Figure 2
Figure 2. EPCR overexpression and aPC administration protect mice from B16-F10 melanoma metastases. (A) Survival curves comparing WT and Tie2-EPCR mice over the 2 weeks after intrasplenic injection of B16-F10 melanoma cells, P = .03. For each group, n = 10. (B) Calculated percentage of total liver surface area involved with tumor nodules and corresponding photographs of representative liver surfaces for WT and Tie2-EPCR mice, ***P < .001, n = 10 per group. Images were visualized with a Leica S6D dissecting microscope equipped with a 10×/0.32 Plan Apo lens (Leica, Richmond Hill, ON) and a QImaging Micropublisher 3.3 RTV digital camera (QImaging, Surrey, BC). Images were acquired with QCapture Pro 5.0 software (QImaging) and were analyzed with the SimplePCI Imaging System (Compix). (C) Representative intravital images and cell counts per high-power field (HPF) of CFDA-SE–labeled B16-F10 melanoma cells in WT and Tie2-EPCR livers, **P < .01, n = 6 per group. One hour after intrasplenic B16-F10 inoculation, mice were anesthetized, and their livers were exteriorized for image capture. Images were visualized using a Leica DM5000B fluorescence microscope and Hamamatsu C9100 EM-CCD camera (Hamamatsu Photonics, Bridgewater, NJ). Images were acquired with Volocity 5.0.0 software (Improvision, Lexington, MA). Ten HPFs were captured per liver. Cell counts per HPF were performed by 2 blinded observers. Bar = 100 μm. (D) Calculated percentage of total lung surface area involved with tumor nodules and corresponding photographs of representative lung surfaces for WT and Tie2-EPCR mice, ***P < .001. A similar difference was observed if lung metastasis was measured with total nodule number or lung surface area (data not shown). For each group, n = 12. (E) Quantitation of E-selectin, P-selectin, and TNF-α transcript levels from WT and Tie2-EPCR lung specimens harvested 2 weeks after tail vein injection with B16-F10 cells, *P < .05, n = 6 per group. Each RT-PCR reaction was run in duplicate. Transcript levels expressed relative to mean value for nontumor-injected WT animals. In the absence of tumor injection, no significant differences in transcript levels were observed between WT and Tie2-EPCR mice. (F) Representative intravital images and cell counts per HPF of CFDA-SE–labeled B16-F10 melanoma cells in PBS- and rhaPC-treated WT animal livers, **P < .01, n = 6 per group. Bar = 100 μm. (G) Calculated percentage of total lung surface area involved with tumor nodules and corresponding photographs of representative lung surfaces for PBS- and rhaPC-treated WT mice, *P = .02. For PBS-treated group, n = 12; for rhaPC-treated group, n = 10. (H) Quantitation of E-selectin, P-selectin, and TNF-α transcript levels from PBS- and rhaPC-treated WT lung specimens harvested 12 hours after initiation of PBS or rhaPC injections (10 hours after inoculation with B16-F10 cells), *P = .02, n = 6 per group. Each RT-PCR reaction was run in duplicate. Transcript levels expressed relative to mean value for PBS-treated, nontumor-injected mice. In the absence of tumor injection, no significant differences in transcript levels were observed between PBS- and APC-treated mice. All data reported as mean with standard error.

EPCR overexpression and aPC administration protect mice from B16-F10 melanoma metastases. (A) Survival curves comparing WT and Tie2-EPCR mice over the 2 weeks after intrasplenic injection of B16-F10 melanoma cells, P = .03. For each group, n = 10. (B) Calculated percentage of total liver surface area involved with tumor nodules and corresponding photographs of representative liver surfaces for WT and Tie2-EPCR mice, ***P < .001, n = 10 per group. Images were visualized with a Leica S6D dissecting microscope equipped with a 10×/0.32 Plan Apo lens (Leica, Richmond Hill, ON) and a QImaging Micropublisher 3.3 RTV digital camera (QImaging, Surrey, BC). Images were acquired with QCapture Pro 5.0 software (QImaging) and were analyzed with the SimplePCI Imaging System (Compix). (C) Representative intravital images and cell counts per high-power field (HPF) of CFDA-SE–labeled B16-F10 melanoma cells in WT and Tie2-EPCR livers, **P < .01, n = 6 per group. One hour after intrasplenic B16-F10 inoculation, mice were anesthetized, and their livers were exteriorized for image capture. Images were visualized using a Leica DM5000B fluorescence microscope and Hamamatsu C9100 EM-CCD camera (Hamamatsu Photonics, Bridgewater, NJ). Images were acquired with Volocity 5.0.0 software (Improvision, Lexington, MA). Ten HPFs were captured per liver. Cell counts per HPF were performed by 2 blinded observers. Bar = 100 μm. (D) Calculated percentage of total lung surface area involved with tumor nodules and corresponding photographs of representative lung surfaces for WT and Tie2-EPCR mice, ***P < .001. A similar difference was observed if lung metastasis was measured with total nodule number or lung surface area (data not shown). For each group, n = 12. (E) Quantitation of E-selectin, P-selectin, and TNF-α transcript levels from WT and Tie2-EPCR lung specimens harvested 2 weeks after tail vein injection with B16-F10 cells, *P < .05, n = 6 per group. Each RT-PCR reaction was run in duplicate. Transcript levels expressed relative to mean value for nontumor-injected WT animals. In the absence of tumor injection, no significant differences in transcript levels were observed between WT and Tie2-EPCR mice. (F) Representative intravital images and cell counts per HPF of CFDA-SE–labeled B16-F10 melanoma cells in PBS- and rhaPC-treated WT animal livers, **P < .01, n = 6 per group. Bar = 100 μm. (G) Calculated percentage of total lung surface area involved with tumor nodules and corresponding photographs of representative lung surfaces for PBS- and rhaPC-treated WT mice, *P = .02. For PBS-treated group, n = 12; for rhaPC-treated group, n = 10. (H) Quantitation of E-selectin, P-selectin, and TNF-α transcript levels from PBS- and rhaPC-treated WT lung specimens harvested 12 hours after initiation of PBS or rhaPC injections (10 hours after inoculation with B16-F10 cells), *P = .02, n = 6 per group. Each RT-PCR reaction was run in duplicate. Transcript levels expressed relative to mean value for PBS-treated, nontumor-injected mice. In the absence of tumor injection, no significant differences in transcript levels were observed between PBS- and APC-treated mice. All data reported as mean with standard error.

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