Abstract
Background: Cell fusion and the subsequent aneuploidy are commonly observed in different kinds of tumor. In glioma, cell fusion and the number of the polyploid giant cells were found to be augmented with the tumor grades (WHO Ⅰ-Ⅳ) and closely related to poor prognosis. Phytohemagglutinin (PHA) holds an ability to induce cell-cell membrane contact and accelerates the cell fusion process mediated by the fusogenic agent polyethylene glycol (PEG). Dimethyl sulfoxide (DMSO) is well known as a cryoprotective agent involving in cell cryopreservation. In this study, we aim to obtain the glioma fusion hybrids by the modified fusion method in vitro, and then investigate the pathological consequences and the related molecular mechanism with the cell hybrids.Methods: Glioma cells were labelled by lentiviruses infection. The PEG fusion efficiency was respectively improved by the addition of PHA and DMSO, and quantified by flow cytometry. Then, fusion hybrids were obtained by puromycin screening and fluorescence-activated cell sorting (FACS). Furthermore, DNA content was analyzed through flow cytometry. Cell proliferation rate and cell viability under temozolomide (TMZ) was detected by CCK-8 assay. Lastly, the related gene expression was measured through qRT-PCR and Western blotting. Results: Glioma cell-cell contact was achieved by adding certain concentration of PHA in vitro. Tumor-tumor cell fusion efficiency was improved by PHA and DMSO. Glioma fusion hybrids were successfully obtained after puromycin screening and FACS. Cell size, DNA content and chromosome numbers of the fusion hybrids were almost twice that of the parental glioma cells. Moreover, glioma fusion hybrids showed an enhanced TMZ resistance potential compared to the parental cells, and also the MGMT expression was up-regulated in the hybrids.Conclusions: We successfully obtained the glioma tumor-tumor cell fusion hybrids through the modified PHA-DMSO-PEG fusion method. Cell fusion may contribute to TMZ resistance in glioma, thus inhibition of cell fusion could be a promising orientation to improve TMZ resistance. Moreover, combining TMZ and MGMT inhibitor could be a beneficial approach in patients with glioma polyploid giant cells.
Tumor Cell Fusion and Multipolar Trivision