Human Adipose-Derived Mesenchymal Stromal Cells May Promote Breast Cancer Progression and Metastatic Spread.

BACKGROUND: Stem cell-enriched fat grafting has been proposed as a potential therapy for reconstructive, restorative, or enhancement-related procedures of the breast. Its role in postoncologic breast reconstruction is still emerging, with concerns about safety. The authors investigated the dose-dependent interaction between human adipose-derived mesenchymal stromal cells (AD-MSCs) and human breast cancer cell (BCC) lines [MDA-MB-231 (MDA) and MCF-7 (MCF)] focusing on tumor microenvironment, tumor growth, and metastatic spread. METHODS: Adipose-derived mesenchymal stromal cell influence on viability and factor expression [regulated on activation, normal T cell expressed and secreted (RANTES), tumor necrosis factor-α, and eotaxin) of breast cancer cells was studied in vitro using direct and indirect co-culture systems. Groups were formed according to adipose-derived mesenchymal stromal cell-to-cancer cell number ratio [MDA/MCF only, AD-MSC/(MDA/MCF), and AD-MSC/(MDA/MCF)]. A humanized orthotopic murine cancer model was used to evaluate breast cancer progression and metastasis (n = 10/group). Cells were injected into the mammary pad in different ratios and animals were monitored over 42 days. Microdialysis was performed to analyze RANTES levels in the tumor microenvironment (days 21 and 42). Primary and metastatic tumors were weighed and analyzed for oncogene, growth factor, and metastatic marker expression. RESULTS: MDA cell viability increased from 45.5 percent to 95.5 percent in presence of adipose-derived mesenchymal stromal cells in vitro. In vivo, animals with AD-MSC showed increased mean tumor weight (MDA, p < 0.01; MCF versus controls, p < 0.05) and metastatic occurrence (40 percent in MDA; 30 percent in MCF versus 0 percent in controls). Cytokine analysis revealed switching of MCF tumor phenotype to a more malignant type in the presence of adipose-derived mesenchymal stromal cells. CONCLUSION: Human adipose-derived mesenchymal stromal cells may promote progression and metastatic spread in breast cancer through a switch to a more malignant phenotype with worse prognosis.

Overexpression of EphB4 in the mammary epithelium shifts the differentiation pathway of progenitor cells and promotes branching activity and vascularization.

Postnatally, the mammary gland undergoes continuous morphogenesis and thereby is especially prone to malignant transformation. Thus, the maintenance of the epithelium depends on a tight control of stem cell recruitment. We have previously shown that epithelial overexpression of the EphB4 receptor results in defective mammary epithelial development and conferred a metastasizing tumor phenotype on experimental mouse mammary tumors accompanied by a preponderance of progenitor cells. To analyze the effect of EphB4 overexpression on mammary epithelial cell fate, we have used Fluorescence Activated Cell Sorting (FACS) analyses to quantify epithelial sub-populations and repopulation assays of cleared fat pads to investigate their regenerative potential. These experiments revealed that deregulated EphB4 expression leads to an augmentation of bi-potent progenitor cells and to a shift of the differentiation pathway towards the luminal lineage. The analyses of the ductal outgrowths indicated that EphB4 overexpression leads to enforced branching activity, impedes ductal differentiation and stimulates angiogenesis. To elucidate the mechanisms forwarding EphB4 signals, we have compared the expression profile of defined cell populations between EphB4 transgene and wild type mammary glands concentrating on the wnt signaling pathway and on genes implicated in cell migration. With respect to wnt signaling, the progenitor cell population was the most affected, whereas the stem cell-enriched population showed the most pronounced deregulation of migration-associated genes. Thus, the luminal epithelial EphB4 signaling contributes, most likely via wnt signaling, to the regulation of migration and cell fate of early progenitors and is involved in the determination of branching points along the ductal tree.

The multifaceted roles of Eph/ephrin signaling in breast cancer.

Eph receptors and their membrane-bound ligands are intimately involved in the control of morphogenic processes during embryonic development and adult tissue homeostasis. By their ability to orchestrate cell migration, pattern formation and tissue integrity they are also prone to be involved in carcinogenic growth. In this review we concentrate on their involvement in the normal and carcinogenic development of the breast. In this context we summarize their multi-faceted functions as tumor suppressors, tumor promoters, angiogenic inducers and regulators of stem cell homeostasis.

