Effects of polystyrene nano- and microplastics on human breast epithelial cells and human breast cancer cells.

The continuous littering of the environment with plastic and the resulting nano- and microplastics produced from various processes are ever-increasing problems. These materials also affect humans, as the absorption and accumulation of nano- and microplastics and their effects on health have thus far been rarely researched, which also applies to cancer. In the present study, the absorption of different sizes of polystyrene (PS) nano- and microplastics (PS particles) into human breast epithelial cells and human breast cancer cells was investigated. Subsequently, how the proliferation, colony and mammosphere formation abilities, cell fusion and migration of the cells were influenced by the PS particles were investigated. Our data revealed granularity-, dose- and cell line-dependent absorption of the PS particles, with the highest absorption observed in the MDA-MB-231-DSP1-7 cells and the lowest in the M13SV1_Syn1-DSP8-11 cells. Neither the colony-forming ability nor the cell fusion activity increased with the addition of PS particles. In contrast, slight, partially significant stimulatory effects on both proliferation and cell migration were observed, although these effects depended on the particle quantity and size and the cell line used. In summary, PS particles are absorbed by human breast epithelial and human breast cancer cells and influence cells that may be associated with cancer progression.

How Much Do You Fuse? A Comparison of Cell Fusion Assays in a Breast Cancer Model.

Cell fusion is a biological process that is crucial for the development and homeostasis of different tissues, but it is also pathophysiologically associated with tumor progression and malignancy. The investigation of cell fusion processes is difficult because there is no standardized marker. Many studies therefore use different systems to observe and quantify cell fusion in vitro and in vivo. The comparability of the results must be critically questioned, because both the experimental procedure and the assays differ between studies. The comparability of the fluorescence-based fluorescence double reporter (FDR) and dual split protein (DSP) assay was investigated as part of this study, in which general conditions were kept largely constant. In order to be able to induce both a high and a low cell fusion rate, M13SV1 breast epithelial cells were modified with regard to the expression level of the fusogenic protein Syncytin-1 and its receptor ASCT2 and were co-cultivated for 72 h with different breast cancer cell lines. A high number of fused cells was found in co-cultures with Syncytin-1-overexpressing M13SV1 cells, but differences between the assays were also observed. This shows that the quantification of cell fusion events in particular is highly dependent on the assay selected, but the influence of fusogenic proteins can be visualized very well.

Altered Phenotypes of Breast Epithelial × Breast Cancer Hybrids after ZEB1 Knock-Out.

ZEB1 plays a pivotal role in epithelial-to-mesenchymal transition (EMT), (cancer) cell stemness and cancer therapy resistance. The M13HS tumor hybrids, which were derived from spontaneous fusion events between the M13SV1-EGFP-Neo breast epithelial cells and HS578T-Hyg breast cancer cells, express ZEB1 and exhibit prospective cancer stem cell properties. To explore a possible correlation between the ZEB1 and stemness/ EMT-related properties in M13HS tumor hybrids, ZEB1 was knocked-out by CRISPR/Cas9. Colony formation, mammosphere formation, cell migration, invasion assays, flow cytometry and Western blot analyses were performed for the characterization of ZEB1 knock-out cells. The ZEB1 knock-out in M13HS tumor cells was not correlated with the down-regulation of the EMT-related markers N-CADHERIN (CDH2) and VIMENTIN and up-regulation of miR-200c-3p. Nonetheless, both the colony formation and mammosphere formation capacities of the M13HS ZEB1 knock-out cells were markedly reduced. Interestingly, the M13HS-2 ZEB1-KO cells harbored a markedly higher fraction of ALDH1-positive cells. The Transwell/ Boyden chamber migration assay data indicated a reduced migratory activity of the M13HS ZEB1-knock-out tumor hybrids, whereas in scratch/ wound-healing assays only the M13SH-8 ZEB1-knock-out cells possessed a reduced locomotory activity. Similarly, only the M13HS-8 ZEB1-knock-out tumor hybrids showed a reduced invasion capacity. Although the ZEB1 knock-out resulted in only moderate phenotypic changes, our data support the role of ZEB1 in EMT and stemness.

Why do certain cancer cells alter functionality and fuse?

Cancer cell fusion represents a rare event. However, the surviving cancer hybrid cells after a post-hybrid selection process (PHSP) can overgrow other cancer cells by exhibiting a proliferation advantage and/or expression of cancer stem-like properties. Addition of new tumor properties during hetero-fusion of cancer cells e.g. with mesenchymal stroma-/stem-like cells (MSC) contribute to enhanced tumor plasticity via acquisition of new/altered functionalities. This provides new avenues for tumor development and metastatic behavior. Consequently, the present review article will also address the question as to whether cancer cell fusion represents a general and possibly evolutionary-conserved program or rather a random process?

