Spontaneous Fusion of MSC with Breast Cancer Cells Can Generate Tumor Dormancy.

Direct cellular interactions of MDA-MB-231cherry breast cancer cells with GFP-transduced human mesenchymal stroma/stem-like cells (MSCGFP) in a co-culture model resulted in spontaneous cell fusion by the generation of MDA-MSC-hyb5cherry GFP breast cancer hybrid cells. The proliferative capacity of MDA-MSC-hyb5 cells was enhanced about 1.8-fold when compared to the parental MDA-MB-231cherry breast cancer cells. In contrast to a spontaneous MDA-MB-231cherry induced tumor development in vivo within 18.8 days, the MDA-MSC-hyb5 cells initially remained quiescent in a dormancy-like state. At distinct time points after injection, NODscid mice started to develop MDA-MSC-hyb5 cell-induced tumors up to about a half year later. Following tumor initiation, however, tumor growth and formation of metastases in various different organs occurred rapidly within about 10.5 days. Changes in gene expression levels were evaluated by RNA-microarray analysis and revealed certain increase in dormancy-associated transcripts in MDA-MSC-hyb5. Chemotherapeutic responsiveness of MDA-MSC-hyb5 cells was partially enhanced when compared to MDA-MB-231 cells. However, some resistance, e.g., for taxol was detectable in cancer hybrid cells. Moreover, drug response partially changed during the tumor development of MDA-MSC-hyb5 cells; this suggests the presence of unstable in vivo phenotypes of MDA-hyb5 cells with increased tumor heterogeneity.

Cell culture media notably influence properties of human mesenchymal stroma/stem-like cells from different tissues.

BACKGROUND AIMS: Mesenchymal stroma/stem-like cells (MSCs) are a popular cell source and hold huge therapeutic promise for a broad range of possible clinical applications. However, to harness their full potential, current limitations in harvesting, expansion and characterization have to be overcome. These limitations are related to the heterogeneity of MSCs in general as well as to inconsistent experimental protocols. Here we aim to compare in vitro methods to facilitate comparison of MSCs generated from various tissues. METHODS: MSCs from 3 different tissues (bone marrow, dental pulp, adipose tissue), exemplified by cells from 3 randomly chosen donors per tissue, were systematically compared with respect to their in vitro properties after propagation in specific in-house standard media, as established in the individual laboratories, or in the same commercially available medium. RESULTS: Large differences were documented with respect to the expression of cell surface antigens, population doubling times, basal expression levels of 5 selected genes and osteogenic differentiation. The commercial medium reduced differences in these parameters with respect to individual human donors within tissue and between tissues. The extent, size and tetraspanin composition of extracellular vesicles were also affected. CONCLUSIONS: The results clearly demonstrate the extreme heterogeneity of MSCs, which confirms the problem of reproducibility of results, even when harmonizing experimental conditions, and questions the significance of common parameters for MSCs from different tissues in vitro.

Anti-Tumor Effects of Exosomes Derived from Drug-Incubated Permanently Growing Human MSC.

Similar to growth-limited human primary cultures of mesenchymal stroma/stem-like cells (MSC), the continuously proliferating human MSC544 cell line produced extracellular vesicles as characterized by expression of the tetraspanin molecules CD9, CD63, and CD81. Release of these particles was predominantly detectable during continuous cell growth of MSC544 in contrast to confluency-mediated transient growth arrest. For therapeutic use, these particles were isolated from proliferating MSC544 after taxol treatment and applied to different cancer cell cultures. A pronounced cytotoxicity of lung, ovarian, and breast cancer cells was observed primarily with taxol-loaded exosomes, similar to the effects displayed by application of taxol substance. While these findings suggested pronounced cancer cell targeting of MSC544 exosomes, a tumor therapeutic approach was performed using a mouse in vivo breast cancer model. Thus, intravenous injection of taxol-loaded MSC544 exosomes displayed superior tumor-reducing capabilities as compared to application of taxol exosomes by oral gavage. To broaden this therapeutic spectrum, epirubicin was applied to MSC544, and the derived exosomes likewise exhibited significant cytotoxic effects in different cancer cell cultures. These findings suggest an unlimited source for large-scale exosome production with reproducible quality to enable variable drug targeting of tumors or other diseases.

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.

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.