Deregulated ephrin-B2 signaling in mammary epithelial cells alters the stem cell compartment and interferes with the epithelial differentiation pathway.

Cancer most probably originates from stem/progenitor cells and exhibits a similar cell hierarchy as normal tissues. Moreover, there is growing evidence that only the stem cells are capable of metastasis formation. We have previously shown that overexpression of a dominant negative ephrin-B2 mutant interferes with mammary gland differentiation and confers a metastatic phenotype to NeuT-induced mammary tumors with an increase in cells with stem/progenitor characteristics. To investigate the role of ephrin-B2 in the control of the mammary stem cell niche, we analyzed the mammary stem and progenitor cell populations in transgenic mice overexpressing the mutant ephrin-B2. Quantification by FACS analysis revealed a significant increase of cells in the basal/alveolar cell-, the bi-potent progenitor- and the stem cell-enriched fractions. Moreover, the supposed precursors of estrogen receptor-positive cells were elevated in the stem cell-enriched fraction. In contrast, the epithelium from transgenic mice overexpressing the native ephrin-B2 gene showed an augmentation of the luminal cell- and the bi-potent progenitor-enriched fractions. Repopulation assays revealed that the epithelial cells of truncated ephrin-B2 transgenic epithelial cells have a higher regeneration capacity than those of controls and of native ephrin-B2 transgenic mice, confirming the augmentation of stem cells. Morphologically, these outgrowths exhibited impaired basal/luminal compartmentalization and epithelial polarization. These results demonstrate that deregulated ephrin-B2 expression interferes with the regulation of the stem cell niche and leads to a shift of the differentiation pathway and may thereby contribute to the acquisition of the metastatic phenotype long before carcinogenic growth becomes apparent.

Preponderance of cells with stem cell characteristics in metastasising mouse mammary tumours induced by deregulated EphB4 and ephrin-B2 expression.

We have previously shown that EphB4 and ephrin-B2 are differentially expressed in the mammary gland and that their deregulated expression in the mammary epithelium of transgenic mice leads to perturbations of the mammary parenchyma and vasculature. In addition, overexpression of EphB4 and expression of a truncated ephrin-B2 mutant, capable of receptor stimulation but incapable of reverse signalling, confers a metastasising phenotype on NeuT initiated mouse mammary tumours. We have taken advantage of this transgenic tumour model to compare stem cell characteristics between the non-metastasising and metastasising mammary tumours. We analysed the expression of the proliferation attenuating p21(waf) gene, which was significantly increased in the metastasising tumours. Moreover, we compared the expression of CK-19, Sca-1, CD24 and CD49f as markers for progenitor cells exhibiting a decreasing differentiation grade. Sca-1 expressing cells were the earliest progenitors detected in the non-metastasising NeuT induced tumours. The metastasising NeuT/EphB4 tumours were enriched in CD24 expressing cells, whereas the metastasising NeuT/truncated ephrin-B2 tumours contained in addition significant amounts of CD49f expressing cells. The same cell populations were also enriched in mammary glands of single transgenic MMTV-EphB4 and MMTV-truncated ephrin-B2 females indicating that deregulated EphB4-ephrin-B2 signalling interferes with the homeostasis of the stem/progenitor cell pool before tumour formation is initiated. Since the same cell populations are enriched in the normal tissue, primary mammary tumours and metastases we conclude that these progenitor cells were the origin of tumour formation and that this change in the tumour origin has led to the acquisition of the metastatic tumour phenotype.

The mammary gland vasculature revisited.

