Juliane von der Ohe

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

BackgroundThe 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 bodySeveral 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.ConclusionA better knowledge of CSC niche physiology may increase the chances that cancer stemness-depleting interventions ultimately result in arrest of tumor growth and metastasis.

In vitro and in vivo therapeutic approach for a small cell carcinoma of the ovary hypercalcaemic type using a SCCOHT-1 cellular model

BackgroundThe small cell ovarian carcinoma of the hypercalcemic type (SCCOHT) which preferably affects young women during regenerative age represents a rare and aggressive form of ovarian tumors with poor prognosis and lacks an efficient therapy.Methods and resultsIn vitro chemotherapy testing in a fluorescence assay using a recently developed cellular model from a recurrent SCCOHT revealed sensitivity for certain epothilones, methotrexate and topotecan whereas little if any cytotoxicity was observed with other chemotherapeutics including platin-based compounds. In particular, epothilone B demonstrated a high sensitivity in contrast to ixabepilone with only little detectable effects. Western blot and cell cycle analysis revealed that the epothilone B sensitivity was associated with increased Ser15 phosphorylation of p53, a significant G1 and G2 cell cycle accumulation and subsequent cell death in subG1 phase. Moreover, tubulinβ3 expression in SMARCA4/BRG1-defective SCCOHT-1 in contrast to other ovarian cancer cells was also affected during chemotherapy treatment. Increased extracellular Ca2+ levels further enhanced the epothilone B cytotoxicity in SCCOHT-1 cells. These in vitro effects were also confirmed in vivo in NOD/scid mouse xenografts demonstrating an attenuated tumor growth in epothilone B / Ca2+-treated mice. After 4d of subsequent treatment, the tumor sizes were reduced by about 90% as compared to continuously growing control tumors. In parallel, a hypercalcemia in control tumor-carrying mice was reverted to normal serum Ca2+ levels after epothilone B / Ca2+ therapy.ConclusionsTaken together, these data demonstrated anti-tumorigenic effects of epothilone B / Ca2+ in SCCOHT providing a focused therapeutic approach against this rare disease and arising recurrent tumors.

Conditioned umbilical cord tissue provides a natural three-dimensional storage compartment as in vitro stem cell niche for human mesenchymal stroma/stem cells

BackgroundThe use of large amounts of human multipotent mesenchymal stroma/stem cells (MSC) for cell therapies represents a desirable property in tissue engineering and banking in the field of regenerative medicine.Methods and resultsWhereas cryo-storage of umbilical cord (UC) tissue pieces in liquid nitrogen without ingredients was associated with predominant appearance of apoptotic cells after thawing and re-culture, progressive growth of MSC was observed following use of cryo-medium. Moreover, conditioning of UC tissue pieces by initial explant culture and subsequent cryo-storage with cryo-medium accelerated a further MSC culture after thawing. These findings suggested that conditioning of UC tissue pieces provides an in vitro stem cell niche by maintenance of a 3-dimensional natural microenvironment for continuous MSC outgrowth and expansion. Indeed, culture of GFP-labeled UC tissue pieces was accompanied by increased outgrowth of GFP-labeled cells which was accelerated in conditioned UC tissue after cryo-storage. Moreover, cryopreserved conditioned UC tissue pieces in cryo-medium after thawing and explant culture could be cryopreserved again demonstrating renewed MSC outgrowth after repeated thawing with similar population doublings compared to the initial explant culture. Flow cytometry analysis of outgrowing cells revealed expression of the typical MSC markers CD73, CD90, and CD105. Furthermore, these cells demonstrated little if any senescence and cultures revealed stem cell-like characteristics by differentiation along the adipogenic, chondrogenic and osteogenic lineages.ConclusionsExpression of MSC markers was maintained for at least 10 freeze/thaw/explant culture cycles demonstrating that repeated cryopreservation of conditioned UC tissue pieces provided a reproducible and enriched stem cell source.

c-Met inhibitors attenuate tumor growth of small cell hypercalcemic ovarian carcinoma (SCCOHT) populations

