Microscopic Analysis of Cell Fate Alteration Induced by Cell Fusion.

In mammals, differentiated cells generally do not de-differentiate nor undergo cell fate alterations. However, they can be experimentally guided toward a different lineage. Cell fusion involving two different cell types has long been used to study this process, as this method induces cell fate alterations within hours to days in a subpopulation of fused cells, as evidenced by changes in gene-expression profiles. Despite the robustness of this system, its use has been restricted by low fusion rates and difficulty in eliminating unfused populations, thereby compromising resolution. In this study, we address these limitations by isolating fused cells using antibody-conjugated beads. This approach enables the microscopic tracking of fused cells starting as early as 5 hours after fusion. By taking advantage of species-specific FISH probes, we show that a small population of fused cells resulting from the fusion of mouse ES and human B cells, expresses OCT4 from human nuclei at levels comparable to human induced pluripotent stem cells (iPSCs) as early as 25 hours after fusion. We also show that this response can vary depending on the fusion partner. Our study broadens the usage of the cell fusion system for comprehending the mechanisms underlying cell fate alterations. These findings hold promise for diverse fields, including regenerative medicine and cancer.

Tspan5 promotes the EMT process to regulate the syncytialization of trophoblast cells by activating Notch signalling.

Placental trophoblastic cells play important roles in placental development and fetal health. However, the mechanism of trophoblastic cell fusion is still not entirely clear. The level of Tspan5 in the embryo culture medium was detected using enzyme-linked immunosorbent assay (ELISA). Fusion of BeWo cells was observed by immunofluorescence. Cell fusion-related factors and EMT-related factors were identified by qRT-PCR and western blotting. Notch protein repressor DAPT was used to verify the role of Tspan5 in BeWo cells. The expression of Tspan5 was significantly increased in embryo culture medium. The fusion of BeWo cells was observed after treatment with forskolin (FSK). Cell fusion-related factors (i.e. ß-hCG and syncytin 1/2) and Tspan5 were significantly increased after FSK treatment. In addition, FSK treatment promoted EMT-related protein expression in BeWo cells. Knockdown of Tspan5 inhibited cell fusion and EMT-related protein levels. Notch-1 and Jagged-1 protein levels were significantly upregulated, and the EMT process was activated by overexpression of Tspan5 in FSK-treated BeWo cells. Interestingly, blocking the Notch pathway by the repressor DAPT had the opposite results. These results indicated that Tspan5 could promote the EMT process by activating the Notch pathway, thereby causing cell fusion. These findings contribute to a better understanding of trophoblast cell syncytialization and embryonic development. Tspan5 may be used as a therapeutic target for normal placental development.

Could the Human Endogenous Retrovirus-Derived Syncytialization Inhibitor, Suppressyn, Limit Heterotypic Cell Fusion Events in the Decidua?

Proper placental development relies on tightly regulated trophoblast differentiation and interaction with maternal cells. Human endogenous retroviruses (HERVs) play an integral role in modulating cell fusion events in the trophoblast cells of the developing placenta. Syncytin-1 (ERVW-1) and its receptor, solute-linked carrier family A member 5 (SLC1A5/ASCT2), promote fusion of cytotrophoblast (CTB) cells to generate the multi-nucleated syncytiotrophoblast (STB) layer which is in direct contact with maternal blood. Another HERV-derived protein known as Suppressyn (ERVH48-1/SUPYN) is implicated in anti-fusogenic events as it shares the common receptor with ERVW-1. Here, we explore primary tissue and publicly available datasets to determine the distribution of ERVW-1, ERVH48-1 and SLC1A5 expression at the maternal-fetal interface. While SLC1A5 is broadly expressed in placental and decidual cell types, ERVW-1 and ERVH48-1 are confined to trophoblast cell types. ERVH48-1 displays higher expression levels in CTB and extravillous trophoblast, than in STB, while ERVW-1 is generally highest in STB. We have demonstrated through gene targeting studies that suppressyn has the ability to prevent ERVW-1-induced fusion events in co-culture models of trophoblast cell/maternal endometrial cell interactions. These findings suggest that differential HERV expression is vital to control fusion and anti-fusogenic events in the placenta and consequently, any imbalance or dysregulation in HERV expression may contribute to adverse pregnancy outcomes.

