Potential Effect of Exosomes Derived from Cancer Stem Cells and MSCs on Progression of DEN-Induced HCC in Rats

Abstract

Cross talk, mediated by exosomes, between normal stem cells and cancer stem cells (CSCs) in the tumor microenvironment has been given less attention so far. In addition, no publications are available in the literature that address the in vivo impact of exosomes derived from CSCs and mesenchymal stem cells (MSCs) on progression of long-term hepatocellular carcinoma (HCC). Herein, we hypothesized that transfer of exosomes among the cells in the HCC microenvironment could either induce or inhibit tumor growth and metastasis depending on their source. To check this hypothesis, we investigated the effect of exosomes coming from two different stem cell populations, hepatic CSCs and bone marrow (BM) MSCs, on progression of long-term DEN-induced HCC in rats and the involved underlying mechanisms. CSCs-exosomes induced a significant increase in liver relative weight and serum levels of cancer markers (AFP and GGT) and liver enzymes (ALT, AST, and ALP), intensive immunostaining for the HCC marker GST-P, and an increased number and area of tumor nodules as compared to HCC rats injected by PBS. CSCs-exosomes also decreased apoptosis (marked by downregulation of Bax and p53 and upregulation of Bcl2, and increased immunostaining of PCNA), increased angiogenetic activity (revealed by upregulation of VEGF), enhanced metastasis and invasiveness (indicated by upregulation of P13K and ERK proteins and their downstream target MMP9 and downregulation of TIMP1), and induced epithelial mesenchymal transition (marked by increased serum and hepatic level of TGFβ1 mRNA and protein). Notably, CSCs-exosomes also elevated HCC exosomal microRNA (miR) 21, exosomal long noncoding (lnc) RNA Tuc339, lncHEIH, and the HCC lncHOTAIR and decreased liver miR122 and HCC miRs (miR148a, miR16, and miR125b). All these cellular, functional, and molecular changes were reversed following injection of BM-MSCs-exosomes. However, both CSCs- and MSCs-exosomes failed to change the elevated oxidative stress or the inhibited antioxidant activities induced by HCC. Collectively, our results revealed a tumor stimulatory effect (induction of tumor growth, progression, and metastasis) for exosomes derived from CSCs and an inhibitory effect for exosomes derived from MSCs. These results provide valuable insight on the effect of CSCs- and MSCs-exosomes on HCC growth and progression in vivo, which may be helpful to understand the mechanism of HCC development.

Figure 1

Identification of cultured CSCs and MSCs. MSCs with their characteristic fusiform (fibroblast-like) shape were grown after passage 2 (a) and passage 8 (b). After 2 weeks of plating HCC explants, stem-like cancer cells were grown (c), with characteristic clonal expansion (d) and a very notable proliferative ability (e), and later on few floating cells forming spheres appeared (f). The isolated CSCs and MSCs confirmed by detection of stem cell-specific genes Nanog (120 bp) and Oct3/4 (165 bp) using RT-PCR (g), and by flow cytometry which revealed that CSCs were CD24⁺, CD44⁺, CD133⁺, EpCam⁺, and CD45⁻, while MSCs were CD90⁺, CD44⁺, CD45⁻, and CD34⁻ (h). Scale bars: 35 μm (c–e) and 50 μm (a, b, f).

Figure 2

Characterization and labeling of CSC- and MSC-derived exosomes. Transmission electron microscopic examination shows small nanovesicles (30–100 nm) in the sample isolated from the BM-MSCs (a) and hepatic CSCs (b) culture media by ultracentrifugation. Western blot analysis of the culture medium, and exosomes shows presence of CD63 and CD81 in the exosomes of CSC-Ex and MSCs-Ex groups (c). β-Actin was used as internal loading control.

Figure 3

Serum levels of cancer markers (AFP, GGT), TGFβ, liver damage enzymes (ALT, AST, and ALP), and albumin in normal rats (Nor) and DEN-induced HCC rats treated by PBS or exosomes derived from CSCs (CSC-Ex) or MSCs (MSC-Ex). Values are expressed as mean ± SEM (n = 9/group). Different letters on columns mean significant differences. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, and ^∗∗∗∗P < 0.0001 versus PBS group; ^####P < 0.0001 versus CSC group.

Figure 4

Antioxidant/ROS status of HCC rats after treatment by exosomes derived from CSCs and MSCs showed unchanged levels of MDA, SOD, CAT, and GPX in the liver. Values are expressed as mean ± SEM (n = 9/group). Different letters on columns mean significant difference at P < 0.0001.

Figure 5

Photomicrographs of H&E-stained liver sections of the normal group (a), the PBS group (b), the CSC-Ex group (c), and the MSC-Ex group (d). Scale bar: a, b, 50 μm; c, d, 30 μm.

Figure 6

Immunostaining for GST-P (A–D) and PCNA (E–H) in livers of rats in the normal group (A, E), PBS group (B, F), CSC-Ex group (C, G), and MSC-Ex group (D, H). Arrowhead points to PCNA-positive nucleus. Scale bar: (A) E–H, 50 μm, B–D, 30 μm. The graphs show mean number and area of GST-P-positive foci and number of PCNA-positive nuclei in different rat groups. Data was presented as mean ± SEM (n = 9). Different letters on columns mean significant difference. ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗∗P < 0.0001 versus PBS group; ^####P < 0.0001 versus CSC group.

Figure 7

qPCR analysis shows changes in relative gene expression of apoptotic genes (p53 and Bax), antiapoptotic gene (Bcl2), EMT-related gene (TGFβ1), inflammation-related gene (NFκB), angiogenesis-related genes (VEGF), and metastasis-related genes (MMP9 and TIMP1) in livers of normal rats (Nor) and DEN-induced HCC rats treated by PBS or exosomes derived from CSCs (CSC-Ex) or MSCs (MSC-Ex). Data presented as fold change from the normal (Nor) control group. Values are expressed as fold change mean ± SEM (n = 9). Different letters on columns mean significant difference. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, and ^∗∗∗∗P < 0.0001 versus PBS group; ^####P < 0.0001 versus CSC group.

Figure 8

qPCR analysis shows changes in relative gene expression of miR21, miR122, miR148a, miR16, miR125b, lncTuc339, lncHEIH, and lncHOTAIR in livers of DEN-induced HCC rats treated by PBS (PBS) or exosomes derived from CSCs (CSC-Ex) or MSCs (MSC-Ex). Data presented as fold change from the PBS group. Values are expressed as mean ± SEM (n = 9). Different letters on columns mean significant difference. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001, and ^∗∗∗∗P < 0.0001 versus PBS group; ^####P < 0.0001 versus CSC group.

Figure 9

Effect of exosomes derived from CSCs and MSCs on the expression of TGFb, P13K, and ERK proteins in livers of DEN-induced HCC rats using Western blotting. β-Actin was used as a control for internal protein loading. Band intensity revealed relative changes in protein expression using the normal group as a control. Values were expressed as mean ± SEM, n = 9, from two separate experiments. Different letters on columns mean significant difference. ^∗∗P < 0.01, ^∗∗∗P < 0.001, and ^∗∗∗∗P < 0.0001 versus PBS group; ^####P < 0.0001 versus CSC group.