RNAseq and Single Cell sequencing Analysis of KSHV-infected Mesenchymal stem cells in a pro-angiogenic environment show KS sarcomagenic mechanisms through KSHV oncogenic gene expression, endothelial differentiation, and cytokine induction

JULIÁN NAIPAUER; LACUNZA, EZEQUIEL; AHUJA, ANUJ; COSO, OMAR A.; ABBA MARTIN; ENRIQUE A. MESRI

Conferencia; 24th International KSHV Conference 2022; 2022

Cumulative evidence shows the importance of bone marrow-derived humanMesenchymal Stem Cells (hMSC) in Kaposi’s sarcoma (KS) Herpesvirus(KSHV) induced tumorigenesis. These data reinforce the hypothesis that KSmay derive from KSHV-infected hMSCs migrating into an inflammatory andangiogenic site enhancing the reprogramming and transformation capacity ofKSHV. However, the KS spindle-cell progenitor subpopulation identity,defining markers and specific host conditions of KSHV-driven oncogenesisupon de novo infection are not well defined yet. We carried out a KSHV infectionof primary hMSCs then subjected to MSC cell culture conditions or KS-likepro-angiogenic culture conditions to uncover specific cell subpopulations,mechanisms and conditions involved in KSHV-induced transformation andreprograming. Whole and single-cell RNA-sequencing analysis of theDifferentially Expressed Genes (DEGs) between these different environmentalconditions showed processes such as extracellular matrix organization,angiogenesis, cell differentiation, cytokine activity, cell proliferation, MAPKand PI3K signaling is overrepresented in cells grown in KS-like conditions.Using a KS gene signature we showed that KSHV infection in pro-angiogenicenvironmental conditions reprogram human MSCs closer to the KS geneexpression profiles compared to infection in MSC cell culture conditions. Singlecell RNA-sequencing analysis of KSHV-infected hMSC revealed that differentpopulations of cells express different amounts of host and viral oncogenes depending on the environmental conditions the cells are grown, leading to aa subpopulation of infected cells in a proangiogenic environment expressing bothviral and host oncogenes. Increased expression of cytokines and endothelialmarkers such as: CXCL1, CXCL8, CCL2, PDPN/PECAM1, VEGFR1,ROBO4, XDH, PLAU, HMGA2, PLAT, DUSP6, HMOX1 and ESM1 wereupregulated in this subpopulation of KSHV-infected hMSCs. These highlightsthe importance of this condition in KSHV reprogramming of hMSC towardsendothelial differentiation and transformation and closer to KS gene expressionprofiles, reinforcing the notion of these cell subpopulations as KS precursors.