Lymphangiogenesis is increased by TβR-I inhibitor in pancreatic adenocarcinoma BxPC3 xenograft models. (A-D) Effects of TβR-I inhibitor on lymphangiogenesis were examined in a xenograft model using a human pancreatic cancer cell line, BxPC3. BxPC3 cells mixed with or without VEGF-C (1 μg/mL) were subcutaneously inoculated in BALB/c nude mice. After tumors had formed, the mice were injected intraperitoneally with TβR-I inhibitor (LY364947, 1 mg/kg) 3 times a week for 3 weeks. They were killed at the end of the experiment, and excised tumors were examined histologically. (A) Immunostaining of BxPC3 xenograft sections by LYVE-1 antibody (shown in green). Bars represent 50 μm. (B) LYVE-1–positive areas in the BxPC3 xenograft sections were determined in the presence and absence of VEGF-C and TβR-I inhibitor (n = 3 for each group). Error bars represent SE. n.s. indicates not significant (*P < .05). (C) Immunostaining of BxPC3 xenograft sections for Prox1 (left panel, red), podoplanin (right panel, red), and LYVE-1 (green). Bars represent 20 μm. (D) Immunostaining of BxPC3 xenograft sections for PECAM1 (red), LYVE-1 (left panel, green), Prox1 (right panel, green), and TOTO-3 (blue). Bars represent 100 μm. (E,F) Effects of TβR-I inhibitor on lymphangiogenesis were examined in a xenograft model using BxPC3 cells overexpressing VEGF-C by infection of the VEGF-C-lentivirus. BxPC3 cells infected with a lentivirus containing GFP were used as a control. (E) Up-regulation of VEGF-C mRNA in BxPC3 cells after infection of the VEGF-C-lentivirus was determined by real-time PCR. (F) LYVE-1-positive areas in the GFP- and VEGF-C–expressing BxPC3 xenograft sections treated with or without TβR-I inhibitor were determined (n = 3 for each group). Error bars represent SE. n.s. indicates not significant (***P < .001).