Fig 2: CYR61, YAP, and CD31 expression in mouse xenografts and in triple negative human breast cancer. (A) Thick paraffin sections of breast obtained from tumor xenografts of mice injected with MDA-MB-231 or sh-WISP2 human breast cell lines were subjected to CYR61/PAS and YAP/PAS double staining (magnification 40×). PAS staining revealed VM channels which are lined by tumor cells positive for Periodic Acid-Schiff and CYR61 or YAP (Black Arrow). (B) Thick paraffin sections obtained from triple negative human breast tumors were subjected to CYR61/PAS, YAP/PAS and CD31/PAS double staining (magnification 40×). Representative images of IHC staining show strong expression of CYR61 and YAP proteins in cells that form vascular mimicry tubules revealed by PAS staining, whereas CD31 is not detected in these structures (black arrow). CD31-positive endothelial vessel (yellow arrow) indicates regular vascular channels positive for CD31 staining. A protein expression score was expressed according to the intensity of the observed labeling. Scale bar = 50 µm.

Fig 3: PADI4 promotes breast cancer angiogenesis and tumor growth.(A) Immunohistochemistry for HIF-1a and PADI4 was performed with consecutive sections of human breast cancer tissue, each of which included both tumor (T) and adjacent nontumor (NT) tissue. Magnification: ×100 for top panels and ×400 for bottom panels. (B) The stained area in five fields from each tumor section was quantified by ImageJ software and represented as the percentage of total area that was positive for HIF-1a or PADI4 staining for each case (n = 45). Data were analyzed using Pearson’s correlation test. (C) SMA and CD31 immunohistochemistry was performed with consecutive sections of human breast cancer tissue. The 45 cases were divided into low (n = 23) and high (n = 22) PADI4 groups based on the median percentage of total area that was positive for PADI4 (left, magnification: ×200). The stained area in five fields from each tumor section was quantified by ImageJ software and represented as the mean percentage of total area positive for SMA or CD31. Staining for SMA (middle) or CD31 (right) was compared in the low and high PADI4 groups (mean ± SD). ***P < 0.001 (Student’s t test). (D) Data were analyzed using Pearson’s correlation test to compare PADI4-positive staining percentage with SMA-positive (left) or CD31-positive (right) staining percentage in tumor sections (n = 45). (E) Summary of results: PADI4 recruitment drives HIF transcriptional activity and tumor vascularization. Hypoxia induces HIF-dependent PADI4 expression. PADI4 protein is recruited by HIFs to HREs and deiminates histone arginine residues to citrulline, leading to chromatin decondensation. PADI4 also stimulates HRE occupancy by HIFs and trimethylation of H3K3 and H3K36. The integrated effect of this chromatin remodeling is to activate transcription of hundreds of HIF target genes.

Fig 4: 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/a-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 5: 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).