Single cell sequencing unveils endothelial alterations after cisplatin treatment

Abstract Introduction Cisplatin, one of the most potent anti-cancer chemotherapy drugs, is still broadly used in first-line chemotherapy regimens, which are subscribed to about 10 to 20% of total cancer patients. Nephrotoxicity is a particularly limiting factor for cancer patients to remain in the effective treatment due to the acute kidney injury. It has been reported that as early as 24 hours post treatment, testicular cancer patients receiving cisplatin developed endothelial dysfunction and vascular injury. A cross-sectional follow-up study published in 2008 suggested that testicular cancer survivors continued to experience endothelial dysfunction and vascular injury. A recent 30-year follow-up study on testicular cancer survivors suggested that testicular cancer survivors experienced worse diastolic function. Purpose This study is focused on identifying the specific genes altered in cardiac endothelial cells after the cisplatin treatment to unveil the molecular mechanisms of injury for potential new therapeutic development. Methods Cisplatin induced AKI mouse model was generated by i.p. injecting 25 mg/kg cisplatin to C57BL/6 mice. Saline injection was served as control. To evaluate blood vessel damage induced by cisplatin, mice were sacrificed 48 hours post injection. Hearts were collected and single cell suspensions were produced by using Multi Tissue Dissociation Kit 2. Freshly prepared single cell suspensions were used to created libraries by using 10X genomics kits, before sequencing. The CellRanger (10X genomics) was used for processing Single cell RNASeq outputs, before secondary Seurat and DE pathway analysis. Results The GO enrichment analysis suggested that, in endothelial cells, cisplatin treatment significantly altered cellular anatomical entity, intracellular anatomical structure, apical part of the cell, cell junction, and anchoring junction. Consequently, increased vascular permeability, signaling regulating monocyte differentiation, macrophage cytokine production, and cardiac muscle cell apoptosis were observed. At molecular level, cisplatin treatment significantly upregulated DNA damage (Ddit4, Acer2), hypoxia (Phlda3, Mt1, Slc3a2, Ier3, Klf9, Adipor2, UCP2), inflammatory responses (Timp4, Tns1, Gdf15, Neat1), cellular senescence (Cdkn1a), Cell cycle arrest (Trp53inpl), intrinsic and extrinsic apoptosis (Fas, Bax, Ei24, Tgm2), blood vessel remodeling (Pim-3), and angiogenesis (Timp3, Flt1). These results indicated that cisplatin treatment likely not only result in acute endothelial dysfunction, injury, and death, but also accelerated aging, which could contribute the cardiovascular complications in the cancer survivors. Conclusions Protecting endothelial cells from oxidative stress and inflammation caused by cisplatin treatment might prevent their irreversible injury and entering into premature cellular senescence, consequently, mitigating anti-cancer treatment induced cardiovascular complications in cancer survivors. Funding Acknowledgement Type of funding sources: Other. Main funding source(s): NIH