Evidence for Kaposi Sarcoma Originating from Mesenchymal Stem Cell through KSHV-induced Mesenchymal-to-Endothelial Transition

Abstract

The major transmission route for Kaposi sarcoma-associated herpesvirus (KSHV) infection is the oral cavity through saliva. Kaposi sarcoma (KS) frequently occurs in the oral cavity in HIV-positive individuals and is often the first presenting sign of AIDS. However, the oral target cells for KSHV infection and the cellular origin of Kaposi sarcoma remain unknown. Here we present clinical and experimental evidences that Kaposi sarcoma spindle cells may originate from virally modified oral mesenchymal stem cells (MSC). AIDS-KS spindle cells expressed neuroectodermal stem cell marker (Nestin) and oral MSC marker CD29, suggesting an oral/craniofacial MSC lineage of AIDS-associated Kaposi sarcoma. Furthermore, oral MSCs were highly susceptible to KSHV infection, and infection promoted multilineage differentiation and mesenchymal-to-endothelial transition (MEndT). KSHV infection of oral MSCs resulted in expression of a large number of cytokines, a characteristic of Kaposi sarcoma, and upregulation of Kaposi sarcoma signature and MEndT-associated genes. These results suggest that Kaposi sarcoma may originate from pluripotent MSC and KSHV infection transforms MSC to Kaposi sarcoma-like cells through MEndT.Significance: These findings indicate that Kaposi sarcomas, which arise frequently in AIDS patients, originate from neural crest-derived mesenchymal stem cells, with possible implications for improving the clnical treatment of this malignancy. Cancer Res; 78(1); 230-45. ©2017 AACR.

Fig. 1. AIDS-KS spindle cells exhibit oral MSC-specific markers

(A) Paraffin-embedded sections of KS lesions from five AIDS-KS patients were subjected to immunohistochemical analyses for LANA, Nestin and CD29. (B) Two cases, one carrying the sarcoma in the palate and the other in the palm, are illustrated. H&E staining reveals typical KS histological feathers such as spindle shaped KS cells and slit-like vascular channels with erythrocytes. Immunohistochemical staining shows LANA, Nestin and CD29 in KS spindle cells. (C) Oral MSCs from periodontal ligament (PDLSC), dental pulp (DPSC) and gingiva (GMSC), along with lymphatic endothelial cells (LECs) and KSHV-infected LECs, were examined for their expression of Nestin using IFA and DAPI. KSHV infection of LECs was confirmed by IFA with an anti-LANA antibody.

Fig. 2. Human oral MSCs are highly susceptible for KSHV infection and viral latent infection leads to morphological and cell marker changes of MSCs

(A) Primary oral MSCs of different origins (PDLSCs, GMSCs and DPSCs) were infected with GFP-KSHV in an MOI of 50 (KSHV genome equivalent) for 48 hours and analyzed by GFP fluoresces. (B) Infected cells were drug-selected for a week followed by two weeks culture without selection and analyzed by IFA with anti-Vimentin antibody. Images show the phase-contrast, the antibody staining and GFP fluorescence of the cells. (C) Flow cytometric analysis of mock- and KSHV-infected PDLSCs with mesenchyme markers (CD29, CD73, CD90, VD105 and CD166, Y-axis) and GFP (KSHV-infected cells, X-axis). (D) The expression of mesenchymal and endothelial markers in mock- and KSHV-infected PDLSCs.

Fig. 3. KSHV infection promotes multi-lineage differentiation

PDLSCs were mock- and KSHV-infected in an MOI of 50 (viral genomic DNA equivalent). (A) Cells were induced under osteogenic culture condition for 4 weeks and assayed for their osteogenic differentiation by Alizarin staining. (B) PDLSCs were subjected to adipogenic induction and adipogenic differentiation was analyzed by oil Red staining. (C) Mock- and virally infected PDLSCs were loaded on the top of Matrigel and the ability of the cells in formation of capillary-like tubules was analyzed under a ZEISS fluorescence microscope. (D) The effect of KSHV infection on angiogenesis property of PDLSCs was also examined ex vivo using the Matrigel plug assay. Matrigel containing mock- or KSHV-infected PDLSCs were subcutaneously implanted into C57BL/6 mice. After 7 days, Matrigel plug were removed and photographed. After Matrigel plugs were homogenized and centrifuged, their supernatant was used to quantitate the haemoglobin content using Drabkin’s reagent.

Fig. 4. Kidney capsule implantation of KSHV-infected MSCs

KSHV infected-PDLSCs (1×10⁶ cells) were implanted into kidney capsule. Spindle-like cells, sieve-like pattern, and mononuclear cells, with slit-like vascular spaces containing red blood cells were observed (B in comparison to A). Immunohistofluorescence staining showed expression of LANA and vascular endothelial marker CD31 in KSHV-infected human PDLSC transplant (B in comparison to A). Bar: 50 μm.

Fig. 5. Expression and secretion of chemokines and cytokines in PDLSCs and endothelial cells upon KSHV infection

(A) Chemokines and cyckines that are up-regulated in KSHV-infected PDLSCs identified in our RNA-seq analysis are listed. Genes that were reported to be over-expressed in KS lesions are marked in red. The cytokines that are up-regulated in HDMEC and HMVECs upon KSHV infection are included as comparison. (B-G) PDLSCs and LECs were infected with KSHV in a MOI of 50 (viral genomic DNA equivalent). The infectivity rates of PDLSCs and LECs were determined using GFP fluorescence and were 92.1% and 88.5% respectively. Ninety-six hours post-infection, mock- and KSHV-infected cells were seeded in a relative low density (1×10⁵ per mL) with α-MEM containing 1% FBS. Supernatants were collected after 6 hours, and subjected to ELISA for bFGF, VEGF-A and VEGF-D, IGF1, TGFβ3 and ANGPT2.

Fig. 6. Relationship of KSHV-infected MSCs and endothelial cells to KS in gene expression profile

(A) Genes listed in KS signature were picked from uninfected and KSHV-infected MSC, LEC and BEC, and shown by heatmap. RNA-seq Data of KSHV-PDLSC and PDLSC were normalized with the Microarray data of MSC (31). Unsupervised clustering of samples (X-axis) and genes (Y-axis) were performed by average linkage method. (B) Linkage distance between KS and each cell group was determined by Pearson correlation coefficient. (C) First two principal components of these data were identified and shown in multipledimensional scaling (MDS) plot. (D) DEGs from KSHV-infected MSCs and ECs are compared with KS signature with venny diagram. (E) The genes that are consistently regulated in KS and KSHV-infected MSCs or ECs were assorted according to their functions, and compared respectively. “NS”, no significance. “*”, p<0.05.

Fig. 7. Expression of MEndT and EndMT related gene in PDLSCs and LECs in response to KSHV infection

PDLSCs and LECs were infected with KSHV in an MOI of 50 (viral genomic DNA equivalent). Ninety-six hours post-infection, mock- and KSHV-infected cells were collected and analyzed by Western Blotting (A and B) or seeded in coverslips and subjected to IFA (C and D) with antibodies specified.