In Vitro Fusion of Normal and Neoplastic Breast Epithelial Cells with Human Mesenchymal Stroma/Stem Cells Partially Involves Tumor Necrosis Factor Receptor Signaling.

Formation of hybrid cells by "accidental cell fusion" of normal and neoplastic breast epithelial cells with local tissue-associated mesenchymal stroma/stem-like cells (MSC) in an inflammatory microenvironment can generate new cancer cell populations whereby molecular signaling mechanisms of this process remain unclear. Fusions of lentiviral enhanced green fluorescent protein-labeled MSC with mcherry-labeled breast epithelial cells were quantified and effects of tumor necrosis factor alpha (TNF-α) and receptor downstream signaling were investigated. Cocultures of MSC with normal human mammary epithelial cells, with neoplastic MCF10A, or with MDA-MB-231 or MCF7 breast cancer cells demonstrated hybrid cell formation between 0.1% and about 2% of the populations within 72 hours, whereby the fusion process occurred in less than 5 minutes. Addition of the pro-inflammatory cytokine TNF-α significantly enhanced MCF10A-MSC cell fusion. Small-interfering RNA (siRNA) knockdown experiments revealed an involvement of tumor necrosis factor (TNF) receptor-1 and -2 in this process. This was also substantiated by siRNA knockdown of tumor necrosis factor receptor type 1-associated death domain which abolished TNF-α-stimulated fusion. While TNF receptor signaling can be relayed via the Mitogen-activated protein kinase 8 (MAPK8), NF-κB or cell death pathways, examination of further downstream signaling exhibited little if any effects of MAPK8 or RelA (p65) on TNF-α-mediated cell fusion, respectively. These data suggested that cell fusion between MSC and MCF10A breast epithelial cells can be stimulated by TNF-α involving TNF receptor-activated cell death pathways or additional NF-κB signaling. Stem Cells 2018;36:977-989.

Concise Review: Crosstalk of Mesenchymal Stroma/Stem-Like Cells with Cancer Cells Provides Therapeutic Potential.

Various direct and indirect cellular interactions between multi-functional mesenchymal stroma/stem-like cells (MSCs) and cancer cells contribute to increasing plasticity within the tumor tissue and its microenvironment. Direct and tight communication between MSC and cancer cells is based on membrane protein interactions and the exchange of large plasma membrane fragments also known as trogocytosis. An ultimate but rare direct interaction resumes in fusion of these two cellular partners resulting in the formation of new cancer hybrid cell populations. Alternatively, indirect interactions are displayed by the release of membranous vesicle-encapsulated microRNAs and proteins or soluble components such as molecular growth factors, hormones, chemo-/cytokines, and metabolites. Released single molecules as well as multivesicular bodies including exosomes and microvesicles can form local concentration gradients within the tumor microenvironment and are incorporated not only by adjacent neighboring cells but also affect distant target cells. The present Review will focus on vesicle-mediated indirect communication and on cancer cell fusion with direct contact between MSC and cancer cells. These different types of interaction are accompanied by functional interference and mutual acquisition of new cellular properties. Consequently, alterations in cancer cell functionalities paralleled by the capability to reorganize the tumor stroma can trigger changes in metastatic behavior and promote retrodifferentiation to develop new cancer stem-like cells. However, exosomes and microvesicles acting over long distances may also provide a tool with therapeutic potential when loaded with anti-tumor cargo. Stem Cells 2018;36:951-968.

Involvement of Actin Cytoskeletal Components in Breast Cancer Cell Fusion with Human Mesenchymal Stroma/Stem-Like Cells.

Cell fusion as a rare event was observed following the co-culture of human MDA-MB-231cherry breast cancer cells or benign neoplastic MCF10Acherry breast epithelial cells together with different mesenchymal stroma/stem-like cells (MSCGFP) cultures, respectively, resulting in the generation of double-fluorescing hybrid cells. Analysis of potential molecular mechanisms for the formation of cancer hybrid cells revealed cytoskeletal components, including F-actin. Thus, a sub-lethal concentration of cytochalasin D, which blocks elongation of actin filaments, was able to significantly reduce cancer hybrid cell formation. Simultaneously, cell cycle progression of the different co-cultures remained unaffected following treatment with cytochalasin D, indicating continued proliferation. Moreover, exposure to 50 nM cytochalasin D revealed little if any effect on the expression of various integrins and cell adhesion molecules in the different co-cultures. However, LC-MS proteome analysis of the different control co-cultures compared to corresponding cytochalasin-treated co-cultures demonstrated predominant differences in the expression of actin-associated cytoskeletal proteins. In addition, the requirement of structured actin to provide an appropriate cytoskeletal network for enabling subsequent fusion processes was also substantiated by the actin filament disrupting latrunculin B, which inhibits the fusion process between the breast cancer populations and mesenchymal stroma/stem-like cells (MSC). Together, these findings suggest an important role of distinct actin structures and associated cytoskeletal components during cell fusion and the formation of breast cancer hybrid cells.