Evolutionarily related small viral fusogens hijack distinct but modular actin nucleation pathways to drive cell-cell fusion.

Fusion-associated small transmembrane (FAST) proteins are a diverse family of nonstructural viral proteins. Once expressed on the plasma membrane of infected cells, they drive fusion with neighboring cells, increasing viral spread and pathogenicity. Unlike viral fusogens with tall ectodomains that pull two membranes together through conformational changes, FAST proteins have short fusogenic ectodomains that cannot bridge the intermembrane gap between neighboring cells. One orthoreovirus FAST protein, p14, has been shown to hijack the actin cytoskeleton to drive cell-cell fusion, but the actin adaptor-binding motif identified in p14 is not found in any other FAST protein. Here, we report that an evolutionarily divergent FAST protein, p22 from aquareovirus, also hijacks the actin cytoskeleton but does so through different adaptor proteins, Intersectin-1 and Cdc42, that trigger N-WASP-mediated branched actin assembly. We show that despite using different pathways, the cytoplasmic tail of p22 can replace that of p14 to create a potent chimeric fusogen, suggesting they are modular and play similar functional roles. When we directly couple p22 with the parallel filament nucleator formin instead of the branched actin nucleation promoting factor N-WASP, its ability to drive fusion is maintained, suggesting that localized mechanical pressure on the plasma membrane coupled to a membrane-disruptive ectodomain is sufficient to drive cell-cell fusion. This work points to a common biophysical strategy used by FAST proteins to push rather than pull membranes together to drive fusion, one that may be harnessed by other short fusogens responsible for physiological cell-cell fusion.

Use of cell fusion proteins to enhance adenoviral vector efficacy as an anti-cancer therapeutic.

Oncolytic viruses are designed to replicate in and kill cancer cells, and have shown tremendous promise in preclinical and clinical studies. Indeed, several oncolytic viruses are available to patients in a number of different countries around the world. However, most oncolytic viruses show a poor ability to spread throughout the tumor mass, frequently leading to only a partial response and regrowth of the tumor. One approach to improve spread of the viral effect throughout the tumor mass is to arm the oncolytic virus with a fusogenic protein. In this manner, a single infected cell can fuse with many adjacent uninfected cells, essentially amplifying the anti-tumor effects. In this review, we discuss the development and use of fusogenic proteins to enhance the efficacy of human adenovirus-based vectors for cancer therapy.

Endocannabinoid signaling impairs syncytialization: Using flow cytometry to evaluate forskolin-induced cell fusion.

Cytotrophoblast cells fuse to form the syncytiotrophoblast, the main structure responsible for the placenta's specialized functions. This complex process denominated syncytialization is fundamental for a correct pregnancy outcome. We observed that the endocannabinoid anandamide disrupts syncytialization employing traditional techniques and flow cytometry in BeWo cell line.

B7-1 and GM-CSF enhance the anti-tumor immune effect of DC-tumor fusion vaccine in the treatment of prostate cancer.

The treatment of castration-resistant prostate cancer (CRPC) is always a difficulty in the clinic. Most patients with localized tumor eventually develop CRPC, even if hormone therapy is initially effective. Increasing evidence shows immunotherapy has special advantages compared with traditional therapy in cancer treatment. In this study, we constructed the DC-PC-3 fusion vaccine with B7-1- and GM-CSF-specific modification, and studied its ability to stimulate specific immune response and anti-tumor effect in vitro. The results showed that fusion of DC and tumor cells can improve the expression of associated antigens of DCs. DC-tumor fusion vaccine can strongly promote T cell proliferation and IFN-γ secretion and induce a significant tumor-specific cytotoxic T lymphocyte response. In addition, the B7-1/GM-CSF-modified fusion vaccine showed a more significant anti-tumor effect and greater ability to stimulate the immune response than that without specific modification in vitro. Thus, GM-CSF/B7-1-modified fusion vaccine might be used as a potential therapy strategy for prostate cancer.

