vessel density, assessed via staining of CD31+ cells, did not vary between groups, however vessel length and diameter decreased in the irradiated group. Vessel perfusion, assessed by tracing lectin, increased dramatically in the irradiated group with the level of perfusion dropping rapidly between 1 and 8 days post irradiation. Pimonidazole staining demonstrated much reduced levels of hypoxia in the irradiated group, returning to levels in the control group approximately 8 days post irradiation. Irradiation induced blood vessel maturation as assessed via pericyte coverage and activation for migration of T cells as assessed via ICAM-1. CD8+ and regulatory T cell infiltration increased due to irradiation, with their ratio >1 and increasing with time from irradiation. Radiotherapy increased the expression of genes associated with inflammatory response and creation of an immunologically “hot” TME. Immune deconvolution from bulk RNA sequencing also revealed cytotoxic T cell infiltration peaked 4 days post radiotherapy.

A radiotherapy dose fractionation of 2Gy x 5 daily doses improved the blood vessel structure and perfusion of a solid tumour environment. Hypoxia was reduced and T cell infiltration increased, with TME changes showing strong and rapid temporal variations. Increased gene expression and enriched gene sets associated with inflammatory responses suggest the low-dose fractionated radiotherapy was able to remodel the tumour vessels and inflame the tumour microenvironment, with the potential to improve immunotherapy efficacy.