Concomitant with the extensive growth and differentiation of the mammary epithelium during pregnancy and lactation, and epithelial involution after weaning, the vasculature of the mammary gland undergoes repeated cycles of expansion and regression. Vascular expansion is effected by sprouting angiogenesis, intussusception and conceivably also vasculogenesis. The capacity of the epithelial cells to stimulate vascular growth and differentiation is dependent on the constellation of systemic and local hormones and growth factors as well as the changing demands for oxygenation and nutrient supply. This results in the release of angiogenic factors which stimulate endothelial cell growth and regulate vascular architecture. In contrast to the angiogenic phase of the mammary gland cycle, little is known about the control of vascular regression although this would possibly offer new insights into therapeutic possibilities against breast cancer. In this review we summarize knowledge regarding the mechanisms regulating the vasculature of the mammary gland and delineate the importance of the vasculature in the attainment of organ function. In addition, we discuss the angiogenic mechanisms observed during mammary carcinogenesis and their consequences for breast cancer therapy.

In vitro investigation into the potential of a mistletoe extract to alleviate adverse effects of cyclophosphamide.

OBJECTIVES: Mistletoe extracts have been shown to provide deoxyribonucleic acid (DNA)-stabilizing effects in human peripheral blood mononuclear cells (PBMC) in vitro. We investigated the effect of a mistletoe extract on PBMC with and without concomitant treatment with cyclophosphamide and compared mitochondrial activity and replication of normal PBMC with that of a T-cell leukemia cell line. DESIGN: The experiments were performed with PBMC of healthy blood donors and the T-cell leukemia Jurkat cell line. Cells were pre-incubated with mistletoe extract for 60 to 65 hours. 4-hydroperoxycyclophosphamide (4-hpc, precursor of 4-hydroxycyclophosphamide) was added for 2 hours, after which mitochondrial activity and replication were measured. All experiments were randomized and blinded. MAIN OUTCOME MEASURES: Cell mitochondrial activity and replication were assessed with spectrophotometric analysis of WST-1 reduction and BrdU incorporation. RESULTS: The application of 4-hpc consistently reduced mitochondrial activity and replication of PBMC and Jurkat cells. Mistletoe extract strongly enhanced PBMC mitochondrial activity and replication (with or without 4-hpc) and partially inhibited Jurkat cell replication (with 4-hpc only). Compared to mistletoe untreated cells, enhancement ofPBMC mitochondrial activity by mistletoe extract was independent of treatment with 4-hpc, but enhancement of PBMC replication by mistletoe extract was stronger when treated with 4-hpc. CONCLUSIONS: Mistletoe extract strongly stimulated healthy PBMC but not malignant Jurkat cells. In addition, mistletoe extract seemed to partially protect healthy PBMC-but not malignant Jurkat cells-from the cytostatic effect of 4-hpc. The results motivate further preclinical and clinical investigations of mistletoe extracts as an adjuvant medication in cancer therapy to alleviate side effects of conventional therapy.

Mammary epithelial-specific knockout of the ephrin-B2 gene leads to precocious epithelial cell death at lactation.

The family of Eph receptor tyrosine kinases and their membrane bound ligands, the ephrins, are involved in a wide variety of morphogenic processes during embryonic development and adult tissue homeostasis. Receptor-ligand interaction requires direct cell-cell contact and results in forward and reverse signaling originating from the receptor and ligand, respectively. We have previously shown that EphB4 and ephrinB2 are differentially expressed during the development of the adult mammary parenchyma. Overexpression of EphB4 in the mammary epithelium of transgenic mice leads to perturbations in mammary epithelial morphology, motility and growth. To investigate the role of ephrinB2 signaling in mammary gland biology, we have established transgenic mice exhibiting conditional ephrinB2 knockout in the mammary epithelium. In homozygote double transgenic CreLox mice, specific knockout of ephrinB2 occurred in the mammary epithelium during the first pregnancy-lactating period. Abolishing ephrinB2 function led to severe interference with the architecture and functioning of the mammary gland at lactation. The morphology of the transgenic lactating glands resembled that of involuting controls, with decreased epithelial cell number and collapsed lobulo-alveolar structures. Accordingly, massive epithelial cell death and expression of involution-specific genes were observed. Interestingly, in parallel to cell death, significant cell proliferation was apparent, suggestive of tissue regeneration.

Deregulated ephrin-B2 expression in the mammary gland interferes with the development of both the glandular epithelium and vasculature and promotes metastasis formation.

