Fig 2: Intra-tumor heterogeneity of the coagulome of malignant cells from individual OSCC. Violin plots showing the expression of the core coagulome genes (F3, PLAU, PLAT, PLAUR, F2R and SERPINE1) in cancer cells from different OSCC tumors analyzed in GSE103322. Each individual tumor was identified according to the study by Puram et al. [28]. The red dots represent the median value for the expression of each gene.

Fig 3: Single-cell analysis of the coagulome in different cell subpopulations in OSCC. Violin plots showing the mRNA expression of the core coagulome genes (F3, PLAU, PLAT, PLAUR, F2R and SERPINE1) in different cell populations present in OSCC (malignant cancer cells, T cells, dendritic cells, macrophages, endothelial cells and fibroblasts). Data are shown from OSCC tumors analyzed by scRNA-seq in Puram et al. [28] (GSE103322). The red dots represent the median values. The indicated p values were obtained with the Kruskal–Wallis test.

Fig 4: SERPINE2 is a direct target of miR-199a-3p. (A) Sequence alignment of miR-199a-3p with the SERPINE2 3'-UTR. The seed-recognizing sites (underlined) in the SERPINE2 sequence matched with the seed regions of miR-199a-3p. (B) Following transfection of CRL-4025 cells with a miR-199a mimic, the expression of miR-199a was upregulated, whereas transfection with an inhibitor effectively suppressed the expression of miR-199a-3p. **P<0.01 vs. NC. (C) Luciferase assay in CRL-4025 cells demonstrated that miR-199a-3p mimics significantly suppressed luciferase activity in wild type reporter constructs, compared with NC. (D) miR-199a-3p inhibitor significantly promoted luciferase activity in wild type constructs, compared with NC. *P<0.05 vs. NC. (E) Reverse transcription-quantitative polymerase chain reaction demonstrated that the level of SERPINE2 mRNA in CRL-4025 cells was not affected by transfection with miR-199a-3p mimics or miR-199a-3p inhibitor. (F) Immunohistochemistry revealed that SERPINE2 was downregulated in DN microvasculature compared with volunteers. (G) Transfection of CRL-4025 cells with miR-199a-3p mimics or miR-199a-3p inhibitor affects SERPINE2 and tPA protein expression levels. Protein expression of SERPINE2 was negatively associated with the expression of miR-199a-3p. All data are presented as the mean ± standard deviation. SERPINE2, serine protease inhibitor E2; miR, microRNA; UTR, untranslated region; NC, negative control; DN, diabetic neuropathy; tPa, tissue plasminogen activator.

Fig 5: Immunoblot analysis of the coagulome in OSCC and the lack of a direct effect of thrombin on the growth of OSCC cells. (A) Immunoblot analysis of the expression of the Tissue Factor (F3), uPA (PLAU), tPA (PLAT) and PAI-1 (SERPINE1) in OSCC cell lines (PE/CA-PJ34, PE/CA-PJ41, SCC9) compared to GBM cell lines (A172, SW1088, U118MG). Actin was provided as a control. Protein extracts were prepared from the indicated cell lines as indicated. Note that the blots are representative of at least 3 independent experiments. (B) Lack of a direct effect of thrombin on the growth of OSCC cells in vitro. Thrombin (10 NIHU/L) and dabigatran (1 µM) were maintained for 72 h, and cancer cell growth was measured with crystal violet and normalized by taking the control condition as reference. (C) Lack of a direct effect of thrombin on the expression of the immune checkpoint molecule CD274/PD-L1 and ERK phosphorylation in OSCC cells. Thrombin (10 NIHU/L) was applied at the same time as Interferon-Gamma (10 ng/mL), used here as an inducer of PD-L1 expression. The indicated markers were analyzed after 18 h of incubation.