Fig 1.
Co-expression of mesenchymal and endothelial markers in KS tissue.
(A) Representative immunofluorescence images of AIDS-KS lesion tissues (lower) and their adjacent normal skin tissues (upper) stained with antibodies against mesenchymal (Nestin, PDGFRA, or α-SAM in green), endothelial (PDPN, CD31 or VEGFR2 in red), and KSHV (LANA in yellow) markers. The nuclei were counterstained with Hoechst 33342 (blue). Scale bars, 50 μm. Images of each individual channel for mesenchymal, endothelial and KSHV marker, respectively, are shown in S1 Fig. (B) Triple labeling for mesenchymal, endothelial, and KSHV markers, demonstrating the colocalization of these three fluorescent signals in the same KS tumor cell, as revealed by the plot of fluorescence intensity profiles across a white arrow in panel A. (C) Co-expression of mesenchymal (Nestin, PDGFRA, and α-SAM), endothelial (PDPN, CD31, and VEGFR2), and KSHV (LANA) markers in KS early (patch and plaque) and late lesions (nodular). Boxed areas are enlarged. Scale bar, 50 μm. (D) Number of LANA, Nestin, CD31, PDGFRA, PDPN, α-SAM, and VEGFR2 positive cells was counted from 4–6 individual fields (180μm x 150μm) composed mostly of spindle tumor cells and vessels in KS early (n = 7 samples) or late lesions (n = 5 samples). Numbers were compared by Chi-2 test. (E) Spearman’s test shows a correlation between LANA expression and cells positive for PDPN in early and late KS tumors. (F) Percentage of Nestin-/CD31-, Nestin+/CD31-, Nestin+/CD31+, Nestin-/CD31+ and PDGFRA-/PDPN-, PDGFRA+/PDPN-, PDGFRA+/PDPN+, PDGFRA-/PDPN+, and α-SAM-/VEGFR2-, α-SAM+/VEGFR2-, α-SAM+/VEGFR2+, α-SAM-/VEGFR2+ cells in LANA+ spindle tumor cells in early or late KS lesions.
Fig 2.
Kaposi’s sarcoma abnormal vessels are made of lined LANA-positive cells with mesenchymal and endothelial markers.
(A) The difference in morphology and structure between normal vessels (left) and KS specialized vessels (right). Higher magnifications of the black-boxed areas are shown underneath. Scale bar, 200 μm (upper), 50 μm (lower). Asterisk, KS abnormal vessels. (B) Expression patterns of mesenchymal and endothelial markers in normal and KS abnormal vessels. Samples were stained with antibodies against Nestin/PDGFRA/α-SAM (green), CD31/PDPN /VEGFR2 (red), and LANA (yellow), and the nuclei were counterstained with Hoechst 33342 (blue). Scale bar, 50 μm. Asterisk, KS abnormal vessels. (C) LANA+ abnormal hybrid vascular density in early and late KS tumors. The number of vessels was quantified from 4–6 individual fields (354μm x 246μm) for each KS tumor sample. Error bars represent mean ± SEM. All statistical analyses were performed using the Mann-Whitney U test. *p < 0.05, **p < 0.01, ***p < 0.001, NS, not significant.
Fig 3.
KSHV infection initiates a differentiation process converting MSCs from mesenchymal phenotype to M/E hybrid state.
(A) Cell lysates from mock- and KSHV-infected PDLSCs at indicated time points were immunoblotted for PDGFRA, COL1A1, α-SAM, TAGLN, PROX1, PDPN, VEGFA, and β-actin. (B) Relative mRNA levels of mesenchymal and endothelial related genes in mock- and KSHV-infected PDLSCs (K-PDLSCs) after 4 days infection. (C) Mock- and KSHV-infected PDLSCs (2-D) were immunostained for LANA, TAGLN, PDPN, vWF, and CD31 at 4 days post-infection. Scale bar, 50 μm. (D) A time course of K-PDLSC aggregating to form spheroid in non-adherent plates. Scale bar, 500 μm. (E) Expression of LANA, TAGLN, PDPN, vWF, and CD31 in mock- and KSHV-infected PDLSC spheroid (3-D) at 4 days post-infection. Scale bar, 50 μm. (F) The expression of endothelial markers in mock- and KSHV-infected PDLSCs under 2-D or 3D cell culture for 4d. (G) The mRNA expression level of TAGLN, α-SAM, Nestin, PDGFRA, PDPN, ICAM, PROX1, CD31, and VEGFA was analyzed by RT- qPCR in K-PDLSC spheroids in comparison with their parallel 2D culture.
