EARLY PHENOTYPIC AND PARACRINE CHANGES IN CARDIAC MESENCHYMAL STROMAL CELLS AFTER THORACIC RADIOTHERAPY AS A HALLMARK OF CARDIOTOXICITY

Background. Radiotherapy (RT)-induced cardiac toxicity and cardiovascular diseases represent potential late complications for cancer survivors who underwent therapeutic thoracic irradiation. Thoracic RT often leads to heart irradiation, increasing the risk of heart diseases. The mechanisms involved in cardiotoxicity induced by therapeutic radiation doses are still largely unknown. Purpose. To assess the phenotypic and paracrine features of cardiac mesenchymal stromal cells (CMSCs) at early follow-up after the end of thoracic irradiation of the heart, as an early sign and/or mechanism of cardiac toxicity anticipating late organ dysfunction. Methods. Resident CMSCs were isolated from a rat model of thoracic irradiation with clinically relevant heart dosimetry (sham, 0.04, 0.3, or 1.2 Gy for 23 fraction), that develops delayed dose-dependent cardiac dysfunction after 1 year. Cells were isolated 6 or 12 weeks after the end of RT before any detectable phenotype in vivo, and fully characterized at transcriptional, paracrine, and functional levels. Results. CMSCs displayed several altered features in a dose- and time-dependent trend, with the most impaired characteristics observed in those exposed in situ to the highest radiation dose with time. CMSCs isolated 12 weeks after the end of RT from the 1.2Gy groups displayed a significant reduction in the migration capacity versus sham (1.2Gy 0.84±0.05 vs sham 0.74±0.06 normalized scratch area at 14h, p<0.05), and a significantly reduced spheroid-forming capacity versus both sham and 0.04Gy (1.2Gy 1.03±0.11 vs sham 2.13±0.21 vs 0.04Gy 2.35±0.30 number/mm², p<0.001 ANOVA). CMSCs exposed to 1.2Gy expressed significantly higher mRNA levels of profibrotic genes (TGFB1 1.76-fold, GATA4 2.24-fold, and COL1A1 1.78-fold, p<0.05 vs sham or 0.04Gy). Transcriptomic analysis by deep RNA sequencing of CMSCs at 6- and 12-week follow-up evidenced a group of 49 genes significantly modulated in the 1.2Gy group at 12-week follow-up (adj p<0.05 vs sham). Gene ontology (GO) analysis on this gene set returned 3 significant terms related to the key fibrotic pathway of TGFβ1 signaling (GO:0007179, GO:0071560, GO:0071559; adj p<0.01). Screening of CMSCs conditioned media from the 1.2Gy group at 12-week follow-up revealed modulation of multiple cytokines; when used to culture the monocyte THP1 cell line, the mRNA levels of 3 classical markers of pro-inflammatory M1 phenotype increased (IL6 1.92-fold, CCL2 1.73-fold, p<0.05 vs sham; IL8 2.38-fold, p<0.01 vs sham). The same conditioned media had also reduced angiogenic support to endothelial cells (2.1±0.6x10³ vs 7.3±0.1x10³ mesh area, p<0.001 vs sham). Conclusion. Data collected on a clinically relevant rat model of heart irradiation simulating thoracic radiotherapy suggest that early pro-fibrotic specification and paracrine alterations of cardiac stromal cells may represent a dose-dependent biological substrate for the cardiac dysfunction phenotype observed in vivo at long follow-up.