A cellular model (SCCOHT-1) of the aggressive small cell hypercalcemic ovarian carcinoma demonstrated constitutive chemokine and growth factor production including HGF. A simultaneous presence of c-Met in 41% SCCOHT-1 cells suggested an autocrine growth mechanism. Expression of c-Met was also observed at low levels in the corresponding BIN-67 cell line (6.5%) and at high levels in ovarian adenocarcinoma cells (NIH:OVCAR-3 (84.4%) and SK-OV-3 (99.3%)). Immunohistochemistry of c-Met expression in SCCOHT tumors revealed a heterogeneous distribution between undetectable levels and 80%. Further characterization of SCCOHT-1 and BIN-67 cells by cell surface markers including CD90 and EpCAM demonstrated similar patterns with differences to the ovarian adenocarcinoma cells. HGF stimulation of SCCOHT-1 cells was associated with c-Met phosphorylation at Tyr1349 and downstream Thr202/Tyr204 phosphorylation of p44/42 MAP kinase. This HGF-induced signaling cascade was abolished by the c-Met inhibitor foretinib. Cell cycle analysis after foretinib treatment demonstrated enhanced G2 accumulation and increasing apoptosis within 72 h. Moreover, the IC50 of foretinib revealed 12.4 nM in SCCOHT-1 cells compared to 411 nM and 481 nM in NIH:OVCAR-3 and SK-OV-3 cells, respectively, suggesting potential therapeutic effects. Indeed, SCCOHT-1 and BIN-67 tumor xenografts in NODscid mice exhibited an approximately 10-fold and 5-fold reduced tumor size following systemic application of foretinib, respectively. Furthermore, foretinib-treated tumors revealed a significantly reduced vascularization and little if any c-Met-mediated signal transduction. Similar findings of reduced proliferative capacity and declined tumor size were observed after siRNA-mediated c-Met knock-down in SCCOHT-1 cells demonstrating that in vivo inhibition of these pathways contributed to an attenuation of SCCOHT tumor growth.

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

This article focusses on the role of TGF-β 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-β 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-β 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-β and, ultimately, the metastatic capacity of the tumor.

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

BackgroundFusion 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.MethodsFunctional 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.ResultsDouble 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.ConclusionTogether, 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.

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.

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

Interference of Ca2+ with the proliferation of SCCOHT-1 and ovarian adenocarcinoma cells

A recently established cellular model for the rare small cell carcinoma of the ovary hypercalcemic type (SCCOHT-1) was characterized in comparison to ovarian adenocarcinoma cells (NIH:OVCAR-3 and SK-OV-3). The different cancer populations exhibited a common sensitivity in acidic pH milieu and a continuous proliferation in alkaline medium of pH 8.0-9.0. In the presence of elevated Ca2+ concentrations, the ovarian cancer cells demonstrated a progressively reduced proliferation within 72 h in contrast to other tumor types such as breast cancer cells. This significant growth inhibition was calcium-specific since the proliferation was unaffected after culture of the ovarian cancer cells in the presence of similar concentrations of other cations. The Ca2+ effects on the ovarian cancer cells were associated with marked differences in the activation of intracellular signaling pathways including enhanced phosphorylation of the p42/44 MAP kinase (Thr202/Tyr204). Further analysis of the signaling pathway revealed a significantly enhanced Ca2+-dependent and p42/44 MAP kinase activation-mediated prostaglandin E2 (PGE2) production in SK-OV-3 and SCCOHT-1 and to a lesser extent in NIH:OVCAR-3 cells. Vice versa, exogenous PGE2 did not affect the proliferative capacity of the ovarian cancer cells and inhibition of the Ca2+-mediated MAP kinase activation did not abolish the Ca2+-mediated cytotoxicity. Collectively, these data suggest that multiple pathways are activated by exogenous Ca2+ in the different ovarian cancer cells, including a specific MAP kinase signaling cascade with subsequent PGE2 production and a parallel pathway for the induction of cell death.