Intrinsic signalling factors associated with cancer cell-cell fusion.

Cellular fusion e.g. between cancer cells and normal cells represents a stepwise process that is tightly regulated. During a pre-hybrid preparation program somatic cells and/or cancer cells are promoted to a pro-fusogenic state as a prerequisite to prepare a fusion process. A pro-fusogenic state requires significant changes including restructure of the cytoskeleton, e.g., by the formation of F-actin. Moreover, distinct plasma membrane lipids such as phosphatidylserine play an important role during cell fusion. In addition, the expression of distinct fusogenic factors such as syncytins and corresponding receptors are of fundamental importance to enable cellular mergers. Subsequent hybrid formation and fusion are followed by a post-hybrid selection process. Fusion among normal cells is important and often required during organismal development. Cancer cells fusion appears more rarely and is associated with the generation of new cancer hybrid cell populations. These cancer hybrid cells contribute to an elevated tumour plasticity by altered metastatic behaviour, changes in therapeutic and apoptotic responses, and even in the formation of cancer stem/ initiating cells. While many parts within this multi-step cascade are still poorly understood, this review article predominantly focusses on the intracellular necessities for fusion among cancer cells or with other cell populations of the tumour microenvironment. Video Abstract.

Extracellular Events Involved in Cancer Cell-Cell Fusion.

Fusion among different cell populations represents a rare process that is mediated by both intrinsic and extracellular events. Cellular hybrid formation is relayed by orchestrating tightly regulated signaling pathways that can involve both normal and neoplastic cells. Certain important cell merger processes are often required during distinct organismal and tissue development, including placenta and skeletal muscle. In a neoplastic environment, however, cancer cell fusion can generate new cancer hybrid cells. Following survival during a subsequent post-hybrid selection process (PHSP), the new cancer hybrid cells express different tumorigenic properties. These can include elevated proliferative capacity, increased metastatic potential, resistance to certain therapeutic compounds, and formation of cancer stem-like cells, all of which characterize significantly enhanced tumor plasticity. However, many parts within this multi-step cascade are still poorly understood. Aside from intrinsic factors, cell fusion is particularly affected by extracellular conditions, including an inflammatory microenvironment, viruses, pH and ionic stress, hypoxia, and exosome signaling. Accordingly, the present review article will primarily highlight the influence of extracellular events that contribute to cell fusion in normal and tumorigenic tissues.

Impact of Cross-Linked Hyaluronic Acid on Osteogenic Differentiation of SAOS-2 Cells in an Air-Lift Model.

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.

Human Gingival Fibroblast Adhesion and Proliferation on Hydroxyapatite-Coated Zirconia Abutment Surfaces.

Applying antibacterial coatings to dental implant materials seems reasonable but can have negative influences on desired cell adhesion and healing. In this study, zirconia abutment specimens interacting with gingival tissue were used. The aim was to compare the influence of machined or coated zirconia surfaces on the adhesion and proliferation of human gingival fibroblasts (HGF-1). Surface modifications were performed using atmospheric plasma coating with hydroxyapatite, zinc, and copper. Zirconia specimens were divided into four groups: hydroxyapatite, hydroxyapatite with zinc oxide (ZnO), hydroxyapatite with copper (Cu), and an untreated machined surface. After the characterization of the surface conditions, the morphology of adhered HGF-1 was determined by fluorescence staining and subjected to statistical evaluation. The visual analysis of cell morphology by SEM showed flat, polygonal, and largely adherent fibroblast cells in the untreated group, while round to partially flat cells were recorded in the groups with hydroxyapatite, hydroxyapatite + ZnO, and hydroxyapatite + Cu. The cell membranes in the hydroxyapatite + ZnO and hydroxyapatite + Cu groups appeared porous. The results show that HGF-1 adhere and proliferate well on machined zirconia, while plasma coating with hydroxyapatite or hydroxyapatite mixtures does not lead to increased adhesion or proliferation.

Generation of Cancer Stem/Initiating Cells by Cell-Cell Fusion.