Fig 4.
The hybrid M/E state is enriched through KSHV-mediated MEndT and displays partial endothelial characteristics.
(A) Tubule formation assays were performed with mock- and KSHV-infected PDLSCs (K-PDLSCs). Scale bar, 500 μm. (B) Mock- and KSHV-infected PDLSCs were incubated with DiI-acLDL for 4 hours. DiI-acLDL uptake (red), as well as KSHV-GFP infection (green), were analyzed with a fluorescence microscope. Scale bar, 100 μm. (C) Mock- and KSHV-infected PDLSC spheroids were embedded into Matrigel, and the sprouting length was analyzed by a Zeiss fluorescence microscope. Scale bar, 200 μm. (D) H&E staining of PDLSC and K-PDLSC spheroid sections. Scale bar, 50 μm. (E) The expression of CD31 in the spheroids of PDLSC and K-PDLSC was detected by IFA. Scale bar, 50 μm. (F) Mock- and KSHV-infected PDLSCs, along with HUVECs, were examined for Weibel–Palade bodies (WPBs) under a transmission electron microscope. Scale bar, 1 μm. Original magnification, ×2 (enlarged insets).
Fig 5.
Characterization of KSHV-infected PDLSCs of distinct states in the mesenchymal-to-endothelial differentiation spectrum.
(A) Mock- and KSHV-infected PDLSCs and LECs were examined for PDGFRA and PDPN expression profile by flow cytometry analysis. Three subpopulations (xM, hybrid M/E, and xE) were quantified based on the PDGFRA and PDPN profiles (n = 3 independent experiments). Statistical analyses were performed using two-tailed Student’s test and P-values were calculated by GraphPad Prism. *p < 0.05, **p < 0.01, ***p < 0.001. (B) PDGFRA and PDPN expression in PDLSC and K-PDLSC spheroids at 4 days post-infection were analyzed by IFA. Scale bar, 50 μm. (C) K-PDLSCs were stained for PDGFRA and PDPN and sorted by flow cytometry. The purified xM and M/E populations were cultured for 10 days and their PDGFRA/PDPN profiles were examined for their phenotypic plasticity. (D) Western blot analysis of xM, M/E, and LECs for their mesenchymal and endothelial markers. (E) The expression profiles of mesenchymal and endothelial markers in hybrid M/E and xM state cells were analyzed at the mRNA level by RT-qPCR. (F) IFA analysis of LANA, TAGLN, and VCAM in xM and M/E state cells. Scale bar, 50 μm.
Fig 6.
KSHV endows PDLSCs with tumorigenic Properties and M/E exhibits high tumorigenicity.
(A) Soft agar colony formation assay to determine anchorage-independent cell growth in PDLSCs, K-PDLSCs, xM, and M/E state cells. Representative cell colonies in soft agar are shown. Scale bar, 200 μm. (B) Images of PDLSC and K-PDLSC spheroids 48h after transferring onto nonadherent and adherent plates. Scale bar, 200 μm. (C) FASC analysis of the migrated cells detached from K-PDLSC spheroids for PDGFRA/PDPN profiles. The cells that were stained with mouse and rat IgG were used as a control for creating flow cytometry gates. (D) Illustration of spheroids Transwell invasion assay. (E) Quantitation of the number of invaded cells from PDLSC and K-PDLSC spheroids. (F) PDPN/PDGFRA expression profiles of the invaded cells from K-PDLSC spheroids. (G) Transwell migration and invasion assays of PDLSC, K-PDLSC, xM, and M/E state cells. Quantitation of cell migration and invasion was shown on the right. (H) Tubule formation assays were performed with xM and M/E cells. HUVECs were included as a positive control. Error bars represent mean ± SEM (n = 3). Statistical analyses were performed using the two-tailed Student’s test. * p < 0.05, ** p < 0.01, *** p < 0.001.
Fig 7.
Ectopic transplantation of PDLSC and K-PDLSC spheroids in mice under the kidney capsule.
