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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. [ABSTRACT FROM AUTHOR]

There is a growing list of data indicating that cancer (stem) cells could functionally adapt foreign tissue features, such as endothelial-like cells or neuroendocrine cells, express lineage markers or could differentiate into various lineages in response to appropriate differentiation criteria. The finding that cancer (stem) cells may possess some kind of differentiation capacity poses the question whether this might be an inherent or acquired property. Cancer stem cells share stem cell characteristics and may thus possess an inherent differentiation capacity enabling the cells to respond to various differentiation stimuli. Considering the plasticity of cancer (stem) cells, even non-tumorigenic (and putatively non-differentiable) tumor cells could give rise to tumorigenic tumor stem cells, exhibiting stem cell characteristics including an inherent differentiation capacity. On the contrary, cancer (stem) cells may have acquired differentiation capacity as a consequence of a previous cell fusion event with cell types exhibiting differentiation potential and being fusogenic, such as macrophages or stem cells. Of pivotal interest in a tumor context are macrophages, which chiefly foster the chronically inflamed tumor microenvironment. Because chronically inflamed tissue is a well-known trigger for cell fusion and both macrophages and stem cells are highly fusogenic we conclude that cell fusion events between these cell types and cancer (stem) cells should frequently occur, thereby giving rise to hybrid cells exhibiting not only novel properties, like an enhanced metastatogenic phenotype, but also parental characteristics, such as differentiation capacity. Conceivably, the combination of both properties might be advantageous for metastasizing cancer (stem) cells to adapt better and faster to a foreign organ tissue environment. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

The biological phenomenon of cell fusion has been linked to tumor progression because several data provided evidence that fusion of tumor cells and normal cells gave rise to hybrid cell lines exhibiting novel properties, such as increased metastatogenic capacity and an enhanced drug resistance. Here we investigated M13HS hybrid cell lines, derived from spontaneous fusion events between M13SV1-EGFP-Neo breast epithelial cells exhibiting stem cell characteristics and HS578T-Hyg breast cancer cells, concerning CCL21/CCR7 signaling. Western Blot analysis showed that all cell lines varied in their CCR7 expression levels as well as differed in the induction and kinetics of CCR7 specific signal transduction cascades. Flow cytometry-based calcium measurements revealed that a CCL21 induced calcium influx was solely detected in M13HS hybrid cell lines. Cell migration demonstrated that only M13HS hybrid cell lines, but not parental derivatives, responded to CCL21 stimulation with an increased migratory activity. Knockdown of CCR7 expression by siRNA completely abrogated the CCL21 induced migration of hybrid cell lines indicating the necessity of CCL21/CCR7 signaling. Because the CCL21/CCR7 axis has been linked to metastatic spreading of breast cancer to lymph nodes we conclude from our data that cell fusion could be a mechanism explaining the origin of metastatic cancer (hybrid) cells. [ABSTRACT FROM AUTHOR]