CS/ICs have raised great expectations in cancer research and therapy, as eradication of this key cancer cell type is expected to lead to a complete cure. Unfortunately, the biology of CS/ICs is rather complex, since no common CS/IC marker has yet been identified. Certain surface markers or ALDH1 expression can be used for detection, but some studies indicated that cancer cells exhibit a certain plasticity, so CS/ICs can also arise from non-CS/ICs. Another problem is intratumoral heterogeneity, from which it can be inferred that different CS/IC subclones must be present in the tumor. Cell-cell fusion between cancer cells and normal cells, such as macrophages and stem cells, has been associated with the generation of tumor hybrids that can exhibit novel properties, such as an enhanced metastatic capacity and even CS/IC properties. Moreover, cell-cell fusion is a complex process in which parental chromosomes are mixed and randomly distributed among daughter cells, resulting in multiple, unique tumor hybrids. These, if they have CS/IC properties, may contribute to the heterogeneity of the CS/IC pool. In this review, we will discuss whether cell-cell fusion could also lead to the origin of different CS/ICs that may expand the overall CS/IC pool in a primary tumor.

A Human In Vitro Model to Study Adenoviral Receptors and Virus Cell Interactions.

To develop adenoviral cell- or tissue-specific gene delivery, understanding of the infection mechanisms of adenoviruses is crucial. Several adenoviral attachment proteins such as CD46, CAR and sialic acid have been identified and studied. However, most receptor studies were performed on non-human cells. Combining our reporter gene-tagged adenovirus library with an in vitro human gene knockout model, we performed a systematic analysis of receptor usage comparing different adenoviruses side-by-side. The CRISPR/Cas9 system was used to knockout CD46 and CAR in the human lung epithelial carcinoma cell line A549. Knockout cells were infected with 22 luciferase-expressing adenoviruses derived from adenovirus species B, C, D and E. HAdV-B16, -B21 and -B50 from species B1 as well as HAdV-B34 and -B35 were found to be CD46-dependent. HAdV-C5 and HAdV-E4 from species E were found to be CAR-dependent. Regarding cell entry of HAdV-B3 and -B14 and all species D viruses, both CAR and CD46 play a role, and here, other receptors or attachment structures may also be important since transductions were reduced but not completely inhibited. The established human knockout cell model enables the identification of the most applicable adenovirus types for gene therapy and to further understand adenovirus infection biology.

Cell-Cell Fusion Mediated by Viruses and HERV-Derived Fusogens in Cancer Initiation and Progression.

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 Role of MSCs and Cell Fusion in Tissue Regeneration.

Regenerative medicine is concerned with the investigation of therapeutic agents that can be used to promote the process of regeneration after injury or in different diseases. Mesenchymal stem/stromal cells (MSCs) and their secretome-including extracellular vesicles (EVs) are of great interest, due to their role in tissue regeneration, immunomodulatory capacity and low immunogenicity. So far, clinical studies are not very conclusive as they show conflicting efficacies regarding the use of MSCs. An additional process possibly involved in regeneration might be cell fusion. This process occurs in both a physiological and a pathophysiological context and can be affected by immune response due to inflammation. In this review the role of MSCs and cell fusion in tissue regeneration is discussed.

Cancer Cell Fusion and Post-Hybrid Selection Process (PHSP).

Fusion of cancer cells either with other cancer cells (homotypic fusion) in local vicinity of the tumor tissue or with other cell types (e.g., macrophages, cancer-associated fibroblasts (CAFs), mesenchymal stromal-/stem-like cells (MSC)) (heterotypic fusion) represents a rare event. Accordingly, the clinical relevance of cancer-cell fusion events appears questionable. However, enhanced tumor growth and/or development of certain metastases can originate from cancer-cell fusion. Formation of hybrid cells after cancer-cell fusion requires a post-hybrid selection process (PHSP) to cope with genomic instability of the parental nuclei and reorganize survival and metabolic functionality. The present review dissects mechanisms that contribute to a PHSP and resulting functional alterations of the cancer hybrids. Based upon new properties of cancer hybrid cells, the arising clinical consequences of the subsequent tumor heterogeneity after cancer-cell fusion represent a major therapeutic challenge. However, cellular partners during cancer-cell fusion such as MSC within the tumor microenvironment or MSC-derived exosomes may provide a suitable vehicle to specifically address and deliver anti-tumor cargo to cancer cells.

Hybrid Formation and Fusion of Cancer Cells In Vitro and In Vivo.

The generation of cancer hybrid cells by intra-tumoral cell fusion opens new avenues for tumor plasticity to develop cancer stem cells with altered properties, to escape from immune surveillance, to change metastatic behavior, and to broaden drug responsiveness/resistance. Genomic instability and chromosomal rearrangements in bi- or multinucleated aneuploid cancer hybrid cells contribute to these new functions. However, the significance of cell fusion in tumorigenesis is controversial with respect to the low frequency of cancer cell fusion events and a clonal advantage of surviving cancer hybrid cells following a post-hybrid selection process. This review highlights alternative processes of cancer hybrid cell development such as entosis, emperipolesis, cannibalism, therapy-induced polyploidization/endoreduplication, horizontal or lateral gene transfer, and focusses on the predominant mechanisms of cell fusion. Based upon new properties of cancer hybrid cells the arising clinical consequences of the subsequent tumor heterogeneity after cancer cell fusion represent a major therapeutic challenge.