Letrozole Suppresses the Fusion of Osteoclast Precursors through Inhibition of p38-Mediated DC-STAMP Pathway.

Letrozole is a reversible nonsteroidal aromatase inhibitor that is widely used in postmenopausal breast cancer patients. It is well established that letrozole decreases bone density owing to estrogen depletion; however, few studies have reported its direct effect on bone cells in vitro. Therefore, we investigated the effect of letrozole on bone metabolism, focusing on osteoclastogenesis. Letrozole did not affect the viability, proliferation, or migration of bone marrow-derived macrophages (BMMs); however, it reduced the multinucleation of immature osteoclasts and subsequent bone resorption in vitro. Overall, letrozole inhibited the expression of dendritic cell-specific transmembrane protein (DC-STAMP), tartrate-resistant acid phosphatase, calcitonin receptor, and cathepsin K. Among them, the reduced expression of DC-STAMP was the most prominent. However, this downregulation of DC-STAMP expression following letrozole treatment was not related to the inhibition of major osteoclastogenesis pathways, such as the nuclear factor-κB (NF-κB), c-Fos, and nuclear factor of activated T cell c1 (NFATc1) pathways, but was attributed to the inhibition of p38, which is known to reside upstream of DC-STAMP expression. Notably, the anti-osteoclastogenic effect of letrozole was abolished following treatment with the p38 activator anisomycin. Contrary to our expectations, these results strongly suggest a previously unknown anti-osteoclastogenic activity of letrozole, mediated by the downregulation of the p38/DC-STAMP pathway.

Altered Tumor Plasticity after Different Cancer Cell Fusions with MSC.

While cell fusion demonstrates an important pathway during tissue development and regeneration of distinct organs, this process can also contribute to pathophysiological phenotypes during tumor progression. Hybrid cell formation after heterofusion between cancer cells and various other cell types within the tumor microenvironment is observed in vitro and in vivo. In particular, mesenchymal stroma/stem-like cells (MSC) perform diverse levels of communication with cancer cells by exhibiting anti- and pro-tumorigenic effects. During these cellular interactions, MSC can eventually fuse with cancer cells. Thereby, the newly generated disparate hybrid populations display aneuploidy associated with chromosomal instability. Based upon a subsequent post-hybrid selection process (PHSP), fused cancer cells can undergo apoptosis/necroptosis, senescence, dormancy, or a proliferative state by acquisition of new properties. Consequently, PHSP-surviving hybrid cancer cells demonstrate altered functionalities within the tumor tissue. This is accompanied by changes in therapeutic responsiveness and a different metastatic behavior. Accordingly, enhanced tumor plasticity interferes with successful therapeutic interventions and aggravates patient prognoses. The present review article focusses on fusion of MSC with different human cancer cells, in particular breast cancer populations and resulting characteristics of various cancer hybrid cells. Moreover, some mechanisms of cancer cell fusion are discussed together with multiple PHSP pathways.

Novel enhanced GFP-positive congenic inbred strain establishment and application of tumor-bearing nude mouse model.

Transgenic GFP gene mice are widely used. Given the unique advantages of immunodeficient animals in the field of oncology research, we aim to establish a nude mouse inbred strain that stably expresses enhanced GFP (EGFP) for use in transplanted tumor microenvironment (TME) research. Female C57BL/6-Tg(CAG-EGFP) mice were backcrossed with male BALB/c nude mice for 11 generations. The genotype and phenotype of novel inbred strain Foxn1nu .B6-Tg(CAG-EGFP) were identified by biochemical loci detection, skin transplantation and flow cytometry. PCR and fluorescence spectrophotometry were performed to evaluate the relative expression of EGFP in different parts of the brain. Red fluorescence protein (RFP) gene was stably transfected into human glioma stem cells (GSC), SU3, which were then transplanted intracerebrally or ectopically into Foxn1nu .B6-Tg(CAG-EGFP) mice. Cell co-expression of EGFP and RFP in transplanted tissues was further analyzed with the Live Cell Imaging System (Cell'R, Olympus) and FISH. The inbred strain Foxn1nu .B6-Tg(CAG-EGFP) shows different levels of EGFP expression in brain tissue. The hematological and immune cells of the inbred strain mice were close to those of nude mice. EGFP was stably expressed in multiple sites of Foxn1nu .B6-Tg(CAG-EGFP) mice, including brain tissue. With the dual-fluorescence tracing transplanted tumor model, we found that SU3 induced host cell malignant transformation in TME, and tumor/host cell fusion. In conclusion, EGFP is differentially and widely expressed in brain tissue of Foxn1nu .B6-Tg(CAG-EGFP), which is an ideal model for TME investigation. With Foxn1nu .B6-Tg(CAG-EGFP) mice, our research demonstrated that host cell malignant transformation and tumor/host cell fusion play an important role in tumor progression.

