Journal: Cancer Science

Article Title: Low-dose paclitaxel improves the therapeutic efficacy of recombinant adenovirus encoding CCL21 chemokine against murine cancer

doi: 10.1111/cas.12537

Figure Lengend Snippet: In vitro and in vivo expression and biological activity of murine CCL21. (a) Western blot analysis of murine CCL21 expression after infection of Hela cells. (b) 4T1 cells were infected with Ad-mCCL21 or Ad-LacZ at varying MOIs, yielding dose-dependent supernatant mCCL21 concentrations up to 31.9 ± 2.7 ng/mL after Ad-mCCL21 infection. (c) Chemotactic assay of conditioned medium from infected 4T1 cells. Supernatants from Ad-mCCL21 infected 4T1 cells (MOI = 100) clearly attracted lymphocytes, with a superior capacity for chemotaxis compared with purchased mCCL21 in vitro . (d) Tumor mCCL21 levels 0–8 days after a single intratumoral injection of Ad-mCCL21. Levels were 4220.1 ± 303.5 pg/mg at 4 days and remained significantly higher than controls at day 8. Bars, ±SD. * P < 0.01, significantly different from the controls.

Article Snippet: Forty-eight hours after infection, cells were harvested and analyzed by Western blotting using a rabbit anti-murine mCCL21 monoclonal IgG antibody (Peprotech EC, London, UK).

Techniques: In Vitro, In Vivo, Expressing, Activity Assay, Western Blot, Infection, Chemotaxis Assay, Injection

Journal: Cancer Science

Article Title: Low-dose paclitaxel improves the therapeutic efficacy of recombinant adenovirus encoding CCL21 chemokine against murine cancer

doi: 10.1111/cas.12537

Figure Lengend Snippet: Tumor suppression and survival advantage in mice. Immunocompetent C57BL/6 and BALB/c mice bearing B16-F10 melanoma and 4T1 breast carcinoma were treated with normal saline (NS), paclitaxel (Pac), Ad-mCCL21, Ad-LacZ+Pac, or Ad-mCCL21+Pac. (a) Suppression of s.c. tumor growth in mice. Ad-mCCL21+Pac treatment resulting in significant tumor growth inhibition versus NS controls ( P < 0.01), Pac ( P < 0.01), Ad-mCCL21 ( P < 0.05), or Ad-LacZ+Pac ( P < 0.01) from day 20 after initiation of treatment. Points, average tumor volume; bars, ±SD. (b) A significant increase was observed in the survival rates of Ad-mCCL21+Pac treatment mice compared with the other groups ( P < 0.01, by log–rank test).

Article Snippet: Forty-eight hours after infection, cells were harvested and analyzed by Western blotting using a rabbit anti-murine mCCL21 monoclonal IgG antibody (Peprotech EC, London, UK).

Techniques: Saline, Inhibition

Journal: Cancer Science

Article Title: Low-dose paclitaxel improves the therapeutic efficacy of recombinant adenovirus encoding CCL21 chemokine against murine cancer

doi: 10.1111/cas.12537

Figure Lengend Snippet: Histochemical analysis of tumors. (a) Tumor angiogenesis was determined by staining paraffin-embedded sections with anti-CD31 antibody. The number of vessels per ×200 field was counted. Occasionally, isolated microvessels could be found in Ad-mCCL21 + low-dose paclitaxel (Ad-mCCL21+Pac)-treated tumors. Ad-mCCL21, Pac, or Ad-LacZ treatment also has an angiostatic effect. At the same magnification, the well-formed capillaries surrounding nests of tumor cells could be found in the normal saline (NS) control group. (b) Apoptotic tumor cells within tumor tissues were elevated by TUNEL assays. The apoptotic index was calculated as a ratio of the apoptotic cell number to the total cell number in each field. Sequential analysis showed that Ad-mCCL21+Pac treatment resulted in significant increment of apoptotic index versus NS control. Pac, Ad-mCCL21, and Ad-LacZ+Pac also efficiently induced the apoptosis of tumor cells. The columns in all graphs correspond to the labeled columns in the picture. Bars, ±SD. * P < 0.01 relative to NS control. ▲ P < 0.01 relative to Pac, Ad-mCCL21, or Ad-LacZ+Pac group. MVD, microvessel density, # P < 0.05 relative to NS control.

Article Snippet: Forty-eight hours after infection, cells were harvested and analyzed by Western blotting using a rabbit anti-murine mCCL21 monoclonal IgG antibody (Peprotech EC, London, UK).

Techniques: Staining, Isolation, Saline, Control, TUNEL Assay, Labeling

Journal: Cancer Science

Article Title: Low-dose paclitaxel improves the therapeutic efficacy of recombinant adenovirus encoding CCL21 chemokine against murine cancer

