Current strategies to determine tumor × normal (TN)-hybrid cells among human cancer cells include the detection of hematopoietic markers and other mesodermal markers on tumor cells or the presence of donor DNA in cancer samples from patients who had previously received an allogenic bone marrow transplant. By doing so, several studies have demonstrated that TN-hybrid cells could be found in human cancers. However, a prerequisite of this cell fusion search strategy is that such markers are stably expressed by TN-hybrid cells over time. However, cell fusion is a potent inducer of genomic instability, and TN-hybrid cells may lose these cell fusion markers, thereby becoming indistinguishable from nonfused tumor cells. In addition, hybrid cells can evolve from homotypic fusion events between tumor cells or from heterotypic fusion events between tumor cells and normal cells possessing similar markers, which would also be indistinguishable from nonfused tumor cells. Such indistinguishable or invisible hybrid cells will be referred to as dark matter hybrids, which cannot as yet be detected and quantified, but which contribute to tumor growth and progression.

BackgroundIn addition to physiological events such as fertilisation, placentation, osteoclastogenesis, or tissue regeneration/wound healing, cell fusion is involved in pathophysiological conditions such as cancer. Cell fusion, which applies to both the proteins and conditions that induce the merging of two or more cells, is not a fully understood process. Inflammation/pro-inflammatory cytokines might be a positive trigger for cell fusion. Using a Cre-LoxP-based cell fusion assay we demonstrated that the fusion between human M13SV1-Cre breast epithelial cells and human MDA-MB-435-pFDR1 cancer cells was induced by the pro-inflammatory cytokine tumour necrosis factor-α (TNF-α).MethodsThe gene expression profile of the cells in the presence of TNF-α and under normoxic and hypoxic conditions was analysed by cDNA microarray analysis. cDNA microarray data were verified by qPCR, PCR, Western blot and zymography. Quantification of cell fusion events was determined by flow cytometry. Proteins of interest were either blocked or knocked-down using a specific inhibitor, siRNA or a blocking antibody.ResultsThe data showed an up-regulation of various genes, including claudin-1 (CLDN1), ICAM1, CCL2 and MMP9 in M13SV1-Cre and/or MDA-MB-435-pFDR1 cells. Inhibition of these proteins using a blocking ICAM1 antibody, CLDN1 siRNA or an MMP9 inhibitor showed that only the blockage of MMP9 was correlated with a decreased fusion rate of the cells. Likewise, the tetracycline-based antibiotic minocycline, which exhibits anti-inflammatory properties, was also effective in both inhibiting the TNF-α-induced MMP9 expression in M13SV1-Cre cells and blocking the TNF-α-induced fusion frequency of human M13SV1-Cre breast epithelial cells and human MDA-MB-435-pFDR1 cancer cells.ConclusionsThe matrix metalloproteinase-9 (MMP9) is most likely involved in the TNF-α-mediated fusion of human M13SV1-Cre breast epithelial cells and human MDA-MB-435-pFDR1 cancer cells. Likewise, our data indicate that the tetracycline-based antibiotic minocycline might exhibit anti-fusogenic properties because it inhibits a cell fusion-related mechanism.Electronic supplementary materialThe online version of this article (10.1186/s12964-018-0226-1) contains supplementary material, which is available to authorized users.

Background To date, several studies have confirmed that driving forces of the inflammatory tumour microenvironment trigger spontaneous cancer cell fusion. However, less is known about the underlying factors and mechanisms that facilitate inflammation-induced cell fusion of a cancer cell with a normal cell. Recently, we demonstrated that minocycline, a tetracycline antibiotic, successfully inhibited the TNF-α-induced fusion of MDA-MB-435 cancer cells with M13SV1 breast epithelial cells. Here, we investigated how minocycline interferes with the TNF-α induced signal transduction pathway. Methods A Cre-LoxP recombination system was used to quantify the fusion of MDA-MB-435-pFDR1 cancer cells and M13SV1-Cre breast epithelial cells. The impact of minocycline on the TNF-α signalling pathway was determined by western blotting. The transcriptional activity of NF-κB was characterised by immunocytochemistry, western blot and ChIP analyses. An NF-κB-luciferase reporter assay was indicative of NF-κB activity. Results Minocycline treatment successfully inhibited the TNFR1-TRAF2 interaction in both cell types, while minocycline abrogated the phosphorylation of IκBα and NF-κB-p65 to suppress nuclear NF-κB and its promotor activity only in M13SV1-Cre cells, which attenuated the expression of MMP9 and ICAM1. In MDA-MB-435-pFDR1 cells, minocycline increased the activity of NF-κB, leading to greater nuclear accumulation of NF-κB-p65, thus increasing promoter activity to stimulate the expression of ICAM1. Even though TNF-α also activated all MAPKs (ERK1/2, p38 and JNK), minocycline differentially affected these kinases to either inhibit or stimulate their activation. Moreover, SRC activation was analysed as an upstream activator of MAPKs, but no activation by TNF-α was revealed. The addition of several specific inhibitors that block the activation of SRC, MAPKs, AP-1 and NF-κB confirmed that only NF-κB inhibition was successful in inhibiting the TNF-α-induced cell fusion process. Conclusion Minocycline is a potent inhibitor in the TNF-α-induced cell fusion process by targeting the NF-κB pathway. Thus, minocycline prevented NF-κB activation and nuclear translocation to abolish the target-gene expression of MMP9 and ICAM1 in M13SV1-Cre cells, resulting in reduced cell fusion frequency.