The Histone Variant MacroH2A1 Regulates Key Genes for Myogenic Cell Fusion in a Splice-Isoform Dependent Manner.

MacroH2A histone variants have functions in differentiation, somatic cell reprogramming and cancer. However, at present, it is not clear how macroH2As affect gene regulation to exert these functions. We have parted from the initial observation that loss of total macroH2A1 led to a change in the morphology of murine myotubes differentiated ex vivo. The fusion of myoblasts to myotubes is a key process in embryonic myogenesis and highly relevant for muscle regeneration after acute or chronic injury. We have focused on this physiological process, to investigate the functions of the two splice isoforms of macroH2A1. Individual perturbation of the two isoforms in myotubes forming in vitro from myogenic C2C12 cells showed an opposing phenotype, with macroH2A1.1 enhancing, and macroH2A1.2 reducing, fusion. Differential regulation of a subset of fusion-related genes encoding components of the extracellular matrix and cell surface receptors for adhesion correlated with these phenotypes. We describe, for the first time, splice isoform-specific phenotypes for the histone variant macroH2A1 in a physiologic process and provide evidence for a novel underlying molecular mechanism of gene regulation.

High-Voltage Electrical Pulses in Oncology: Irreversible Electroporation, Electrochemotherapy, Gene Electrotransfer, Electrofusion, and Electroimmunotherapy.

This review summarizes the use of high-voltage electrical pulses (HVEPs) in clinical oncology to treat solid tumors with irreversible electroporation (IRE) and electrochemotherapy (ECT). HVEPs increase the membrane permeability of cells, a phenomenon known as electroporation. Unlike alternative ablative therapies, electroporation does not affect the structural integrity of surrounding tissue, thereby enabling tumors in the vicinity of vital structures to be treated. IRE uses HVEPs to cause cell death by inducing membrane disruption, and it is primarily used as a radical ablative therapy in the treatment of soft-tissue tumors in the liver, kidney, prostate, and pancreas. ECT uses HVEPs to transiently increase membrane permeability, enhancing cellular cytotoxic drug uptake in tumors. IRE and ECT show immunogenic effects that could be augmented when combined with immunomodulatory drugs, a combination therapy the authors term electroimmunotherapy. Additional electroporation-based technologies that may reach clinical importance, such as gene electrotransfer, electrofusion, and electroimmunotherapy, are concisely reviewed. HVEPs represent a substantial advancement in cancer research, and continued improvement and implementation of these presented technologies will require close collaboration between engineers, interventional radiologists, medical oncologists, and immuno-oncologists.

Femtosecond laser-induced blastomere fusion results in embryo tetraploidy by common metaphase plate formation.

The cell fusion is a widespread process, which takes place in many systems in vivo and in vitro. Fusion of cells is frequently related to tetraploidy, which can be found within natural physiological conditions, e.g., placentation, and in pathophysiological conditions, such as cancer and early pregnancy failure in humans. Here we investigate the mechanism of tetraploidization with help of femtosecond laser-induced mouse blastomere fusion by the means of Hoechst staining, GFP, BODIPY dyes and fluorescent species generated intracellularly by a femtosecond laser. We establish diffusive mixing of cytosol, whereas the large components of a cytoplasm (organelles, cytoskeleton) are poorly diffusible and are not completely mixed after cell fusion and a subsequent division. We show that mechanisms which are responsible for the formation of a common metaphase plate triggered tetraploidization in fused mouse embryos and could be a significant factor in polyploidy formation in vivo. Thus, our results suggest that microtubules play a critical role in tetraploidization.

