Figure 4.
Elevated TLT-1 in circulation promotes tumor progression in mice. (A) Representative images of in vivo imaging of luciferase-labeled tumor in mice before (upper panel) and after (lower panel) recTLT-1 or vehicle administration. (B) Quantified weekly measured tumor sizes (volume) are shown for mice induced with tumor and compared between Veh and TLT-1 administered mice (n = 8 per group). The data from both groups at multiple time points were analyzed by repeated two-way ANOVA. (C) Representative images of primary tumors excised from mice for both treatment groups (scale bar = 10 mm). (D) Representative bioluminescence imaging of thoracic region of mice bearing primary subcutaneous tumor after 5 wk of tumor growth (+VEH or +TLT-1) showing metastatic spread of tumor in recTLT-1–injected mice (top panel). The images were captured using PerkinElmer’s in vivo imaging system (IVIS) immediately after luciferin injection (i.p.). The images of excised lungs (upper panel) from mice with or without tumors showing metastasis in recTLT-1–administered group (middle panel). The respective Fontana-Masson–stained sections (original magnification, 100×) are shown to confirm the tumor presence as the black region on the peripheral side of lungs in the recTLT-1 group (bottom panel). (E and F) Representative tumor tissue micrographs (immunofluorescence) compare the CD8+ TILs and IFNγ expression, towards the tumor periphery, showing reduced CD8+ TILs and IFNγ in recTLT-1–treated mice, and CD8+ TILs quantitation (F) is shown. Statistical significance tested by Mann–Whitney test. The anti-luciferase antibody was stained to identify tumor cells (cyan). (G) Representative images (n = 5) of in vivo imaging of luciferase-labeled tumor in mice after 4 wk of indicated administration, which started after 7 d of subcutaneous B16F10 tumor injection. (H) Quantified weekly measured tumor sizes (volume) are shown for mice induced with tumor and the indicated treatment (Veh or recTLT-1 with cAb or anti–TLT-1 antibody) and compared between groups. The data from all four groups (n = 5 per group) at multiple time points were analyzed by repeated two-way ANOVA. The quantitation is presented as mean ± SEM, and the P values correspond to Mann–Whitney test or repeated two-way ANOVA as appropriate. Significant differences are indicated with asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001). All the data represents three independent experiments.

Elevated TLT-1 in circulation promotes tumor progression in mice. (A) Representative images of in vivo imaging of luciferase-labeled tumor in mice before (upper panel) and after (lower panel) recTLT-1 or vehicle administration. (B) Quantified weekly measured tumor sizes (volume) are shown for mice induced with tumor and compared between Veh and TLT-1 administered mice (n = 8 per group). The data from both groups at multiple time points were analyzed by repeated two-way ANOVA. (C) Representative images of primary tumors excised from mice for both treatment groups (scale bar = 10 mm). (D) Representative bioluminescence imaging of thoracic region of mice bearing primary subcutaneous tumor after 5 wk of tumor growth (+VEH or +TLT-1) showing metastatic spread of tumor in recTLT-1–injected mice (top panel). The images were captured using PerkinElmer’s in vivo imaging system (IVIS) immediately after luciferin injection (i.p.). The images of excised lungs (upper panel) from mice with or without tumors showing metastasis in recTLT-1–administered group (middle panel). The respective Fontana-Masson–stained sections (original magnification, 100×) are shown to confirm the tumor presence as the black region on the peripheral side of lungs in the recTLT-1 group (bottom panel). (E and F) Representative tumor tissue micrographs (immunofluorescence) compare the CD8+ TILs and IFNγ expression, towards the tumor periphery, showing reduced CD8+ TILs and IFNγ in recTLT-1–treated mice, and CD8+ TILs quantitation (F) is shown. Statistical significance tested by Mann–Whitney test. The anti-luciferase antibody was stained to identify tumor cells (cyan). (G) Representative images (n = 5) of in vivo imaging of luciferase-labeled tumor in mice after 4 wk of indicated administration, which started after 7 d of subcutaneous B16F10 tumor injection. (H) Quantified weekly measured tumor sizes (volume) are shown for mice induced with tumor and the indicated treatment (Veh or recTLT-1 with cAb or anti–TLT-1 antibody) and compared between groups. The data from all four groups (n = 5 per group) at multiple time points were analyzed by repeated two-way ANOVA. The quantitation is presented as mean ± SEM, and the P values correspond to Mann–Whitney test or repeated two-way ANOVA as appropriate. Significant differences are indicated with asterisks (*, P < 0.05; **, P < 0.01; ***, P < 0.001). All the data represents three independent experiments.

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