Eph receptor tyrosine kinases and their membrane-bound ephrin ligands play key roles during morphogenesis and adult tissue homeostasis. Receptor-ligand interactions result in forward and reverse signalling from the receptor and ligand respectively. To delineate the role(s) of forward and reverse signalling in mammary gland biology we have established transgenic mice exhibiting mammary epithelial-specific overexpression of either the native ephrin-B2 or a dominant negative ephrin-B2 mutant incapable of reverse signalling. During pregnancy and lactation overexpression of the native ephrin-B2 resulted in precocious differentiation, whereas overexpression of mutated ephrin-B2 caused delayed epithelial differentiation and in disturbed tissue architecture. Both transgenes affected also mammary vascularisation. Whereas ephrin-B2 induced superfluous but organised capillaries, mutant ephrin-B2 overexpression resulted in an irregular vasculature with blind-ending capillaries. Mammary tumours were not observed in either transgenic line, however, the crossing with NeuT transgenic animals revealed that mutated ephrin-B2 expression significantly accelerated tumour growth and imposed a metastatic phenotype.

HIC1 tumour suppressor gene is suppressed in acute myeloid leukaemia and induced during granulocytic differentiation.

A hallmark of acute myeloid leukaemia (AML) is a block in differentiation caused by deregulated gene expression. The tumour suppressor Hypermethylated In Cancer 1 (HIC1) is a transcriptional repressor, which is epigenetically silenced in solid cancers. HIC1 mRNA expression was found to be low in 128 patient samples of AML and CD34+ progenitor cells when compared with terminally differentiated granulocytes. HIC1 mRNA was induced in a patient with t(15;17)-positive acute promyelocytic leukaemia receiving all-trans retinoic acid (ATRA) therapy. We therefore investigated whether HIC1 plays a role in granulocytic differentiation and whether loss of function of this gene might contribute to the differentiation block in AML. We evaluated HIC1 mRNA levels in HL-60 and U-937 cells upon ATRA-induced differentiation and in CD34+ progenitor cells after granulocyte colony-stimulating factor-induced differentiation. In both models of granulocytic differentiation, we observed significant HIC1 induction. When HIC1 mRNA was suppressed in HL-60 cells using stably expressed short hairpin RNA targeting HIC1, granulocytic differentiation was altered as assessed by CD11b expression. Bisulphite sequencing of GC-rich regions (CpG islands) in the HIC1 promoter provided evidence that the observed suppression in HL-60 cells was not because of promoter hypermethylation. Our findings indicate a role for the tumour suppressor gene HIC1 in granulocytic differentiation. Low expression of HIC1 may very well contribute to pathogenic events in leukaemogenesis.

Cardiac glycosides initiate Apo2L/TRAIL-induced apoptosis in non-small cell lung cancer cells by up-regulation of death receptors 4 and 5.

Tumor necrosis factor (TNF)-related apoptosis-inducing ligand (Apo2L/TRAIL) belongs to the TNF family known to transduce their death signals via cell membrane receptors. Because it has been shown that Apo2L/TRAIL induces apoptosis in tumor cells without or little toxicity to normal cells, this cytokine became of special interest for cancer research. Unfortunately, cancer cells are often resistant to Apo2L/TRAIL-induced apoptosis; however, this can be at least partially negotiated by parallel treatment with other substances, such as chemotherapeutic agents. Here, we report that cardiac glycosides, which have been used for the treatment of cardiac failure for many years, sensitize lung cancer cells but not normal human peripheral blood mononuclear cells to Apo2L/TRAIL-induced apoptosis. Sensitization to Apo2L/TRAIL mediated by cardiac glycosides was accompanied by up-regulation of death receptors 4 (DR4) and 5 (DR5) on both RNA and protein levels. The use of small interfering RNA revealed that up-regulation of death receptors is essential for the demonstrated augmentation of apoptosis. Blocking of up-regulation of DR4 and DR5 alone significantly reduced cell death after combined treatment with cardiac glycosides and Apo2L/TRAIL. Combined silencing of DR4 and DR5 abrogated the ability of cardiac glycosides and Apo2L/TRAIL to induce apoptosis in an additive manner. To our knowledge, this is the first demonstration that glycosides up-regulate DR4 and DR5, thereby reverting the resistance of lung cancer cells to Apo2/TRAIL-induced apoptosis. Our data suggest that the combination of Apo2L/TRAIL and cardiac glycosides may be a new interesting anticancer treatment strategy.