Cell-Cell Fusion and the Roads to Novel Properties of Tumor Hybrid Cells.

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.

The phospholipase D inhibitor FIPI potently blocks EGF-induced calcium signaling in human breast cancer cells.

BACKGROUND: Phosphotyrosine kinase (PTK)-mediated phospholipase C-γ1 (PLC-γ1) signaling plays a crucial role in the release of the universal second messenger calcium from intracellular stores, which is mandatory for several cellular processes, including cell migration. However, PLC-γ1 could also be activated in a PTK-independent manner by phospholipase D (PLD)-derived phosphatidic acid (PA). Because both higher PLD expression levels and PLD activity have also been associated with breast cancer cell invasion and migration, we wondered whether there might be a link between PLD and PLC-γ1, which was investigated in this study. MATERIALS: MDA-MB-468-NEO (EGFR positive) and MDA-MB-468-HER2 (EGFR and HER2 positive) human breast cancer cells were used in this study. The migratory behavior of the cells in the presence of epidermal growth factor (EGF) and the PLD inhibitor 5-fluoro-2-indolyl-des-chlorohalopemide (FIPI) was analyzed using the 3D collagen matrix migration assay. Changes in cytosolic calcium levels in the presence of EGF, FIPI and Sig-1R agonists and antagonists as well as in PLD1 siRNA knockdown cells were determined by flow cytometry. Western blot analyses were performed to determine the basal expression levels and phosphorylation patterns of EGFR, HER2, AKT, MAPKp42/44, PLC-γ1 and Sig-1R. RESULTS: The EGF-induced migration of MDA-MB-468-NEO and MDA-MB-468-HER2 cells was significantly impaired by FIPI. Likewise, FIPI also significantly abolished EGF-induced calcium release in both cell lines. However, neither the expression levels nor the phosphorylation patterns of EGFR, HER2, AKT, MAPKp42/44 and PLC-γ1 were markedly changed by FIPI. Knockdown of PLD1 expression by siRNA also significantly impaired EGF-induced calcium release in both cell lines. Targeting Sig-1R, which interacts with IP3R, with the antagonist BD1047 also abrogated EGF-induced calcium release. However, EGF-induced calcium release was also impaired if cells were treated with the Sig-1R agonists PRE084 and PPBP maleate. CONCLUSION: In summary, blocking PLD activity with the specific inhibitor FIPI or knocking down PDL1 expression by siRNA significantly impaired EGF-induced calcium release in MDA-MB-468-NEO and MDA-MB-468-HER2 cells, likely indicating a connection between PLD activity and PLC-γ1-mediated calcium signaling. However, how PLD activity interferes with the release of calcium from intracellular stores remains unclear. Video Abstract.

Cell Fusion of Mesenchymal Stem/Stromal Cells and Breast Cancer Cells Leads to the Formation of Hybrid Cells Exhibiting Diverse and Individual (Stem Cell) Characteristics.

Cancer is one of the most common diseases worldwide, and treatment bears many challenges such as drug and radioresistance and formation of metastases. These difficulties are due to tumor heterogeneity, which has many origins. One may be cell fusion, a process that is relevant in both physiological (e.g., wound healing) and pathophysiological (cancer and viral infection) processes. In this study, we examined if cell fusion between mesenchymal stem/stromal cells (MSCs) and breast cancer (BC) cells occurs and if newly generated hybrid cells may exhibit cancer stem/initiating cell (CS/IC) characteristics. Therefore, several methods such as mammosphere assay, AldeRed assay, flow cytometry (CD24, CD44, CD104) and Western blot analysis (of epithelial to mesenchymal transition markers such as SNAIL, SLUG and Twist) were applied. In short, four different hybrid clones, verified by short tandem repeat (STR) analysis, were analyzed; each expressed an individual phenotype that seemed not to be explicitly related to either a more stem cell or cancer cell phenotype. These results show that cancer cells and MSCs are able to fuse spontaneously in vitro, thereby giving rise to hybrid cells with new properties, which likely indicate that cell fusion may be a trigger for tumor heterogeneity.

Reversible Growth-Arrest of a Spontaneously-Derived Human MSC-Like Cell Line.

