Establishment of an in vivo murine model of immune checkPoint inhibitors cardiotoxicity: emerging role of vascular permeability in pembrolizumab-induced cardiotoxicity
Authors: Efentakis P , Choustoulaki A , Kostopoulos Ι , Varela A , Georgoulis A , Tsekenis G , Gakiopoulou C , Ntanasis-Stathopoulos I , Davos C , Tsitsiloni O , Dimopoulos M A , Terpos E , Gavriatopoulou M , Andreadou I 
Published: November 2023

Abstract

Introduction
Advances in oncology have led to the discovery of immune checkpoint inhibitors (ICIs) that exhibit remarkable results in cancer patients’ survival and tumour regression. Despite their antitumor potential, some life-threatening immune-related adverse effects (AEs) including cardiotoxicity have been reported, while the pathomechanism of the cardiac AEs is obscure.

Purpose
We herein investigated the cardiotoxic effects of ipilimumab (IPI, anti-CTLA-4), pembrolizumab (PEM, anti-PD-1) and avelumab (AVE, anti-PD-L1) in primary murine ventricular cardiomyocytes (mAVCs) and spleenocytes in vitro and established the mechanism and progression of Pem’s cardiotoxicity in an in vivo murine model.

Methods
Primary mAVCs and spleenocytes were isolated and incubated with IPI, PEM and AVE (0-100 μg/ml) for 24h. Cell viability was assessed by MTT assay. Subsequently conditioned media from spleenocytes treated with the aforementioned ICIs was transferred onto the mAVCs for 24h. The experiments were repeated for molecular analyses. For the confirmation of PEM binding on the murine PD-1, human and murine PD-1 extracellular domains (ED) were biotechnologically produced and PEM binding was confirmed by circular dichroism (CD). C57BL6/J male mice were randomized into i. IgG4 and ii. Pem groups and treated for 2 (n = 5/group) and 5 weeks (n = 9/group). IgG4 and Pem were administered intraperitoneally once weekly at 2mg/ml. Pem dose was directly translated from humans. Mice underwent weekly echocardiography analysis, while at the endpoint, mice were sacrificed for myocardial sampling and histology analyses.

Results
IPI, PEM and AVE did not induce direct cytotoxicity on primary mAVCs, whereas IPI was excluded from the study due to unexpected spleenocyte cytotoxicity. Incubation of mAVCs with PEM and AVE conditioned media, revealed that only PEM could induce Immune cell (IC)-dependent cytotoxicity at 50 and 100 μg/ml and establishment of inflammation and autophagy in vitro. CD experiments showed that PEM binds on the murine PD-1 ED, which supports for the first time the use of the murine model for the extraction of translational results. In vivo Pem led to %Fractional shortening (FS%) decline, which was significantly reduced versus baseline at 2 weeks and further depressed at 5 weeks. At two weeks, Pem increased ICAM-1 expression as well as iNOS and beclin-1 expression in the myocardium, which was accompanied by intra-cardiac IC infiltration. At five weeks, Pem further increased vascular permeability, as shown by e-selectin and ICAM-1 upregulation and led to increase of cardiac autophagy and inflammation markers.

Conclusions
Herein we established for the first time a murine in vivo model of PEM cardiotoxicity. PEM induces an IC-mediated mAVCs cytotoxicity, via induction of inflammation, autophagy and ER-stress. Increased vascular permeability and dysregulated endothelial homeostasis seem to orchestrate early-on Pem’s cardiotoxicity manifestation in vivo.