Changes in uPA, PAI-1, and TGF-ß Production during Breast Cancer Cell Interaction with Human Mesenchymal Stroma/Stem-Like Cells (MSC).

The interactions of cancer cells with neighboring non-malignant cells in the microenvironment play an important role for progressive neoplastic development and metastasis. Long-term direct co-culture of human MDA-MB-231cherry breast cancer cells with benign human mesenchymal stroma/stem-like cells (MSC) MSC544GFP stably expressing mCherry and eGFP fluorescence proteins, respectively, was associated with the formation of three-dimensional (3D) tumor spheroids in vitro. The quantification of the breast tumor marker urokinase plasminogen activator (uPA) in mono-cultured MDA-MB-231 cells revealed an approximately 14-fold enhanced expression when compared to five different normal human MSC mono-cultures. Moreover, uPA levels in 3D tumor spheroids remained elevated 9.4-fold above the average of five different human MSC cultures. In contrast, the expression of the corresponding plasminogen activator inhibitor type-1 (PAI-1) declined by 2.6-fold in the breast cancer cells and was even further reduced by 3.2-fold in the MDA-MB-231cherry/MSC544GFP 3D co-culture spheroids when compared to the various MSC populations. The supportive data were obtained for the production of TGF-ß1, which is an important growth factor in the regulation of tumor growth and metastasis formation. Whereas, TGF-ß1 release in MDA-MB-231cherry/MSC544GFP co-cultures was elevated by 1.56-fold as compared to MSC544 mono-cultures after 24 h; this ratio further increased to 2.19-fold after 72 h. Quantitative PCR analyses in MSC544 and MDA-MB-231 cells revealed that MSC, rather than the breast cancer cells, are responsible for TGF-ß1 synthesis and that TGF-ß1 contributes to its own synthesis in these cells. These findings suggested potential synergistic effects in the expression/secretion of uPA, PAI-1, and TGF-ß during the co-culture of breast cancer cells with MSC.

MSC stimulate ovarian tumor growth during intercellular communication but reduce tumorigenicity after fusion with ovarian cancer cells.

The tumor microenvironment enables important cellular interactions between cancer cells and recruited adjacent populations including mesenchymal stroma/stem cells (MSC). In vivo cellular interactions of primary human MSC in co-culture with human SK-OV-3 ovarian cancer cells revealed an increased tumor growth as compared to mono-cultures of the ovarian cancer cells. Moreover, the presence of MSC stimulated formation of liver metastases. Further interactions of MSC with the ovarian cancer cells resulted in the formation of hybrid cells by cell fusion. Isolation and single cell cloning of these hybrid cells revealed two differentially fused ovarian cancer cell populations termed SK-hyb1 and SK-hyb2. RNA microarray analysis demonstrated expression profiles from both parental partners whereby SK-hyb1 were attributed with more SK-OV-3 like properties and SK-hyb2 cells displayed more similarities to MSC. Both ovarian cancer hybrid populations exhibited reduced proliferative capacity compared to the parental SK-OV-3 cells. Moreover, the fused populations failed to develop tumors in NODscid mice. Together, these data suggested certain stimulatory effects on ovarian tumor growth in the presence of MSC. Conversely, fusion of MSC with SK-OV-3 cells contributed to the generation of new cancer hybrid populations displaying a significantly reduced tumorigenicity.

Enhanced metastatic capacity of breast cancer cells after interaction and hybrid formation with mesenchymal stroma/stem cells (MSC).

