Fusion Between Bone Marrow-derived Mesenchymal Stem Cells And Lung Cancer Cells Promote Metastasis

Background：Lung cancer, especially non-small-cell lung cancer (NSCLC), remains the leadingcancer-related mortality worldwide. The most deadly phase in cancer progression is theinappropriate acquisition of molecular machinery leading to metastatic transformation.However, the exact mechanisms involved in this process are still elusive.Epithelial-mesenchymal transition (EMT) has been suggested to be a keytrans-differentiation process in cancer cell invasion and metastasis, during which cellsacquire a mesenchymal fibroblastoid phenotype, become motile and disseminate. During theprocess of EMT, disseminated cancer cells may also impart a self-renewal capability toestablish foci at distant sites. However, the mechanisms that defining those processesremains unclear.One possible mechanism underlying metastatic conversion is fusion between bonemarrow derived cells (BMDC) within tumor-associated stroma and cancer cells which couldcontribute to aberrant gene expression patterns associated with highly malignantsubpopulations. The genome of cancer cells might contribute tumorigenicity to the hybrids,whereas myeloid cells could imparts mesenchymal genes and migratory blood cellproperties to hybrids and drive metastatic disease.Spontaneous cell fusion between BMDC and tumor cells has been shown to occur invitro and in vivo. Among bone marrow derived cells within tumor-associated stroma,mesenchymal stem cells (MSCs) have drawn attention for having a role in cancerprogression for their capacity to self-renew and migratory blood cell properties. MSCs arefusogenic and present in most, if not all, NSCLC tissues. Tumor-infiltrating MSCs providevarious functional aids to promote malignancy by direct or indirect effects on tumor cells.Taken together, on the basis of evidence above, we propose the hypothesis that bonemarrow derived MSCs could facilitate lung cancer aquire mesenchymal and stem cell-likeproperties and promote metastasis by transferring distinct cellular capabilities during a fusion event with lung cancer cells. Therefore, this study attempts to observe spontaneousformation of tumorigenic hybrids fused between MSCs and human lung adenocarcinomaA549cell lines, large cell carcinoma H460cell lines, as well as squamous cell carcinomaSK-MES-1cell lines in vitro and in vivo. On this basis, we designed to identify EMT andcancer stem cell-like properties along with increased metastatic capacity of hybrids, in hopeof providing evidence for the design of targeting therapies to prevent the establishment ofdistant metastasis.Objective：To identify whether hybrids formed by fusion between human lung cancer cells andbone marrow-derived MSCs could become a resource for mesenchymal/cancer stem-likecells and to elucidate that fusion between MSCs and lung cancer cells is a potentialmechanism of the generation of invasive/metastatic lung cancer cells, which may providebasis for further study of how to prevent metastasis by interfering fusion event.Methods：(1) Direct co-culture experiments with MSCs-RFP and lung cancer cells-EGFP wereperformed in vitro. Spontaneously-formed heterotypic hybrids double stained with both RFPand EGFP were observed under a fluorescence microscope or a confocal microscope andselected by fluorescence-activated cell sorting. Mixture of gender-mismatched MSCs andHCC827cells were xenografted by subcutaneous injection into immunodeficient mice andheterotypic hybrids were co-cultured or evaluated by FISH of sex chromosomes.(2) The morphological changes of hybrids were observed under an inverted lightmicroscope. Immunofluorescence and QRT-PCR were used to examine the expression ofepithelial markers and mesenchymal markers, QRT-PCR were also used to examine theexpression of EMT-inducing transcription factors in heterotypic hybrids and in theirrespective parental lung cancer cells.(3) The expression of CD133on MSC-lung cancer hybrids was assessed using flowcytometry and QRT-PCR. QRT-PCR was also performed to examine the expression ofgenes responsible for regulating and maintaining the stem cell phenotype. Soft agar andpneumosphere assays were performed to examine the ability to form tumor spheres. Serialdilutions of cells were subcutaneous injected to NOD/SCID mice to evaluate thetumorigenic activity of hybrids in vivo. (4) Transwell migration assays and invasion assays were performed to evaluate themotility of cells. Serial dilutions of cells were injected intravenously into nude mice toexamine metastatic activity of hybrids in vivo.Results：(1) Spontaneously-formed heterotypic hybrids fused between MSCs and lung cancercells were detected both in vitro and in vivo.(2) After fusion with MSCs, lung cancer cells dispersed and assumed a fibroblast-likeappearance with two or more nuclei. Immunofluorescence staining revealed that heterotypichybrids exhibited a strong reduction in protein expression of epithelial markers andsignificantly increased expression of mesenchymal markers. QRT-PCR analyses showedthat the level of E-cadherin mRNA was markedly reduced, while expression of mRNAsencoding mesenchymal markers and EMT-inducing transcription factors were significantlyincreased in hybrids.(3) Flow cytometry analyses showed that hybrids over-expression of CD133.QRT-PCR analyses also revealed that expression of mRNAs encoding CD133and genesresponsible for regulating and maintaining the stem cell phenotype was greatly increased inhybrids. Heterotypic hybrids exhibit increased ability to form tumor spheres in vitro andenhanced tumorigenicity in vivo.(4) Hybrids acquire increased motility and invasiveness as detected by migrationassays and invasion assays in vitro and had an increased metastatic capacity to develop lungmetastases and liver metastases in vivo.Conclusion：(1) Spontaneous formed hybrids between MSCs and lung cancer cells can be foundboth in vitro and in vivo.(2) Cell fusion between lung cancer cells and MSCs give rise to highly malignantsubpopulations both capable of EMT and with properties of cancer stem cells (CSCs).(3) Formation of MSC–lung cancer cell hybrids is a potential mechanism of thegeneration of invasive/metastatic lung cancer cells.