Secreted TRAIL gene-modified adipose-derived stem cells exhibited potent tumor-suppressive effect in hepatocellular carcinoma cells

Abstract

Objective: Considering the potential of adipose-derived stem cells (ADSCs) migrating towards cancer cells, this study was performed to explore the function of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) modified ADSCs on the development and progression of hepatocellular carcinoma (HCC).

Methods: ADSCs were extracted from human adipose tissues and identified through immunofluorescence and flow cytometry. Oil red staining and alizarin red staining were performed to clarify the differentiation potential of ADSCs. AAV-CMV-sTRAIL was transfected into ADSCs before Western blot and Transwell measurements. sTRAIL-ADSCs were cocultured with HCC cells to explore its effect on the proliferation and apoptosis of HCC cells. The possible effect of sTRAIL-ADSCs or ADSCs on tumor growth and metastasis was determined in vivo using xenograft nude mouse models.

Results: ADSCs were successfully extracted from adipose tissues, which were confirmed by cell morphology and positive expressions of CD44 and CD105. ADSCs were found with differentiation potential. After transfection, TRAIL was stably expressed in sTRAIL-ADSCs. Both ADSCs and sTRAIL-ADSCs can migrate towards HCC cells. In addition, sTRAIL-ADSCs can promote the cell apoptosis and inhibit cell proliferation in vitro, on parallel it can also suppress epithelial-mesenchymal transition, tumor growth, and metastasis in vivo.

Conclusion: TRAIL modified ADSCs can migrate towards HCC cells to inhibit tumor growth and the metastasis of implanted HCC tumors, which hints TRAIL modified ADSCs may be a new therapeutic approach for HCC treatment.

Keywords: TNF-related apoptosis-inducing ligand; adipose-derived stem cells; apoptosis; gene modification; hepatocellular carcinoma; metastasis; migration; tumor growth.

Figure 1

Culture, isolation and identification of ADSCs. (A), cell morphologies under microscope after isolated cells being culture for respectively 24 h (left) and 14 days (right); (B), detection of biomarkers of ADSCs, CD44 (left) and CD105 (right) using immunofluorescent staining; (C), FCM measured the expressions of CD105, CD166, CD44, CD116, CD34, and CD45 in the 4th generation of ADSCs; (D), osteogenesis differentiation potential of ADSCs was verified using alizarin red staining; (E), adipogenic differentiation potential of ADSCs was verified using oil red O staining; ADSCs, adipose‐derived stem cells; FCM, flow cytometry

Figure 2

Orientated migration of sTRAIL‐ADSCs toward HCC cells. (A), AAV‐GFP labeled ADSCs or NIH3T3 were cultured with HCC cells for observation of fluorescent under fluorescence microscope; (B), the pattern of Transwell coculture system to induce migration of ADSCs or NIH3T3 cells toward normal liver histolysate, HCC histolysate, Huh7 culture medium or L02 culture medium; (C and D), effect of normal liver histolysate, HCC histolysate, Huh7 culture medium and L02 culture medium on migration of ADSCs; (E and F), effect of normal liver histolysate, HCC histolysate, Huh7 culture medium and L02 culture medium on the migration of NIH3T3; (G), Western blot detecting the expression of TRAIL in ADSCs after AAV‐CMV‐sTRAIL transfection; (H), expressions of TRAIL and CD105 in ADSCs or sTRAIL‐ADSCs; (I and J), effect of normal liver histolysate, HCC histolysate, Huh7 culture medium, and L02 culture medium on migration of sTRAIL‐ADSCs.*p < .05, ***p < .001. ADSCs, adipose‐derived stem cells; FCM, flow cytometry; HCC, hepatocellular carcinoma; TRAIL, tumor necrosis factor‐related apoptosis‐inducing ligand

Figure 3

Coculture with sTRAIL‐ADSCs could induce cell apoptosis of Huh7 cells. (A and B), FCM to measure the effect of sTRAIL on apoptosis of Huh7 cells; (C and D), FCM to measure the effect of sTRAIL on apoptosis of L02 cells; (E and F), FCM to measure the effect of sTRAIL on apoptosis of ADSCs; (G), coculture system of sTRAIL‐ADSCs or ADSCs with Huh7 or L02 cells; (H), CCK‐8 assay to detect the effect of sTRAIL‐ADSCs or ADSCs to proliferation of Huh7 cells; (I), CCK‐8 assay to detect the effect of sTRAIL‐ADSCs or ADSCs to proliferation of L02 cells; (J and K), FCM to measure the effect of coculture of sTRAIL‐ADSCs or ADSCs with Huh7 or L02 cells on apoptosis of Huh7 or L02 cells. ns: p > .05, *p < .05, **p < .01. ADSCs, adipose‐derived stem cells; CCK‐8, Cell Counting Kit‐8; FCM, flow cytometry; HCC, hepatocellular carcinoma; TRAIL, tumor necrosis factor‐related apoptosis‐inducing ligand

Figure 4

sTRAIL‐ADSCs can suppress the growth of subcutaneously implanted HCC in nude mouse. (A), sTRAIL expression in each group was measured by Western blot; (B), tumor size in nude mice of each group; (C), comparison on tumor size; (D), tumor weight; (E), H&E staining for tumor tissues (×400); (F and G), immumohistochemical staining for Ki67 detection (×400); (H and I), Western blot for detection on apoptotic protein Bcl‐2 and Bax. **p < .01, ***p < .001. ADSCs, adipose‐derived stem cells; CCK‐8, Cell Counting Kit‐8; FCM, flow cytometry; HCC, hepatocellular carcinoma; H&E, hematoxylin and eosin; TRAIL, tumor necrosis factor‐related apoptosis‐inducing ligand

Figure 5

Suppressive effect of sTRAIL‐ADSCs on micrometastasis of HCC in nude mouse. (A), distribution of micrometastasis in nude mouse was monitored by in vivo image system; (B), PCR was applied to detect the mRNA expressions of EMT related biomarkers (E‐cadherin, N‐cadherin, Vimentin, and Snail‐1); (C and D), Western blot was applied to detect the mRNA expressions of EMT related biomarkers (E‐cadherin, N‐cadherin, Vimentin, and Snail‐1). *p < .05, **p < .01. ADSCs, adipose‐derived stem cells; EMT, epithelial‐mesenchymal transition; HCC, hepatocellular carcinoma; mRNA, messenger RNA; TRAIL, tumor necrosis factor‐related apoptosis‐inducing ligand