Thrombin is a therapeutic target for non-small-cell lung cancer to inhibit vasculogenic mimicry formation

Figures

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  Fig. 1. Thrombin is overexpressed in NSCLC and the formation of VM in NSCLC is closely related to the level of thrombin and the prognosis of patients. a Score of thrombin expression in adjacent nontumor lung tissue and in NSCLC tissue. b Thrombin expression in different stages (AJCC Tumor-node-metastasis (TNM) stages) of NSCLC. c The association of thrombin expression with tumor size in NSCLC samples, where D is the maximum tumor diameter. d Region showing typical malignant morphology of NSCLC and thrombin expression in corresponding tissues. PAS+/CD31− VM tubes (black arrow) are also shown. e The relationship between the expression level of thrombin and the formation of VM in tumor tissues. f Univariate survival analysis according to VM in NSCLC patients and Kaplan–Meier survival analysis for patients dichotomized by the presence VM. g The expression of thrombin in BEAS-2B, PC9, 95D, A549, and Lewis cells was determined by western blotting. h The expression of thrombin in tumor tissues of mice and normal lung tissues of mice was determined by western blotting. All the results are expressed as the mean ± SD. The error bars indicate the SD. ANOVA followed by Dunnett’s test was applied for…

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  Fig. 2. r-hirudin and DTIP inhibit VM formation of A549 and Lewis cells in vitro. a, b A549 and Lewis cells were pretreated with PBS, 10 nmol/L thrombin, 25 nmol/L r-hirudin, 50 nmol/L DTIP, 10 nmol/L thrombin+25 nmol/L r-hirudin, or 10 nmol/L thrombin+50 nmol/L DTIP, and VM-like network formation on Matrigel in different groups was examined. Left, representative photographs of experiments. Right, quantification of the inhibitory activity of r-hirudin and DTIP on VM tube formation. c The effect of thrombin, r-hirudin and DTIP on the expression of snail and other EMT markers was determined by western blotting analysis. GAPDH was used as the loading control. The summarized western blot data for EMT markers are given (bottom). d Different groups were probed for phosphorylated p65, phosphorylated IκBα and total p65 and IκBα. GAPDH was used as the loading control. Right, summarized western blot data. e The expression of snail and other EMT markers in different groups was determined by western blotting analysis. Right, the summarized western blot data for EMT markers. All the results are expressed as the mean ± SD. The error bars indicate the SD. ANOVA followed by Dunnett’s test was applied…

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  Fig. 3. r-hirudin and DTIP inhibited tumor development and VM formation. LLC cells at a density of 1 × 106 in 0.1 mL serum-free media were injected subcutaneously into the right flank of mice. One week after the injection of LLC cells, mice were administered normal saline, 1.0 mg/kg DTIP, or 0.5 mg/kg r-hirudin for 21 consecutive days. a Volume of resected tumors derived from the normal saline-, r-hirudin-, and DTIP-treated groups at 45 days after cell injection. b Mice were humanely killed and the tumors were resected at 45 days after cell injection. The weights of tumors resected from the normal saline-, r-hirudin-, and DTIP-treated groups. c Number of NS-, r-hirudin-, and DTIP-treated mice with panniculus invasion, lung metastasis, liver metastasis, and colon metastasis. d Number of NS-, r-hirudin-, and DTIP-treated mice with VM. e Representative images of anti-CD31/PAS staining in tumor tissues. Right, the number of VM tubes per tumor tissue sample in different groups. f DAPI and FITC-dextran (dextran inverted)-stained tumor sections from resected tumors. g Leakiness index of resected tumors. h The expression of snail and other EMT markers in tumors was determined by western blotting…

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  Fig. 4. Thrombin with PAR-1 expression is associated with the prognosis of NSCLC patients and the formation of VM. a Different expression statuses of thrombin and PAR-1 detected by immunohistochemical staining in consecutive sections of NSCLC tissues from the same patient. The NSCLC tissues of case 1 were positive for thrombin and had high expression of PAR-1; case 2 had tissues positive for thrombin but with low expression of PAR-1; case 3 had tissues negative for thrombin but with high expression of PAR-1; and case 4 had tissues negative for thrombin with low expression of PAR-1. Based on the score of thrombin or PAR-1 in the central positive staining area in the tumor section as a cutoff value, the scores for thrombin and PAR-1 were classified as positive (thrombin+ ≥ 2 and PAR-1 high expression≥6) or negative (thrombin− < 2 and PAR-1 low expression < 6). b In Kaplan–Meier analyses, an association of thrombin expression with overall survival (OS), were demonstrated in the PAR-1 high-expression subgroup of NSCLC patients. c An association of thrombin expression with overall survival was demonstrated in the PAR-1 low-expression subgroup. d An association of thrombin expression with VM…

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  Fig. 5. PAR-1 is a major determinant in thrombin-promoted metastasis of lung cancer and formation of VM. a, b PAR-1 gRNA constructs targeting the PAR-1 gene via lentiviral transduction were used to construct stable A549PAR-1−/− and LLCPar-1−/− cells. An empty construct was also used as a negative control. VM channel formation in PAR-1-deficient A549 and LLC cells. Left, representative photographs of experiments. Right, quantification of the number of VM tubes. c Cell lysates were probed for phosphorylated p65, phosphorylated IκBα, and total p65 and IκBα. d The expression of snail and other EMT markers in PAR-1-deficient A549 and LLC cells was determined by western blotting analysis. e–j LLC cells infected with gRNA-PAR-1 lentivirus (Par-1−/− group) or LV-negative control (NC, vehicle group) were injected subcutaneously into the right flank of mice. e Volumes of resected tumors derived from injection of LLCPar-1−/− and LLCNC cells into mice at 5 weeks after cell injection. f Photograph of tumors resected from each group at 5 weeks after cell injection. g Number of mice from different groups with panniculus invasion, lung metastasis, liver metastasis, and colon metastasis. h Representative…

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  Fig. 6. Combination therapy with DTIP and gefitinib results in improved antitumor efficacy. LLC cells (1 × 106) were subcutaneously injected into the right dorsal region of mice. One week after LLC inoculation, the mice were randomly divided into four groups: normal saline, DTIP (1 mg/kg per day, s.c.), gefitinib (100 mg/kg per day, i.g.), and combination DTIP with gefitinib treatment groups, and then the mice were administered DTIP, gefitinib, or normal saline for 21 consecutive days. a Tumor growth curve after injection of LLC cells. b Mice were humanely killed and the tumors were resected 45 days after cell injection. c The weight of resected tumors was determined in different groups. d Representative photograph of metastatic nodules in the lungs (upper). H&E-stained sections of representative single lung lobes (bottom). e Number of mice with lung metastasis. f The number of VM tubes per tumor tissue sample was determined in different groups. g Representative images of anti-mouse anti-CD31/PAS staining in tumor tissues (upper). Immunohistochemical analysis was performed to determine the expression of VEGF (bottom). h The expression score for VEGF was determined. All the results are…