(A) Schematic diagram illustrating the process of transplanting PDLSC and K-PDLSC spheroids under the renal capsule of nude mice. (B) The kidneys transplanted with PDLSC and K-PDLSC spheroids were harvested after 28 days. (n = 3–4 mice for each group). (C) Representative images of H&E staining of PDLSC and K-PDLSC spheroids transplants. Scale bar, 200 μm. (D) IHC staining of PDLSC and K-PDLSC spheroids transplants for LANA and Ki-67. (E) Immunofluorescent overview of Nestin, CD31, PDGFRA, PDPN, and LANA expression in PDLSC and K-PDLSC spheroids under the renal capsule. Scale bar, 50 μm.
Fig 8.
vFLIP promotes MEndT of MSCs and enhances the formation of hybrid M/E state cells and tumorigenesis.
(A) PDLSCs were transduced with different viral gene expression vectors as indicated. The expression of some of these viral genes in cells was verified by Western analysis (S2 Fig). The effects of these viral gene products on the expression of PDGFRA, PDPN, ICAM, and VEGFA in PDLSCs were analyzed by Western blotting. (B) vFLIP-, vCyclin- and LANA-expressing PDLSCs were examined for hybrid M/E state cells using FASC. (C) Immunofluorescence staining of vFLIP-expressing PDLSCs for PDPN expression. Scale bar, 20 μm. (D) vFLIP-expressing PDLSCs were subjected to a tubule formation assay. Scale bar, 200 μm. (E) vFLIP-expressing PDLSC spheroids were examined by IFA for PDGFRA/PDPN expression profiles. Scale bar, 50 μm. (F) Representative images of H&E staining of PDLSC and vFLIP-PDLSC spheroids transplanted in mice under the kidney capsule. Scale bar, 100 μm (upper) and 20 μm (lower). (n = 3–4 mice for each group). (G) Immunofluorescent overview of PDGFRA/PDPN expression in PDLSC and vFLIP-PDLSC spheroids under the renal capsule. Scale bar, 50 μm.
Fig 9.
Silencing of vFLIP expression results in the abolishment of KSHV-induced MEndT and tumorigenesis.
(A) Schematic diagram depicting the CRISPR/Cas9 editing sites in the KSHV genome. (B) The CRISPR/Cas9-mediated knockout efficiency of ORF71 (vFLIP) gene from the KSHV genome in KSHV-infected PDLSCs was verified by PCR with primers Pr1 and Pr2. (C) The vFLIP expression in ORF71 knockout cells was determined by RT-qPCR. (D) The effect of vFLIP knockout (vFLIP-KO) on the generation of hybrid M/E state cells, as well as the restoration of vFLIP function by ectopic expression of sgRNA-resistant vFLIP gene (S5 Fig), was quantitated by flow cytometry. (E) The effect of vFLIP-KO on the expression of PDGFRA and PDPN was examined with K-PDLSC spheroids by IFA. Scale bar, 50 μm. (F) The effect of vFLIP-KO on malignant transformation ability was examined using colony formation assay. Scale bar, 200 μm. (G) Images of WT and vFLIP-KO KSKV-infected PDLSC spheroids showing the effect of vFLIP-KO on cell migration capability. Scale bar, 200 μm. (H) The effect of vFLIP-KO on the angiogenic ability of K-PDLSCs was assayed by tubule-formation assay. Scale bar, 200 μm. (I) Representative images of H&E staining of WT and vFLIP-KO KSHV-infected PDLSC spheroid transplants in mice under the kidney capsule. Scale bar, 200 μm. Higher magnifications of the black boxes are shown on the right. Scale bar, 50 μm. (n = 3–4 mice for each group). (J) Immunofluorescence assay of WT and vFLIP-KO KSHV-infected PDLSC transplants for LANA, PDGFRA, and PDPN. Scale bar, 50 μm. (K) Schematic model for the role of KSHV vFLIP in promoting MEndT to generate hybrid M/E state cells for KS tumorigenesis and aberrant angiogenesis. Error bars represent mean ± SEM. n = 3, unless otherwise indicated. Statistical analyses were performed using the two-tailed Student’s test. *p < 0.05, **p < 0.01, ***p < 0.001.