Life cycle limitation hampers the production of high amounts of primary human mesenchymal stroma-/stem-like cells (MSC) and limits cell source reproducibility for clinical applications. The characterization of permanently growing MSC544 revealed some differentiation capacity and the simultaneous presence of known MSC markers CD73, CD90, and CD105 even after continuous long-term culture for more than one year and 32 passages. The expression of CD13, CD29, CD44, and CD166 were identified as further surface proteins, all of which were also simultaneously detectable in various other types of primary MSC populations derived from the umbilical cord, bone marrow, and placenta suggesting MSC-like properties in the cell line. Proliferating steady state MSC544 exhibited immune-modulatory activity similar to a subpopulation of long-term growth-inhibited MSC544 after 189d of continuous culture in confluency. This confluent connective cell layer with fibroblast-like morphology can spontaneously contract and the generated space is subsequently occupied by new cells with regained proliferative capacity. Accordingly, the confluent and senescence-associated beta-galactosidase-positive MSC544 culture with about 95% G0/G1 growth-arrest resumed re-entry into the proliferative cell cycle within 3d after sub-confluent culture. The MSC544 cells remained viable during confluency and throughout this transition which was accompanied by marked changes in the release of proteins. Thus, expression of proliferation-associated genes was down-modulated in confluent MSC544 and re-expressed following sub-confluent conditions whilst telomerase (hTERT) transcripts remained detectable at similar levels in both, confluent growth-arrested and proliferating MSC544. Together with the capability of connective cell layer formation for potential therapeutic approaches, MSC544 provide a long term reproducible human cell source with constant properties.

Spectrum-Wide Exploration of Human Adenoviruses for Breast Cancer Therapy.

Oncolytic adenoviruses (Ads) are promising tools for cancer therapeutics. However, most Ad-based therapies utilize Ad type 5 (Ad5), which displays unsatisfying efficiency in clinical trials, partly due to the low expression levels of its primary coxsackievirus and adenovirus receptor (CAR) on tumor cells. Since the efficacy of virotherapy strongly relies on efficient transduction of targeted tumor cells, initial screening of a broad range of viral agents to identify the most effective vehicles is essential. Using a novel Ad library consisting of numerous human Ads representing known Ad species, we evaluated the transduction efficiencies in four breast cancer (BC) cell lines. For each cell line over 20 Ad types were screened in a high-throughput manner based on reporter assays. Ad types featuring high transduction efficiencies were further investigated with respect to the percentage of transgene-positive cells and efficiencies of cellular entry in individual cell lines. Additionally, oncolytic assay was performed to test tumor cell lysis efficacy of selected Ad types. We found that all analyzed BC cell lines show low expression levels of CAR, while alternative receptors such as CD46, DSG-2, and integrins were also detected. We identified Ad3, Ad35, Ad37, and Ad52 as potential candidates for BC virotherapy.

Comparison of hybrid clones derived from human breast epithelial cells and three different cancer cell lines regarding in vitro cancer stem/ initiating cell properties.

BACKGROUND: Several physiological (fertilization, placentation, wound healing) and pathophysiological processes (infection with enveloped viruses, cancer) depend on cell fusion. In cancer it was postulated that the fusion of cancer cells with normal cells such as macrophages or stem cells may not only give rise to hybrid cells exhibiting novel properties, such as an increased metastatic capacity and drug resistance, but possibly also cancer stem/ initiating cell properties. Hence, hybrid clone cells (M13HS, M13MDA435 and M13MDA231) that were derived from spontaneous fusion events of human M13SV1-EGFP-Neo breast epithelial cells and HS578T-Hyg, MDA-MB-435-Hyg and MDA-MB-231-Hyg cancer cells were investigated regarding potential in vitro cancer stem/ initiating cell properties. METHODS: CD44/CD24 expression pattern and ALDH1 activity of parental cells and hybrid clones was determined by flow cytometry. A colony formation and mammosphere formation assay was applied to determine the cells' capability to form colonies and mammospheres. Sox9, Slug and Snail expression levels were determined by Western blot analysis. RESULTS: Flow cytometry revealed that all hybrid clone cells were CD44+/CD24-/low, but differed markedly among each other regarding ALDH1 activity. Likewise, each hybrid clone possessed a unique colony formation and mammosphere capacity as well as unique Snail, Slug and Sox9 expression patterns. Nonetheless, comparison of hybrid clones revealed that M13HS hybrids exhibited more in vitro cancer stem/ initiating cell properties than M13MDA231 and M13MDA435 hybrids, such as more ALDH1 positive cells or an increased capacity to form colonies and mammospheres. CONCLUSION: The fate whether cancer stem/ initiating cells may originate from cell fusion events likely depends on the specific characteristics of the parental cells.