The biological phenomenon of cell fusion plays a crucial role in several physiological processes, including wound healing and tissue regeneration. Here, it is assumed that bone marrow-derived stem cells (BMSCs) could adopt the specific properties of a different organ by cell fusion, thereby restoring organ function. Cell fusion first results in the production of bi- or multinucleated hybrid cells, which either remain as heterokaryons or undergo ploidy reduction/heterokaryon-to-synkaryon transition (HST), thereby giving rise to mononucleated daughter cells. This process is characterized by a merging of the chromosomes from the previously discrete nuclei and their subsequent random segregation into daughter cells. Due to extra centrosomes concomitant with multipolar spindles, the ploidy reduction/HST could also be associated with chromosome missegregation and, hence, induction of aneuploidy, genomic instability, and even putative chromothripsis. However, while the majority of such hybrids die or become senescent, aneuploidy and genomic instability appear to be tolerated in hepatocytes, possibly for stress-related adaption processes. Likewise, cell fusion-induced aneuploidy and genomic instability could also lead to a malignant conversion of hybrid cells. This can occur during tissue regeneration mediated by BMSC fusion in chronically inflamed tissue, which is a cell fusion-friendly environment, but is also enriched for mutagenic reactive oxygen and nitrogen species.

The phenomenon of cancer cell–cell fusion is commonly associated with the origin of more malignant tumor cells exhibiting novel properties, such as increased drug resistance or an enhanced metastatic capacity. However, the whole process of cell–cell fusion is still not well understood and seems to be rather inefficient since only a certain number of (cancer) cells are capable of fusing and only a rather small population of fused tumor hybrids will survive at all. The low survivability of tumor hybrids is attributed to post-fusion processes, which are characterized by the random segregation of mixed parental chromosomes, the induction of aneuploidy and further random chromosomal aberrations and genetic/epigenetic alterations in daughter cells. As post-fusion processes also run in a unique manner in surviving tumor hybrids, the occurrence of novel properties could thus also be a random event, whereby it might be speculated that the tumor microenvironment and its spatial habitats could direct evolving tumor hybrids towards a specific phenotype.

Cell fusion is a well-known, but still scarcely understood biological phenomenon, which might play a role in cancer initiation, progression and formation of metastases. Although the merging of two (cancer) cells appears simple, the entire process is highly complex, energy-dependent and tightly regulated. Among cell fusion-inducing and -regulating factors, so-called fusogens have been identified as a specific type of proteins that are indispensable for overcoming fusion-associated energetic barriers and final merging of plasma membranes. About 8% of the human genome is of retroviral origin and some well-known fusogens, such as syncytin-1, are expressed by human (cancer) cells. Likewise, enveloped viruses can enable and facilitate cell fusion due to evolutionarily optimized fusogens, and are also capable to induce bi- and multinucleation underlining their fusion capacity. Moreover, multinucleated giant cancer cells have been found in tumors derived from oncogenic viruses. Accordingly, a potential correlation between viruses and fusogens of human endogenous retroviral origin in cancer cell fusion will be summarized in this review.

The aim of this study was to investigate the impact of cross-linked hyaluronic acid on osteoblast-like cells seeded on top of two collagen substrates, native porcine pericardium membrane (substrate A) and ribose cross-linked collagen membranes (substrate B), in an air-lift model. Substrates A or B, saturated with three hyaluronic acid concentrations, served as membranes for SAOS-2 cells seeded on top. Cultivation followed for 7 and 14 days in the air-lift model. Controls used the same substrates without hyaluronic pre-treatment. Cells were harvested, and four (Runx2, BGLAP, IBSP, Cx43) different osteogenic differentiation markers were assessed by qPCR. Triplicated experiment outcomes were statistically analyzed (ANOVA, t-test; SPSS). Supplementary histologic analysis confirmed the cells’ vitality. After seven days, only few markers were overexpressed on both substrates. After 14 days, targeted genes were highly expressed on substrate A. The same substrate treated with 1:100 diluted xHyA disclosed statistically significant different expression level vs. substrate B (p = 0.032). Time (p = 0.0001), experimental condition as a function of time (p = 0.022), and substrate (p = 0.028) were statistically significant factors. Histological imaging demonstrated vitality and visualized nuclei. We conclude that the impact of hyaluronic acid resulted in a higher expression profile of SAOS-2 cells on substrate A compared to substrate B in an air-lift culture after two weeks.

death by apoptosis, necrosis and autophagy, in DNA damage and heat shock proteins/unfolded protein response. Results and discussions Viability tests indicated that cell death arise within 6 hours post-PDT, but massive cell death occurs more than 48 hours post-PDT. The singlet oxygen burst triggered an early activation of some protective mechanisms against oxidative stress (full signature of the cytoprotective NRF2), hypoxia (SERPINE1) and osmotic stress (HSPA4L). Apoptosis (TNFa mediated), necrosis, autophagy and DNA damage (DDIT3) responses were highlighted. Interestingly, a complex inflammatory network was triggered by PDT (CXCL8, TNF, IL1), possibly associated to oxidative stress through NF-kB. These protective responses could be responsible for the delayed massive death of cells subjected to PDT. Conclusion In response to PDT, glioblastoma and colon carcinoma cells developed in vitro various protective mechanisms against a complex web of stressors. Genes overexpressed in response to PDT might be candidates for co-therapies aiming to increase PDT efficacity. It seems reasonable that inhibition of endogenous NRF2-mediated antioxidant mechanisms could be an option for improving oxidative stress-mediated PDT.