Mesenchymal Stem Cells in Inflammation Microenvironment Accelerates Hepatocellular Carcinoma Metastasis by Inducing Epithelial-Mesenchymal Transition

<div><p>In response to inflammation, mesenchymal stem cells (MSCs) are known to migrate to tissue injury sites to participate in immune modulation, tissue remodeling and wound healing. Tumors apply persistent mechanical and pathological stress to tissues and causes continual infiltration of MSCs. Here, we demonstrate that MSCs promote human hepatocellular carcinoma (HCC) metastasis under the influence of inflammation. The metastasis promoting effect could be imitated with the supernatant of MSCs pretreated with IFNγ and TNFα. Interestingly, treatment of HCC cells with the supernatant leads to epithelial-mesenchymal transition (EMT), an effect related to the production of TGFβ by cytokines stimulated MSCs. Importantly, the levels of MSCs expressing SSEA4 in clinical HCC samples significantly correlated with poor prognosis of HCC, and EMT of HCC was strongly associated with a shorter cancer-free interval (CFI) and a worse overall survival (OS). Therefore, our results suggest that MSCs in tumor inflammatory microenvironment could promote tumor metastasis through TGFβ-induced EMT.</p> </div

Correlations of MSCs and EMT markers expression with Clinicopathologic Characteristics of HCC.

<p>(A) SSEA-4 expression was seen high in 69/114 of HCC tissues (upper left×200, upper right×400), and low in 45/114 (bottom left×200, bottom right×400); (B) Estimated overall survival according to the expression of SSEA-4 in 114 cases of HCCs (the Kaplan-Meier method). Log-rank test shows that HCC patients in the high SSEA-4 expression group have poorer overall survival than those in the low SSEA-4 expression group (<i>P</i> = 0.002). (C) Cancer-free survival was analyzed in the same cohort of HCC patients and the results showed that HCC patients in the high SSEA-4 expression group also have poorer cancer-free survival than those in the low SSEA-4 expression group (<i>P</i> = 0.025). (D) Immunohistochemistry of E-cadherin and Vimentin in two representative cases without (Case 1) or with (Case 2) EMT change. Membranous expression of E-cadherin was down-regulated and cytoplasmic translocation of Vimentin was up-regulated in case with EMT change (×200); (E) Kaplan-Meier survival analysis of CFI in HCC cases with preserved versus down-regulated E-cadherin expression; (F) Kaplan-Meier survival analysis of CFI in HCC cases with preserved versus up-regulated Vimentin expression.</p

MSCs pretreated by proinflammatory cytokines lead EMT of HCC cells and up-regulation of TGF-β in MSCs.

<p>(A) qPCR was used to detect changes in expression of EMT genes in SMMC-7721 HCC cells following co-culture with MSC after being stimulated by IFNγ and TNFα. Results presented represent mean ± SEM (n = 3); (B) Immunofluorescent staining of E-cadherin and Vimentin was performed in SMMC-7721 cells, nuclei were counterstained with DAPI (×200); (C) Western blot was used to detect the expression of N-cadherin, Twist and β-catenin in SMMC-7721 cells. PCR (D) and western-blot (E) were used to detected TGFβ expression in MSCs, and the results showed that TGFβ was overexpressed in MSCs stimulated by IFNγ and TNFα both on mRNA and protein levels. (F) Immunofluorescent staining was used to confirm that up-regulation of TGFβ in MSCs stimulated by combination of IFNγ and TNFα (×200).</p

MSCs pretreated by proinflammatory cytokines promote the metastasis of HCC cell.

<p>(A) The wound healing assay was employed to determine the migration of SMMC-7721 cells, cells were monitored every 24 h for 48 h to determine the rate of migration into the scratched area (*<i>P</i><0.05; ×200). (B) Invasiveness of cells was determined using Transwell assay. Cells were co-cultured with MSCs after being stimulated by IFNγ and TNFα, and then plated in the upper chamber of the Transwell and allowed to grow for 24 h in serum-free medium, 5% fetal bovine serum was placed in the lower chamber. Number of cells that invaded through the Matrigel was counted in 10 fields under the ×20 objective lens (*<i>P</i><0.05; ×200). (C) Pictures of metastatic liver nodules in nude mice by splenic-vein injection of SMMC-7721 cells. The arrows indicate the metastatic tumor on the surface of the liver. (D) H&E staining was performed on serial sections of metastatic tumors and normal liver (×100), the arrow indicate the metastatic tumor in the liver issue; The number (E) and the volume (F) of nodules were quantified on livers of nude mice (n = 10 per group) 6 weeks after splenic vein injection of SMMC-7721 cells co-cultured with MSC after being stimulated by IFNγ and TNFα. Values for individual mice are shown above the bars. (G) Survival rate of nude mice 6 weeks after splenic vein injection of different treated cells (Control was SMMC-7721 cells group, *<i>P</i><0.05).</p

TGF-β depletion in MSCs reverses the promotive effect on metastasis and EMT of HCC cells induced by MSCs in inflammation microenvironment.

<p>(A) Expression of EMT genes was detected by qPCR (normalized to β-actin); (B) E-cadherin and Vimentin expression was performed by immunofluorescent staining, nuclei were counterstained with DAPI (×200). (C) Western blot was used to detect the expression of N-cadherin, Twist and β-catenin, SMMC-7721 cells co-cultured with MSCs^si-TGFβ stimulated by both IFNγ and TNFα did not present EMT; (D) The wound healing assay was employed to determine the migration of SMMC-7721 cells (×200); (E) Invasiveness of SMMC-7721 cells was determined using Transwell assay; (F) The metastatic liver nodules in nude mice by splenic-vein injection of SMMC-7721 cells. The arrows indicate the metastatic tumor on the surface of the liver (upper). H&E staining was performed on serial sections of metastatic tumors and normal liver (bottom, ×100); (G) The number of nodules were quantified on livers of nude mice (n = 10 per group) 6 weeks after splenic vein injection of SMMC-7721 cells. (*<i>P</i><0.05 versus Control group; #<i>P</i><0.05 versus MSCs(IFNγ+TNFα) and MSCs(IFNγ+TNFα)^vector; ×200).</p

Correlations Between SSEA-4 Expression and Clinicopathologic Variables of HCC.

<p>(*P<0.05).</p