Endostatin enhances antitumor effect of tumor antigen-pulsed dendritic cell therapy in mouse xenograft model of lung carcinoma

Abstract

Objective: To investigate the antitumor effect of endostatin combined with tumor antigen-pulsed dendritic cell (DC)-T cell therapy on lung cancer.

Methods: Transplanted Lewis lung cancer (LLC) models of C57BL/6 mice were established by subcutaneous injection of LLC cells in left extremity axillary. Tumor antigen-pulsed DC-T cells from spleen cells and bone of mice were cultured in vitro. Tumor-bearing mice were randomly divided into three groups, including DC-T+endostatin group, DC-T group, and phosphate-buffered saline (PBS) control group. Microvessel density (MVD) of tumor tissue in tumor-bearing mice was determined by immunohistochemistry (IHC). The expressions of vascular endothelial growth factor (VEGF) and hypoxia-inducible factor-1α (HIF-1α) were determined by Western blotting and IHC staining. The proportions of CD8+ T cells, mature dendritic cells (mDC), tumor-associated macrophages [TAM (M1/M2)], and myeloid-derived suppressor cells (MDSC) in suspended cells of tumor tissue were determined by flow cytometry. The expressions of interleukin (IL)-6, IL-10, IL-17, transforming growth factor-β (TGF-β) and interferon-γ (IFN-γ) in suspended cells of tumor tissue were detected by enzyme-linked immune sorbent assay (ELISA).

Results: DC-T cells combined with endostatin remarkably suppressed tumor growth. MVD of mice in DC-T+endostatin group was significantly lower than that of the control group and DC-T monotherapy group. The expressions of VEGF, IL-6 and IL-17 in tumors were markedly decreased, but IFN-γ and HIF-1α increased after treating with DC-T cells combined with endostatin, compared to control group and DC-T group. In the DC-T+endostatin group, the proportions of MDSC and TAM (M2 type) were significantly decreased, mDC and TAM (M1 type) were up-regulated, and CD8+ T cells were recruited to infiltrate tumors, in contrast to PBS control and DC-T monotherapy. DC-T cells combined with endostatin potently reduced the expressions of IL-6, IL-10, TGF-β and IL-17 in tumor tissue, and enhanced the expression of IFN-γ.

Conclusions: The study indicated the synergic antitumor effects between endostatin and tumor antigen-pulsed DC-T cells, which may be a prospective therapy strategy to achieve potent antitumor effects on lung cancer.

Keywords: DC-T cells; Endostatin; cellular therapy; lung cancer; tumor microenvironment.

1

DC-T cells in combination with endostatin augment antitumor effect and strongly inhibit tumor vasculature in LLC. (A) Tumors dissected from three groups of tumor-bearing mice (DC-T alone, DC-T+endostatin, PBS control); (B) Tumor growth curves; (C) Microvessel density (MVD). The data are representative results of independent experiments (n=7 per group for each experiment). ^*, P<0.05 compared with control; ^**, P<0.01 compared with control; (D) MVD was determined by blinded measurement of CD31 expression using IHC (400×).

2

DC-T cells combined with endostatin efficiently decrease multiple proangiogenic factors and augment hypoxia in tumors. (A, B) VEGF and HIF-1α expressions in tumors determined by Western blotting. β-actin served as an internal control. Data are expressed as x±s of three independent experiments. *, P<0.05 compared with control; (C) Expression of VEGF and HIF-1α in tumors determined by IHC (400×); (D) Pro/anti-angiogenic cytokines IL-6, IL-17 and IFN-γ assessed by ELISA. Data are expressed as x±s of three independent experiments. *, P<0.05 compared with control; **, P<0.01 compared with control. VEGF, vascular endothelial growth factor; HIF, hypoxia-inducible factor; IFN, interferon.

3

DC-T cells in combination with endostatin significantly decrease immunosuppressive cells in tumor microenvironment and increase tumor-infiltrated mDC and CD8+ T cells. (A) Effect of DC-T cells (5×10⁶ cells, d 1) in combination with endostatin (15 mg/kg, d 1-14) on immunoinhibitory cells (MDSC, M1 and M2); (B) Effect of DC-T combined with endostatin on mDC and cytotoxic CD8+ T cells. The cell proportions were detected by flow cytometry. Data are expressed as x±s of three independent experiments. *, P<0.05 compared with control; **, P<0.01 compared with control. MDSC, myeloid-derived suppressor cells; M1, tumor-associated macrophages of phenotype 1; M2, tumor-associated macrophages of phenotype 2.

4

DC-T cells combined with endostatin decrease immuno-inhibitory cytokines in tumor microenvironment. The expressions of IL-6, IL-10, IL-17, TGF-β and IFN-γ in suspended tumor cells of tumor tissue were detected by ELISA. Data are expressed as x±s of three independent experiments. *, P<0.05 compared with control; **, P<0.01 compared with control. TGF, transforming growth factor.