Cancer cell's neuroendocrine feature can be acquired through cell-cell fusion during cancer-neural stem cell interaction.

Advanced and therapy-resistant prostate tumors often display neural or neuroendocrine behavior. We assessed the consequences of prostate cancer cell interaction with neural cells, which are rich in the human prostate and resident of the prostate tumor. In 3-dimensional co-culture with neurospheres, red fluorescent human LNCaP cells formed agglomerates on the neurosphere surface. Upon induced neural differentiation, some red fluorescent cells showed morphology of fully differentiated neural cells, indicating fusion between the cancer and neural stem cells. These fusion hybrids survived for extended times in a quiescent state. A few eventually restarted cell division and propagated to form derivative hybrid progenies. Clones of the hybrid progenies were highly heterogeneous; most had lost prostatic and epithelial markers while some had acquired neural marker expression. These results indicate that cancer cells can fuse with bystander neural cells in the tumor microenvironment; and cancer cell fusion is a direct route to tumor cell heterogeneity.

Adipose-derived stem cells and cancer cells fuse to generate cancer stem cell-like cells with increased tumorigenicity.

Adipose-derived stem cells (ADSCs) are a type of mesenchymal stem cells isolated from adipose tissue and have the ability to differentiate into adipogenic, osteogenic, and chondrogenic lineages. Despite their great therapeutic potentials, previous studies showed that ADSCs could enhance the proliferation and metastatic potential of breast cancer cells (BCCs). In this study, we found that ADSCs fused with BCCs spontaneously, while breast cancer stem cell (CSC) markers CD44+ CD24-/low EpCAM+ were enriched in this fusion population. We further assessed the fusion hybrid by multicolor DNA FISH and mouse xenograft assays. Only single nucleus was observed in the fusion hybrid, confirming that it was a synkaryon. In vivo mouse xenograft assay indicated that the tumorigenic potential of the fusion hybrid was significantly higher than that of the parent tumorigenic triple-negative BCC line MDA-MB-231. We had compared the fusion efficiency between two BCC lines, the CD44-rich MDA-MB-231 and the CD44-poor MCF-7, with ADSCs. Interestingly, we found that the fusion efficiency was much higher between MDA-MB-231 and ADSCs, suggesting that a potential mechanism of cell fusion may lie in the dissimilarity between these two cell lines. The cell fusion efficiency was hampered by knocking down the CD44. Altogether, our findings suggest that CD44-mediated cell fusion could be a potential mechanism for generating CSCs.

Electro Cell Fusion for Hybridoma Production.

Once a good immune response has developed in an animal and an appropriate screening procedure has been developed, the construction of hybridomas is ready to begin. The electro cell fusion (electrofusion) method uses an electrical field in the form of short, intense pulses to increase the permeability of the membrane. The resulting local perforation of the cell membrane induces the cells to fuse, forming hybridomas. Electrofusion is accomplished in three steps: Prealignment of the cells (convergence and cell contact), membrane fusion, and postalignment (rounding off the fused cells). This method has been applied successfully to hybridoma production with higher efficiency than routine polyethylene glycol fusion, allowing production of more hybrid cells.

TRIM34 facilitates the formation of multinucleated giant cells by enhancing cell fusion and phagocytosis in epithelial cells.

Persistent microbial infection promotes the fusion of several kinds of somatic cells, such as macrophages and endothelial cells, leading to the formation of multinucleated giant cells (MGCs). However, the molecular mechanisms of MGCs formation are still poorly understood. By laser confocal microscope, we discovered that TRIM34 increased the efficiency of cell fusion in Human Embryonic Kidney cells (HEK293T). By means of DiD cell membrane probes, LysoTracker Deep Red or MitoTracker Deep Red staining, we also demonstrated that TRIM34 stimulated cell fusion in paraformaldehyde fixed or living HEK293T cells. Moreover, we discovered that the nuclei shapes of MGCs induced by TRIM34 were diversiform, such as horseshoe shape, ring like shape etc. Through 3D reconstruction of confocal z-stacks images, we found that TRIM34-EGFP proteins could form macromolecule aggregates in the central area of MGCs, while the nuclei were arranged in ring like shape and distributed around the plasma membrane. Cell fusion assay showed that cocultured TRIM34-EGFP+ cells and TRIM34-DsRed1+ cells could fuse to form MGCs. We speculate that the formation of MGCs can be divided into two phase: primary multinucleated cells (PMCs) and secondary multinucleated cells (SMCs). Firstly, TRIM34 induced fusion of multiple adjacent cells resulting in PMCs formation, and then PMCs were endowed with the capacity of phagocytosis and turned into SMCs. Collectively, these results suggest that TRIM34 proteins contribute to the formation of MGCs by promoting cell fusion and phagocytosis in epithelial cells.