BACKGROUND: Fusion of breast cancer cells with tumor-associated populations of the microenvironment including mesenchymal stroma/stem-like cells (MSC) represents a rare event in cell communication whereby the metastatic capacity of those hybrid cells remains unclear. METHODS: Functional changes were investigated in vitro and in vivo following spontaneous fusion and hybrid cell formation between primary human MSC and human MDA-MB-231 breast cancer cells. Thus, lentiviral eGFP-labeled MSC and breast cancer cells labeled with mcherry resulted in dual-fluorescing hybrid cells after co-culture. RESULTS: Double FACS sorting and single cell cloning revealed two different aneuploid male hybrid populations (MDA-hyb1 and MDA-hyb2) with different STR profiles, pronounced telomerase activities, and enhanced proliferative capacities as compared to the parental cells. Microarray-based mRNA profiling demonstrated marked regulation of genes involved in epithelial-mesenchymal transition and increased expression of metastasis-associated genes including S100A4. In vivo studies following subcutaneous injection of the breast cancer and the two hybrid populations substantiated the in vitro findings by a significantly elevated tumor growth of the hybrid cells. Moreover, both hybrid populations developed various distant organ metastases in a much shorter period of time than the parental breast cancer cells. CONCLUSION: Together, these data demonstrate spontaneous development of new tumor cell populations exhibiting different parental properties after close interaction and subsequent fusion of MSC with breast cancer cells. This formation of tumor hybrids contributes to continuously increasing tumor heterogeneity and elevated metastatic capacities.

Cancer stem cell niche models and contribution by mesenchymal stroma/stem cells.

BACKGROUND: The initiation and progression of malignant tumors is driven by distinct subsets of tumor-initiating or cancer stem-like cells (CSCs) which develop therapy/apoptosis resistance and self-renewal capacity. In order to be able to eradicate these CSCs with novel classes of anti-cancer therapeutics, a better understanding of their biology and clinically-relevant traits is mandatory. MAIN BODY: Several requirements and functions of a CSC niche physiology are combined with current concepts for CSC generation such as development in a hierarchical tumor model, by stochastic processes, or via a retrodifferentiation program. Moreover, progressive adaptation of endothelial cells and recruited immune and stromal cells to the tumor site substantially contribute to generate a tumor growth-permissive environment resembling a CSC niche. Particular emphasis is put on the pivotal role of multipotent mesenchymal stroma/stem cells (MSCs) in supporting CSC development by various kinds of interaction and cell fusion to form hybrid tumor cells. CONCLUSION: A better knowledge of CSC niche physiology may increase the chances that cancer stemness-depleting interventions ultimately result in arrest of tumor growth and metastasis.

The role of TGF-ß and its crosstalk with RAC1/RAC1b signaling in breast and pancreas carcinoma.

This article focusses on the role of TGF-ß and its signaling crosstalk with the RHO family GTPases RAC1 and RAC1b in the progression of breast and pancreatic carcinoma. The aggressive nature of these tumor types is mainly due to metastatic dissemination. Metastasis is facilitated by desmoplasia, a peculiar tumor microenvironment and the ability of the tumor cells to undergo epithelial-mesenchymal transition (EMT) and to adopt a motile and invasive phenotype. These processes are controlled entirely or in part by TGF-ß and the small RHO GTPase RAC1 with both proteins acting as tumor promoters in late-stage cancers. Data from our and other studies point to signaling crosstalk between TGF-ß and RAC1 and the related isoform, RAC1b, in pancreatic and mammary carcinoma cells. Based on the exciting observation that RAC1b functions as an endogenous inhibitor of RAC1, we propose a model on how the relative abundance or activity of RAC1 and RAC1b in the tumor cells may determine their responses to TGF-ß and, ultimately, the metastatic capacity of the tumor.

TGF-ß-Dependent Growth Arrest and Cell Migration in Benign and Malignant Breast Epithelial Cells Are Antagonistically Controlled by Rac1 and Rac1b.