Simple Summary: Aberrant fusion of somatic cells or fusion of neoplastic cells may represent one fundamental process among others eventually promoting tumor development. Fusion events are rare, and cancer hybrid cells are further processed in a post-hybrid selection process (PHSP). Although the PHSP-surviving cancer hybrid cells represent a small minority within the tumor tissue, their changed properties may provide a proliferation advantage, eventually overgrowing other cancer cells. These new properties can include cancer stem-cell features such as self-renewal, immune escape, and chemotherapy/necroptosis resistance. Moreover, PHSP-derived cancer hybrid cells can undergo tumor dormancy or contribute to epithelial-mesenchymal transition and enhanced formation of distal organ or tissue metastases. Accordingly, detection of cancer-cell fusions in vivo in a patient's tumor tissues is challenging, and subsequent therapeutic interventions against these processes remain to be elucidated. 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Cell fusion is a cell-biological phenomenon, which was originally described as an event of development and homeostasis. Moreover, recent discoveries give rise to the theory that fusion events between stem cells and tumor cells could act as malignant and crucial originators in cancer stem cell (CSC) generation, besides other theories such as CSCs deriving from tissue stem cells that have accumulated genetic aberrations or CSCs deriving from differentiated progenitor cells that have regained self- renewing capacity. By now CSCs are known as cancer-initiating cells with characteristic features of stem cells, such as self-renewing, differentiation, regeneration of tissues at low cell numbers and drug resistance. This rare population of cancer-initiating cells attracts attention as a target in cancer therapies and, therefore, there is an urgent need for further investigation on CSCs is present. After 24 h co-cultivation of M13SV1 enhanced green fluorescent protein (EGFP)-neo breast stem cells and invasive HS578T-Hyg breast cancer cells under selective conditions, cell clones that emerged from spontaneous fusion events were isolated and cultivated separately. Short tandem repeat analysis of hybrid cells showed an overlap of the parental alleles, indicating that hybrid cells have been originated from real cell fusion events. Despite some varieties concerning the data of the further hybrid cell characterization due to different genetic profile among them, one can detect clear tendencies of similarity between all hybrid cell lines compared with their parental cell lines. XTT-proliferation assays, for example, show a significantly higher proliferation rate of the hybrid cell lines compared with their parental cell lines. Dependent on the hybrid cell line a 5-10-times higher proliferation rate could be detected in comparison with the breast stem cell line M13SV1 EGFP-neo, whereas, in contrast to the breast cancer cell line HS578T Hyg, the proliferation shows an up to four-times higher rate. Moreover, expression pattern analysis by real-time PCR data revealed interesting results in multiple up- and down-regulation of cancer and drug metabolism genes compared with the HS578T cell line and the M13SV1 cell line, for example, upregulation of cell cycle regulators as p16 and p53 in all hybrids, drug resistance transporters as ABCC6, ABCB1 and major vault protein (MVP) in all hybrid cell lines as well as upregulation of androgen receptor in three of four hybrid cell lines, which stands in a reciprocal relationship to downregulation of estrogen receptor in some poor prognosis breast cancers, probably indicating that fusion might cause a switch to an estrogen-independent tumor growth. The real-time PCR data have been confirmed by Western blot analysis. On the basis of spontaneous fusion between M13SV1 breast stem cells and invasive HS578T breast cancer cells we could give a promising insight into characterization of fusion cells as a possible model for CSCs. [ABSTRACT FROM AUTHOR]

Cd2+ induces apoptosis of PT cells. Cd2+ also induces cell cycle arrest, but the underlying mechanisms remain unclear. We hypothesized that Cd2+-+induced stress causes G2 phase arrest by activating the ATM/ATR pathway. This results in activation of two downstream kinases, chk1 and chk2, which turn off the phosphatase cdc25c, thus preventing activation of the cdc2/cyclin B complex that enables cells to enter mitosis. The effects of Cd2+ were investigated in an SV-40 immortalized (p53-inactivated) rat kidney PT cell line. FACS analysis was used for cell cycle analysis, MTT test for cell death quantification and immunoblotting for detection of cdc25c and cdc2. 50µM Cd2+ for 6 h significantly increased PT cells in the G2/M phase by ∼20%. This was increased ∼two-fold in nocodazole synchronized cells. Cd2+ also induced ∼20% cell death, which was further enhanced in synchronized cells. Inhibition of chk1 and chk2 with 300nM UCN-01 significantly increased Cd2+ toxicity by 30%. Immunoblots with antibodies to cdc25c and phospho-cdc2 supported Cd2+ induced activation of the ATM/ATR pathway in the G2 phase. We speculate that the increase in cell death induced by Cd2+ after inhibition of chk1/chk2 with UCN-01 results from damaged PT cells, which progress from the G2 phase to mitosis and then undergo cell death by mitotic catastrophe. [ABSTRACT FROM AUTHOR]