Radiation-induced myocardial damage and its effect on the outcome after ischemia/reperfusion injury in mice
Background: Immunotherapy has revolutionized cancer therapeutics, particularly with the advent of checkpoint inhibitor monoclonal antibodies in 2013, designed to modulate the host immune response and enhance immune surveillance. Despite its transformative potential, challenges such as limited intratumoural T cell infiltration, downregulation of tumour-associated neoantigens, and the absence of tumour-specific immune surveillance in the tumour microenvironment (TME) impede the full efficacy of immunotherapeutic interventions. In solid tumours, insufficient cytotoxic T cell infiltration poses a critical challenge. To address this issue, we propose a novel approach that integrates arenavirus therapy with a CD3-directed T cell engager antibody. This combination aims to introduce viral antigens, enhancing immunosurveillance, and activate T cells at the tumour site through the CD3-targeting antibody. Results: Our study introduces a novel construct termed CD3-EpCAM Fc (CE-Fc), a bispecific entity comprising a CD3-specific single-chain variable fragment (scFv) linked to an EpCAM-specific scFv, featuring a Human Fc tag at the C-terminus. CE-Fc exhibits exceptional affinity for both target antigens. In vitro experiments confirm CE-Fc's ability to induce cytolysis in EpCAM-positive tumour cells, dependent on the Effector-to-Target (E: T) ratio.
In vivo investigations using recombinant LCMV-WE strain in 4T1 and MC38 tumour-bearing mice demonstrate enhanced CD4+ and CD8+ T cell populations in infected tumours compared to uninfected tumours. Further enhancement of T cell activation is observed when CE-Fc is combined with LCMV-WE infection, leading to more pronounced tumour regression. Significantly, the combination therapy surpasses monotherapy in targeting tumours and improving therapeutic efficacy, showcasing profound synergistic effects. Mechanistically, this combination treatment increases immune surveillance in solid tumour models by introducing viral antigens that attract and activate T cells through CD3 antibodies. Thoracic radiotherapy is an important component of multimodal cancer treatment and significantly improves the survival of cancer patients. However, this therapeutic benefit is associated with an increased risk of radiation-induced cardiac injury, leading to late adverse cardiac events. The complex molecular and cellular relationship between thoracic irradiation and induced cardiac damage that compromises cardiovascular health is still not fully understood. The aim of the present study was to investigate whether a single thoracic irradiation leads to increased cardiac vulnerability and worse outcomes in an in vivo model of MI. For this purpose, male mice were exposed to either a single thoracic irradiation (12.5 Gy) or sham-irradiation and were subjected to I/R injury four weeks later. The results showed that mice subjected to single thoracic irradiation had a decreased survival rate within three weeks after I/R compared to sham-irradiated mice + I/R. However, despite this discrepancy in survival outcomes, there were no significant differences in cardiac function or scar size between the two groups. It is important to emphasize that the interpretation of these results may have been biased by the survival effects. Molecular and cellular analysis provided evidence that thoracic irradiation alone resulted in transient fluctuations in circulating immune cells, with recovery observed within four weeks of exposure. In addition, a reduction in relative ATP content and qualitatively increased MitoSOX™ signal were found in the heart, indicating mitochondrial dysfunction. Upon I/R, this pre-damage may predispose cardiac ECs to further damage, as demonstrated by LGE-MRI measurements showing an increased accumulation of gadolinium in the left ventricle of irradiated mice. Taken together, the present study demonstrates that exposure of the heart to ionizing radiation results in significant cellular and molecular changes, including alterations in cardiac mitochondria. These persistent pathological changes are exacerbated by a ‘second hit’, caused by myocardial I/R, resulting in increased susceptibility to the deleterious effects of this injury, enhanced inflammatory response, and impaired survival in mice. Understanding the specific response to I/R in irradiated mice may improve clinical strategies aimed at reducing radiation-induced tissue damage.
RNA-seq analysis of isolated cardiac ECs and subsequent pathway analysis suggested activation of the inflammasome and NET signaling pathway in irradiated mice during the early inflammatory response after I/R. of these pathways could contribute to an enhanced early inflammatory response Activation after I/R as observed by increased plasma concentrations of several pro-inflammatory cytokines, such as MCP-1, IL-1β, MIP-1α and MIP-1β. This effect was accompanied by an increase in the circulating Ly6Chigh monocyte fraction and an enhanced infiltration of labelled-phagocytosing immune cells in the infarcted heart of irradiated mice as assessed by MRI.