doi: 10.1111/cas.12537

Figure Lengend Snippet: Analysis of cytokines in the tumor site. Mice bearing 4T1 breast carcinoma were treated with normal saline (NS), paclitaxel (Pac), Ad-mCCL21, Ad-LacZ+Pac, or Ad-mCCL21+Pac. On day 4 after the completion of treatment, tumor tissues were harvested, cut into small pieces, homogenized, and centrifuged at 12 000 g for 30 min. Tumors were evaluated for the presence of granulocyte–macrophage colony-stimulating factor (GM-CSF), γ-interferon (IFN-γ), monokine induced by interferon-gamma (MIG), interferon-gamma-induced protein 10 (IP-10), interleukin-12 (IL-12), transforming growth factor-β (TGF-β), IL-10, and vascular endothelial growth factor (VEGF) by ELISA. Cytokine concentrations were corrected for total protein by Lowry protein assay. Results are expressed as pg/mg total protein. Compared with tumor tissues from the NS control group, mice treated with Ad-mCCL21+Pac had significant increase in GM-CSF, IFN-γ, MIG, IP-10, and IL-12 (a) but a decrease in TGF-β (b), IL-10 (c), and VEGF (d). Bars, ±SD. * P < 0.01 relative to NS control. # P < 0.05 relative to NS control. ▲ P < 0.01 relative to Pac, Ad-mCCL21, or Ad-LacZ+Pac group. ▵ P < 0.01 relative to Pac or Ad-LacZ+Pac group.

Article Snippet: Forty-eight hours after infection, cells were harvested and analyzed by Western blotting using a rabbit anti-murine mCCL21 monoclonal IgG antibody (Peprotech EC, London, UK).

Techniques: Saline, Enzyme-linked Immunosorbent Assay, Control

Journal: Cancer Science

Article Title: Low-dose paclitaxel improves the therapeutic efficacy of recombinant adenovirus encoding CCL21 chemokine against murine cancer

doi: 10.1111/cas.12537

Figure Lengend Snippet: Expression of at the tumor site attracts the infiltration of immune cells. (a) mCCL21 immunohistochemical peroxidase staining was done on paraffin-embedded tumor sections obtained from each of the five therapy groups: normal saline (NS), paclitaxel (Pac), Ad-mCCL21, Ad-LacZ+Pac, or Ad-mCCL21+Pac. Significant expression of mCCL21 could be detected in both Ad-mCCL21 and Ad-mCCL21+Pac groups. Representative images of immunofluorescence stained with anti-CD4 (b), anti-CD8 (c), and anti-CD11c (d) antibodies. Significant infiltration of CD4-, CD8-, and CD11c-positive cells in the tumor tissues indicate that Ad-mCCL21 or Ad-mCCL21+Pac treatment would lead to the recruitment and maturation of immune cells. The infiltration of different immune cell subsets was microscopically counted at five randomly chosen high-power fields (×200). The columns in all graphs correspond to the labeled columns in the picture. Bars, ±SD. * P < 0.01 relative to NS control. ▲ P < 0.01 relative to Pac, Ad-mCCL21, or Ad-LacZ+Pac group. ▵ P < 0.01 relative to Pac or Ad-LacZ+Pac group.

Article Snippet: Forty-eight hours after infection, cells were harvested and analyzed by Western blotting using a rabbit anti-murine mCCL21 monoclonal IgG antibody (Peprotech EC, London, UK).

Techniques: Expressing, Immunohistochemical staining, Staining, Saline, Immunofluorescence, Labeling, Control

Journal: Cancer Science

Article Title: Low-dose paclitaxel improves the therapeutic efficacy of recombinant adenovirus encoding CCL21 chemokine against murine cancer

doi: 10.1111/cas.12537

Figure Lengend Snippet: Specific antitumor responses were enhanced following treatment with Ad-mCCL21 + low-dose paclitaxel (Ad-mCCL21+Pac) therapy. (a) Enzyme-linked immunospot assay. Mouse γ-interferon-specific assay was done, and spots were quantified with an Immunospot Image Analyzer. Compared with treatment with normal saline (NS), paclitaxel (Pac), Ad-mCCL21, or Ad-LacZ+Pac, T lymphocytes from the Ad-mCCL21+Pac-treated group had significantly greater frequency of specific T cells releasing γ-interferon when restimulated with 4T1 cells. There were minimal responses to the control CT26 cells. Bars, ±SD. * P < 0.01 relative to NS control. (b) CTL-mediated cytotoxicity in vitro . The cytotoxic activity of splenic lymphocytes was measured in 4-h 51 Cr-release assay. The purified T lymphocytes were added to 51 Cr-labeled 4T1 cells immediately after isolation from spleens. T cells derived from the Ad-mCCL21+Pac-treated mice showed higher cytotoxicity against 4T1 cells than those from the Ad-mCCL21 ( P < 0.05), Ad-LacZ+Pac ( P < 0.01), Pac ( P < 0.01), or NS group ( P < 0.01). (c) Abrogation of CTL-mediated cytotoxicity in vivo . Depletion of CD4 + or CD8 + T lymphocytes or natural killer cells by corresponding mAbs. Depletion of CD4 + or CD8 + T lymphocytes significantly impaired the antitumor activity of Ad-mCCL21+Pac therapy, the depletion of natural killer cells showed partly abrogation. Bars, ±SD. * P < 0.01 relative to Ad-mCCL21+Pac therapy. # P < 0.05 relative to Ad-mCCL21+Pac therapy.

Article Snippet: Forty-eight hours after infection, cells were harvested and analyzed by Western blotting using a rabbit anti-murine mCCL21 monoclonal IgG antibody (Peprotech EC, London, UK).

Techniques: Enzyme-linked Immunospot, Saline, Control, In Vitro, Activity Assay, Release Assay, Purification, Labeling, Isolation, Derivative Assay, In Vivo