Syncytin 1, CD9, and CD47 regulating cell fusion to form PGCCs associated with cAMP/PKA and JNK signaling pathway.

BACKGROUND: We have previously reported the formation of polyploid giant cancer cells (PGCCs) through endoreduplication or cell fusion after cobalt chloride (CoCl2 ) induction. Cell fusion plays an important role in development and disease. However, the underlying molecular mechanism concerning cell fusion in PGCCs formation and clinicopathological significances remains unclear. METHODS: We treat HCT116 and LoVo cell with CoCl2 and observed the cell fusion via fluorescent markers of different colors. Western blot and immunocytochemical staining were used to compare the expression and subcellular location of the fusion-related proteins syncytin 1, CD9, and CD47 along with PKA RIα, JNK1, and c-Jun between PGCCs and control cells from the HCT116 and LoVo cell lines. Moreover, 173 cases of colorectal tumor tissue samples were analyzed, including 47 cases of well-differentiated primary colorectal cancer (group I) and 5 cases of corresponding metastatic tumors (group II), 38 cases of moderately differentiated primary colorectal cancer (group III) and 14 cases of corresponding metastatic tumors (group IV), and 42 cases of poorly differentiated primary colorectal cancer (group V) and 27 cases of corresponding metastatic tumors (group VI). RESULTS: The expression of syncytin 1, CD9, and CD47 is higher in PGCCs than in control cells and they are located in the cytoplasm. The expression of PKA RIα and JNK1 decreased, and that of c-Jun increased in PGCCs. The syncytin 1 expression was significantly different between groups I and II (P = 0.000), groups III and IV (P = 0.000), groups V and VI (P = 0.029), groups I and III (P = 0.001), groups III and V (P = 0.000), and groups I, III, and V (P = 0.000). CONCLUSIONS: These data indicate that the cell fusion-related proteins syncytin 1, CD9, and CD47 may be involved in PGCC formation, and that cAMP/PKA and JNK signaling is likely to promote PGCC formation via the regulation of cell fusion processes.

Electrofusion by a bipolar pulsed electric field: Increased cell fusion efficiency for monoclonal antibody production.

The excessive cell death rate caused by electrofusion with unipolar pulses (UPs) has been a bottleneck to increasing cell fusion efficiency in monoclonal antibody technology. Several studies have confirmed that compared with UPs, bipolar pulses (BPs) with microsecond pulse widths can increase electropermeabilization while reducing cell death. Given these characteristics, BPs were used to increase cell fusion efficiency in this study. Cell staining and hybridoma culture experiments were performed using SP2/0 mouse myeloma cells and lymphocytes. Based on the equal energy principle, UPs and BPs were delivered to electrodes at a distance of 3.81 mm, with electric field intensities ranging from 2 kV/cm to 3 kV/cm and pulse duration of 40 µs for the UPs and 20-20 µs for the BPs. The results of cell staining experiments showed that cell fusion efficiency was 3-fold greater with BPs than with UPs. Similarly, the results of the hybridoma culture experiments showed that the hybridoma yields were 0.26‰ and 0.23‰ (2.5 kV/cm and 3 kV/cm, respectively) in the UP groups and increased to 0.46‰ and 0.35‰ in the BP groups. Taken together, the results show that the efficiency of heterologous cell fusion can be greatly increased if BPs are used instead of the commonly applied UPs. This study may provide a promising method for monoclonal antibody technology.