Despite improvements in diagnosis and treatment, breast cancer is still the most common cancer type among non-smoking females. TGF-ß can inhibit breast cancer development by inducing cell cycle arrest in both, cancer cells and, as part of a senescence program in normal human mammary epithelial cells (HMEC). Moreover, TGF-ß also drives cell migration and invasion, in part through the small GTPases Rac1 and Rac1b. Depletion of Rac1b or Rac1 and Rac1b in MDA-MB-231 or MDA-MB-435s breast cancer cells by RNA interference enhanced or suppressed, respectively, TGF-ß1-induced migration/invasion. Rac1b depletion in MDA-MB-231 cells also increased TGF-ß-induced p21WAF1 expression and ERK1/2 phosphorylation. Senescent HMEC (P15/P16), when compared to their non-senescent counterparts (P11/P12), presented with dramatically increased migratory activity. These effects were paralleled by elevated expression of genes associated with TGF-ß signaling and metastasis, downregulated Rac1b, and upregulated Rac1. Our data suggest that acquisition of a motile phenotype in HMEC resulted from enhanced autocrine TGF-ß signaling, invasion/metastasis-associated gene expression, and a shift in the ratio of antimigratory Rac1b to promigratory Rac1. We conclude that although enhanced TGF-ß signaling is considered antioncogenic in HMEC by suppressing oncogene-induced transformation, this occurs at the expense of a higher migration and invasion potential.

Interaction of MSC with tumor cells.

Tumor development and tumor progression is not only determined by the corresponding tumor cells but also by the tumor microenvironment. This includes an orchestrated network of interacting cell types (e.g. immune cells, endothelial cells, fibroblasts, and mesenchymal stroma/stem cells (MSC)) via the extracellular matrix and soluble factors such as cytokines, chemokines, growth factors and various metabolites. Cell populations of the tumor microenvironment can interact directly and indirectly with cancer cells by mutually altering properties and functions of the involved partners. Particularly, mesenchymal stroma/stem cells (MSC) play an important role during carcinogenesis exhibiting different types of intercellular communication. Accordingly, this work focusses on diverse mechanisms of interaction between MSC and cancer cells. Moreover, some functional changes and consequences for both cell types are summarized which can eventually result in the establishment of a carcinoma stem cell niche (CSCN) or the generation of new tumor cell populations by MSC-tumor cell fusion.

Effect of SSRI exposure on the proliferation rate and glucose uptake in breast and ovary cancer cell lines.

Breast cancer is the most prevalent malignancy amongst women worldwide while ovarian cancer represents the leading cause of death among gynecological malignancies. Women suffering from these cancers displayed heightened rates of major depressive disorder, and antidepressant treatment with selective serotonin reuptake inhibitors (SSRIs) is frequently recommended. Recently, narrative reviews and meta-analyses showed increased recurrence risks and mortality rates in SSRI-treated women with breast and ovarian cancer. We therefore examined whether three commonly prescribed SSRIs, fluoxetine, sertraline and citalopram, affect proliferation or glucose uptake of human breast and ovarian cancer cell lines characterized by different malignancies and metastatic potential. SSRI treatment or serotonin stimulation with therapeutically relevant concentrations over various time periods revealed no consistent dose- or time-dependent effect on proliferation rates. A marginal, but significant increase in glucose uptake was observed in SK-OV-3 ovarian cancer cells upon fluoxetine or sertraline, but not citalopram treatment. In three breast cancer cell lines and in two additional ovarian cancer cell lines no significant effect of SSRIs on glucose uptake was observed. Our data suggest that the observed increase in recurrence- and mortality rates in SSRI-treated cancer patients is unlikely to be linked to antidepressant therapies.

In Vivo Cell Fusion between Mesenchymal Stroma/Stem-Like Cells and Breast Cancer Cells.

Cellular communication within the tumor microenvironment enables important interactions between cancer cells and recruited adjacent populations including mesenchymal stroma/stem-like cells (MSC). These interactions were monitored in vivo following co-injection of GFP-labeled human MSC together with mcherry-labeled MDA-MB-231 breast cancer cells in NODscid mice. Within 14 days of tumor development the number of initially co-injected MSC had significantly declined and spontaneous formation of breast cancer/MSC hybrid cells was observed by the appearance of double fluorescing cells. This in vivo fusion displayed a rare event and occurred in less than 0.5% of the tumor cell population. Similar findings were observed in a parallel in vitro co-culture. Characterization of the new cell fusion products obtained after two consecutive flow cytometry cell sorting and single cell cloning revealed two populations, termed MDA-hyb3 and MDA-hyb4. The breast cancer fusion cells expressed both, GFP and mcherry and displayed more characteristics of the MDA-MB-231 cells than of the parental MSC. While little if any differences were determined in the proliferative capacity, a significant delay of MDA-hyb3 cells in tumor formation was observed when compared to the parental MDA-MB-231 cells. Moreover, MDA-hyb3 cells developed an altered pattern of distant organ metastases. These findings demonstrated dynamic tumor changes by in vivo and in vitro fusion with the development of new breast cancer hybrid cells carrying altered tumorigenic properties. Consequently, cancer cell fusion contributes to progressively increasing tumor heterogeneity which complicates a therapeutic regimen.

