PMC

In vivo antibody depletion reduces the antitumor effect of the ADSC-E7’-eGFP–PE(ΔIII)-E7-KDEL3 combined treatment. (A) Time course of the experiment; (B) tumor volume measurements of syngeneic tumor models with different treatments were conducted at indicated days after the subcutaneous injection of CT26 cells; * p < 0.05 using two-way ANOVA.

The tumor inhibition of the combined treatment by subcutaneously inoculated ADSC-E7’-eGFP and the protein vaccine. (A) Time course of the experiment. Representative bioluminescence images of mice subcutaneously injected with (B) 2 × 10⁵ CT26 cells with indicated treatment or (C) 2 × 10⁵ LLC1 cells with indicated treatment. Tumor volume measurements of syngeneic tumor models were conducted at indicated days after subcutaneous injection of (D) CT26 cells or (E) LLC1 cells; ** p < 0.01; *** p < 0.001 using two-way ANOVA.

Evaluation of apoptosis in tumor tissues by TUNEL staining. Representative fluorescence images of (A) the CT26 tumor with different treatments; or (B) the LLC1 tumor with different treatments. Apoptotic-positive cells were shown in green (arrows) and the cellular nucleus was stained by DAPI and shown in blue. (C,D) The number of apoptotic-positive cells in the microscopic fields were calculated. The quantitative results are presented as means + standard error of means (SEM); *** indicates p < 0.001 using unpaired t-test with Welch’s correction. Magnification A,B: 50 µm.

Establishment of ADSC labeled with enhanced green fluorescent protein (eGFP) and combined with modified E7’ (ADSC-E7’-eGFP). (A) Schematic diagram of pLL3.7-E7’-eGFP construction; (B) agarose gel electrophoresis of plasmid pLL3.7-E7’-eGFP (M: 1 kb DNA ladder; lane 1: Undigested plasmid; lane 2: uNheI (8555 bp); lane 3: NheI and BsrGI (1636 bp and 6919 bp); lane 4: bNcoI (4554 bp, 2845 bp, and 1156 bp); lane 5: bSalI (2955 bp, 2514 bp, 2020 bp, and 1066 bp)); (C) illustration of lentiviral transduction of primary ADSCs; and (D) fluorescence microscopy and flow cytometric analysis of ADSC-E7’-eGFP cells.

The tumor inhibition of the combined treatment by the systemic administration of ADSC-E7’-eGFP and the protein vaccine. The GFP immunohistochemical staining of (A) the CT26 tumor with or without the systemic administration of ADSC-E7’-eGFP cells; or (B) the LLC1 tumor with or without the systemic administration of ADSC-E7’-eGFP cells. (C) Time course of the experiment. Two representative bioluminescence images of mice subcutaneously injected with (D) 2 × 10⁵ CT26 cells with indicated treatment; or (E) 2 × 10⁵ LLC1 cells with indicated treatment. Tumor volume measurements of syngeneic tumor models were conducted at indicated days after subcutaneous injection of (F) CT26 cells; or (G) LLC1 cells; * p < 0.05, ** p < 0.01; and *** p < 0.001 using two-way ANOVA.

Histological assessment of tumor angiogenesis. Representative fluorescence images of tumor sections from mice inoculated with (A) CT26 cells or (B) LLC1 cells, which were stained with anti-CD31 (red) after 28 days to detect tumor-associated blood vessels. The quantitative results were determined by the average blood vessel area per microscopic field in tumor sections from mice inoculated with (C) CT26 cells or (D) LLC1 cells. Values are means + SEM; * p < 0.05; ** p < 0.01, *** p < 0.001 using unpaired t-tests (n = 3). Representative fluorescence images of tumor sections from mice inoculated with (E) CT26 cells or (F) LLC1 cells were stained with anti-VEGF (vascular endothelial growth factor) (red) to detect tumor vascularization. The quantitative results were determined by the average VEGF expression area per microscopic field in tumor sections from mice inoculated with (G) CT26 cells or (H) LLC1 cells. Values are means + SEM; ** p < 0.01, *** p < 0.001 using unpaired t-tests (n = 3). Magnification A,B,E,F: 50 µm.