Tumorigenic hybrids between mesenchymal stem cells and gastric cancer cells enhanced cancer proliferation, migration and stemness

Abstract

Background: Emerging evidence indicates that inappropriate cell-cell fusion might contribute to cancer progression. Similarly, mesenchymal stem cells (MSCs) can also fuse with other cells spontaneously and capable of adopting the phenotype of other cells. The aim of our study was to investigate the role of MSCs participated cell fusion in the tumorigenesis of gastric cancer.

Methods: We fused human umbilical cord mesenchymal stem cells (hucMSCs) with gastric cancer cells in vitro by polyethylene glycol (PEG), the hybrid cells were sorted by flow cytometer. The growth and migration of hybrids were assessed by cell counting, cell colony formation and transwell assays. The proteins and genes related to epithelial- mesenchymal transition and stemness were tested by western blot, immunocytochemistry and real-time RT-PCR. The expression of CD44 and CD133 was examined by immunocytochemistry and flow cytometry. The xenograft assay was used to evaluation the tumorigenesis of the hybrids.

Results: The obtained hybrids exhibited epithelial- mesenchymal transition (EMT) change with down-regulation of E-cadherin and up-regulation of Vimentin, N-cadherin, α-smooth muscle actin (α-SMA), and fibroblast activation protein (FAP). The hybrids also increased expression of stemness factors Oct4, Nanog, Sox2 and Lin28. The expression of CD44 and CD133 on hybrid cells was stronger than parental gastric cancer cells. Moreover, the migration and proliferation of heterotypic hybrids were enhanced. In addition, the heterotypic hybrids promoted the growth abilities of gastric xenograft tumor in vivo.

Conclusions: Taken together, our results suggest that cell fusion between hucMSCs and gastric cancer cells could contribute to tumorigenic hybrids with EMT and stem cell-like properties, which may provide a flexible tool for investigating the roles of MSCs in gastric cancer.

Fig. 1

Cell fusion between hucMSCs and gastric cancer cells. a. Cell sorting of HGC27-hucMSC fused cells by flow cytometry. HGC-27 and hucMSCs were labeled with DIO and DID, respectively. DIO-HGC27 and DID-hucMSC was collected respectively. The control group indicates spontaneous fusion while the fusion represents the PEG1500-mediated generation of double positive hybrids. b. The statistical analyses of fusion efficiency. The data represent mean ± SD of three independent experiments. *P < 0.05,***P < 0.001 (c) Microscopic fluorescent images of fused cells. (a) The single double-positive hybrid was detected on the third day after sorting. Scale bar 100 μm, Magnification: ×200; (b) Hybrids with two nuclei were stained with Hoechst 33342 (blue) and cell membrane structures were double-labeled (yellow) were observed on the seventh day after sorting. Scale bar 100 μm, Magnification: ×200. d. Representative images from the cell population gates were tested by imaging cytometer. HGC-27 and hucMSCs were stained with DIO (green) and DID (red), respectively. In the “unfused”group, under the treatment of PEG1500, the two cells formed an adhesion structure but not a hybrid. Also, the hybrids fused with DIO-HGC27 and DID-HucMSCs are yellow and showed in the“fused” group. The double positive cell populations were gated in R2, the population of hybrids was 8.08 %

Fig. 2

Fusion with hucMSCs induces morphological changes and enhanced growth of gastric cancer cells. a. The two gastric cancer cell lines showed epithelial morphology and hucMSCs with fibroblast-like shape. HGC-27 fusion hybrids exhibit elongated shape and front-to-back polarity. SGC-7901 fusion lost the epithelial morphology and assumed a fibroblast-like appearance. Scale bar 100 μm, Magnification: ×100. b. The growth of the parental and hybrid cells was determined by cell counting assay. c. The expression of PCNA and CyclinD1 proteins in parental and hybrid cells was examined by western blot

Fig. 3

Fusion with hucMSCs induces EMT in gastric cancer cells. a. Transwell migration assay of parental and hybrid cells. b. The number of migrated cells in transwell migration assay. ***P < 0.001. c. The expression of mesenchymal markers E-cadherin、N-cadherin and Vimentin was determined by western blot. d. The expression of EMT-related genes was determined by real-time RT-PCR

Fig. 4

Fusion with hucMSCs induces the acquisition of stemness in gastric cancer cells. a. Immunofluorescent staining of CD44 in the parental and the hybrid cells. b. The expression of cancer stem cell marker CD133 in the parental and hybrid cells was determined by flow cytometry. c. The expression of Oct4, Sox2, Nanog proteins in the parental and hybrid cells was determined by western blot. d. The expression of Oct4, Sox2, Nanog, and Lin28 in the hybrid cells related to the parental cells was examined by real-time RT-PCR

Fig. 5

Hybrids of HGC-27 cell promote gastric cancer growth in vivo. a. The representative images of tumor-bearing mice. b. Tumor tissues were photographed 20 days after tumor cell inoculation. c, d. The weight and volume of tumor issues removed from HGC-27 and HGC-27 fusion groups. ***P < 0.0001