RAC1B: A Rho GTPase with Versatile Functions in Malignant Transformation and Tumor Progression.

RAC1B is an alternatively spliced isoform of the monomeric GTPase RAC1. It differs from RAC1 by a 19 amino acid in frame insertion, termed exon 3b, resulting in an accelerated GDP/GTP-exchange and an impaired GTP-hydrolysis. Although RAC1B has been ascribed several protumorigenic functions such as cell cycle progression and apoptosis resistance, its role in malignant transformation, and other functions driving tumor progression like epithelial-mesenchymal transition, migration/invasion and metastasis are less clear. Insertion of exon 3b endows RAC1B with specific biochemical properties that, when compared to RAC1, encompass both loss-of-functions and gain-of-functions with respect to the type of upstream activators, downstream targets, and binding partners. In its extreme, this may result in RAC1B and RAC1 acting in an antagonistic fashion in regulating a specific cellular response with RAC1B behaving as an endogenous inhibitor of RAC1. In this review, we strive to provide the reader with a comprehensive overview, rather than critical discussions, on various aspects of RAC1B biology in eukaryotic cells.

Taxol-Loaded MSC-Derived Exosomes Provide a Therapeutic Vehicle to Target Metastatic Breast Cancer and Other Carcinoma Cells.

MSC-derived exosomes display, among others, an efficient biocompatibility and a reduced intrinsic immunogenicity, representing a valuable vehicle for drug delivery in a tumor-therapeutic approach. Following treatment of several human mesenchymal stroma/stem-like cell (MSC) populations with sub-lethal concentrations of taxol for 24 h, exosomes were isolated and applied to different human cancer populations including A549 lung cancer, SK-OV-3 ovarian cancer, and MDA-hyb1 breast cancer cells. While MSC control exosomes revealed little if any growth inhibition on the tumor cells, exposure to taxol-loaded MSC-derived exosomes was associated with 80-90% cytotoxicity. A similar application of taxol-loaded exosomes from HuVEC displayed much fewer effects. Quantification by LC-MS/MS analysis demonstrated a 7.6-fold reduced taxol concentration in MSC exosomes when compared to equivalent cytotoxic in vitro effects achieved with taxol substances, indicating a specific and more efficient tumor-targeting property. Consequently, MSC-derived taxol exosomes were tested in vivo. Highly metastatic MDA-hyb1 breast tumors were induced in NODscid mice, and systemic intravenous application of MSC-derived taxol exosomes revealed a more than 60% reduction of subcutaneous primary tumors. Moreover, the amount of distant organ metastases observed at least in lung, liver, spleen, and kidney was reduced by 50% with MSC taxol exosomes, similar to the effects observed with taxol, although the concentration of taxol in exosomes was about 1000-fold reduced. Together, these findings in different cancer cell populations and in vivo provide promising future perspectives for drug-loaded MSC-derived exosomes in efficiently targeting primary tumors and metastases by reducing side effects.

Breast Carcinoma: From Initial Tumor Cell Detachment to Settlement at Secondary Sites.

Metastasis represents a multistep cascade of cancer cell alterations accompanied by structural and functional changes within the tumor microenvironment which may involve the induction of a retrodifferentiation program. Major steps in metastatic developments include (A) cell detachment from the primary tumor site involving epithelial-mesenchymal transition (EMT), (B) migration and invasion into surrounding tissue, (C) transendothelial intravasation into the vasculature of blood and/or lymphatic vessels as circulating tumor cells (CTCs), (D) dissemination to distant organs, and (E) extravasation of CTCs to secondary sites as disseminated tumor cells (DTCs). This article highlights some aspects of the metastatic cascade with a focus on breast cancer cells. Metastatic steps critically depend on the capability of cancer cells to adapt to distant tissues and the corresponding new microenvironment. As a consequence, increasing plasticity and developmental changes paralleled by acquisition of new cancer cell functionalities challenge a successful therapeutic approach.