Effect And Molecular Mechanism Of Cell Fusion Between Mesenchymal Stem Cells And Hepatoma Carcinoma Cells In Hepatocellular Carcinoma Metastasis

Hepatocellular carcinoma is one of the most common malignant tumors. And metastasis is the major cause of treatment failure and mortality of hepatocellular carcinoma. So it’s very important to clarify the metastatic mechanism of hepatocellular carcinoma for the treatment of liver cancer. At present, there are several hypotheses about the metastatic mechanism of tumors, such as tumor stem cells, epithelial-mesenchymal transition (EMT), and organ-specific metastasis. These hypotheses are in-depth study, but could not explain the mechanism of hepatocellular carcinoma metastasis satisfactorily. Recently, cell fusion theory has become a hot research hypothesis in tumor metastasis.Cell fusion playes an important role in many critical development processes, including zygote formation and organogenesis of placenta, bone, and skeletal muscle. Cell fusion can be easily recognized in cell culture and animal tumor models by using lineage-specific tracking markers. Because of lacking of available genetic markers in human, it is hard to detect the cell fusion in carcinoma formation and matastasis, therefore, rare articles were reported about cell fusion phenomenon in human cancers. However, Chakraborty and Pawelek, J., have reported two clinical cases of cell fusion. In the first reported case, a renal cell carcinoma (RCC) developed in a child after an HSC transplanting from his cancer-free brother. A lymph node metastasis of this tumor was analysed and it was found that carcinoma cells throughout the tumor contained the donor-specific A allele of the ABO blood group. In the second case, tumor cells from a primary papillary RCC (PRCC) arised after a male-to-female HSC transplant and the donor Y chromosome was found. The two clinical cases above indicated that the tumor cells fusion with stem cells existed in the human body. At the same time, He Xh and Tsang TC introduced the fusion of carcinoma cells with bone marrow-derived stem cells may play an important role in carcinoma metastasis.Bone marrow-derived stem cells (BMDSCs) includes hematopoietic stem cells (HSCs), mesenchymal stem cells (MSCs) and multipotent adult progenitor cells (MAPCs). Mesenchymal stem cells (MSCs) are characterized by multiplex differentiation, self-renewal, infiltration and migration ability. Many reports of MSCs were about tissue repair, neural tissue regeneration, tumor growth and metastasis, but we didn’t check any reports about the cell fusion of MSCs.In this study, PEG was used to facilitate the fusion between hepatocellular carcinoma cell line HepG2and MSCs. The fused cells were selected by flow cytometry, and identified by chromosome analysis and DNA contant analysis. Then the invasion and metastasis of fused cells were detected in vitro and in vivo. The metastasis mechanism of fusion was also studied, and it was expected to provide an innovative way for tumor metastasis.Methods1. Identification, cultivation and red fluorescence label of mesenchymal stem cells.Bone marrow was isolated under sterile conditions from femurs and tibias. Briefly, bones were isolated and marrow was flushed out with L-DMEM. Cells were propagated in the medium at37℃in a5%CO2humidified incubator. Non-adherent cells were removed after4and24hrs and every2-3days thereafter by gently washing with medium. Cultures were grown to80-90%confluency before passing. Morphology of the cells was observed by microscope. The expression of CD34, CD45, CD90, CD105were detected by flow cytometry. Incubation of2ug/ml DiI with MSCs for1h, then red fluorescence was observed by fluorescence microscopy. 2. Fusion, screening and identification of fused cells.Low-metastatic human hepatoma cells HepG2were transfected with pEGFP-N1plasmid, and mesenchymal stem cells were labeled with a red fluorescent dye. The two cell types were combined and treated with polyethylene glycol (PEG) to facilitate cell fusion. The resulting fused cells with both labels were identified and isolated by flow cytometry. Chromosome analysis and DNA contant analysis were performed to identify the fused cells.3. Infiltration, migration and proliferation ability were detected in vitro and in vivo.Transwell chamber was used to detect Infiltration and migration of fused cells; CCK-8(cell counting kit) assay and soft agar colony formation assay was applied to evaluate the cell proliferation; flow cytometry was used to detect cell cycle.All mice were anesthetized by Pentobarbital Sodium. Then the peritoneal cavity was opened and the liver was exposed. For each of the three cell types,3×106cells suspended in30μl of matrigel was injected into left liver lobe. Animals were sacrificed after10weeks and necropsied to assess macro metastatic tumor formation.4. Detection of EMT markers and regulation factors in fused cellsEMT markers (E-cadherin and Vimentin) and EMT regulatory factors (Snail and Twist1) expression were detected by Western blot; The mRNA levels were evaluated by RT-PCR; MMP2and MMP9changes were detected by Gelatin zymography.Results1. The cells isolated and cultured from bone marrow were MSCs, which were identified by the surface markers including CD34-,CD45-, CD90+and CD105+by flow cytometry. Growth curve showed that the1st and4th generation of MSCs has much stronger growth ability than the8th generation. Incubated the3rd to5th generation MSCs with DiI for1h, about99%cells were labeled with red fluorescence. 2. The HepG2cells were transfected with pEGFP-Nl plasmid, and selected by500μg/ml G418, and the cells expressed GFP stably. After the PEG treatment, the HepG2cells with GFP and the MSCs with Dil were fused. The results showed that about6.9%cells contained both green and red fluorescence. Chromosomes analysis showed the average chromosome counts of fused cells was128.9±21.3chromosomes for fused cells,the HepG2was89.6±16.0chromosomes for HepG2and MSCs was41.3±4.1. DNA content analysis showed that aneuploid appeared in fused cells.3. The results of infiltration and migration indicated that the fused cells were more migratory and invasive than the low metastatic HepG2cells; results of CCK-8showed that fused cells grew more slowly than HepG2cells. Soft agar colony formation demonstrated that the clones formation of fused cells were weaker than HepG2cells, but it showed that the MSCs owned no colony formation ability.Each of the three cell types were transplanted into liver of nude mouse, and the liver cancer metastatic model was established. The results showed that the average number of liver metastasis was4.50±1.29in fusion group, and of which was2.0±0.82in HepG2group. Rate of lung metastasis was57.14%in fusion group, and it was28.58%in HepG2group. The number of liver metastasis and lung metastasis showed significantly statistical difference between the2groups (P<0.05). The results indicated that the fused cells enhanced tumor metastasis in vivo.4. Results of western blot detection indicated that low expression of E-cadherin and high expression of Vimentin were in fused cells. Further testing showed the expression of EMT regulatory factors Twist1and Snail were also highly expressed in fused cells. The same results was obtained by RT-PCR. Gelatin zymography results confirmed that MMP2and MMP9in fused cells were highly expressed than in HepG2. Conclusions1. The migration and invasion ability of fused cells were enhanced after the fusion between MSCs and low-metastatic hepatocellular carcinoma cell line HepG2. Cell fusion can promote HepG2cells metastasis in vivo.2. After the fusion between MSCs and low-metastatic hepatocellular carcinoma cell line HepG2, fused cells experienced EMT and highly expressed MMP2and MMP9. That may be the mechanism of enhancement of metastatic ability in vivo.