GPI-specific phospholipase D (GPI-PLD) is expressed during mouse development and is localized to the extracellular matrix of the developing mouse skeleton.

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is abundant in serum and has a well-characterized biochemistry; however, its physiological role is completely unknown. Previous investigations into GPI-PLD have focused on the adult animal or on in vitro systems and a putative role in development has been neither proposed nor investigated. We describe the first evidence of GPI-PLD expression during mouse embryonic ossification. GPI-PLD expression was detected predominantly at sites of skeletal development, increasing during the course of gestation. GPI-PLD was observed during both intramembraneous and endochondral ossification and localized predominantly to the extracellular matrix of chondrocytes and to primary trabeculae of the skeleton. In addition, the mouse chondrocyte cell line ATDC5 expressed GPI-PLD after experimental induction of differentiation. These results implicate GPI-PLD in the process of bone formation during mouse embryogenesis.

Tenascin-W is found in malignant mammary tumors, promotes alpha8 integrin-dependent motility and requires p38MAPK activity for BMP-2 and TNF-alpha induced expression in vitro.

Tenascins represent a family of extracellular matrix glycoproteins with distinctive expression patterns. Here we have analyzed the most recently described member, tenascin-W, in breast cancer. Mammary tumors isolated from transgenic mice expressing hormone-induced oncogenes reveal tenascin-W in the stroma around lesions with a high likelihood of metastasis. The presence of tenascin-W was correlated with the expression of its putative receptor, alpha8 integrin. HC11 cells derived from normal mammary epithelium do not express alpha8 integrin and fail to cross tenascin-W-coated filters. However, 4T1 mammary carcinoma cells do express alpha8 integrin and their migration is stimulated by tenascin-W. The expression of tenascin-W is induced by BMP-2 but not by TGF-beta1, though the latter is a potent inducer of tenascin-C. The expression of tenascin-W is dependent on p38MAPK and JNK signaling pathways. Since preinflammatory cytokines also act through p38MAPK and JNK signaling pathways, the possible role of TNF-alpha in tenascin-W expression was also examined. TNF-alpha induced the expression of both tenascin-W and tenascin-C, and this induction was p38MAPK- and cyclooxygenase-dependent. Our results show that tenascin-W may be a useful diagnostic marker for breast malignancies, and that the induction of tenascin-W in the tumor stroma may contribute to the invasive behavior of tumor cells.

EphB4 receptor tyrosine kinase transgenic mice develop glomerulopathies reminiscent of aglomerular vascular shunts.

We have established transgenic mice over-expressing the EphB4 receptor tyrosine kinase in the kidney. The EphB4 protein was localised to the developing tubular system of both control and transgenic newborn mice. In transgenic adults, transgene expression persisted in the proximal tubules and the Bowman's capsules, structures, which were not stained in control kidneys. The glomeruli of control animals consisted of regular, round vascular baskets with clearly discernable afferent and efferent arterioles. In contrast, approximately 40% of the transgenic glomeruli had an irregular shrivelled appearance and many exhibited fused, horse shoe-like afferent and efferent arterioles bypassing the glomerulus. These abnormal glomerular structures are very reminiscent of aglomerular vascular shunts, a human degenerative glomerulopathy of unknown aetiology.

Eph and ephrin signaling in mammary gland morphogenesis and cancer.

The Eph family of receptor tyrosine kinases and their membrane-bound ligands, the ephrins, play a central role in pattern formation during embryonic development and there is growing evidence that they are also instrumental in the control of tissue dynamics in the adult. The mammary gland is a paradigm for morphogenic processes occurring in the adult, since the gland develops predominantly postnatally and is subjected to continuous cyclic remodeling according to functional demands. Thus, pattern formation and the establishment of a functional organ structure are permanent themes in the mammary gland life cycle. In this paper we summarize the experimental evidence and discuss possible mechanisms by which Ephs and ephrins are modulating mammary epithelial cell adhesion, communication, and migration. Furthermore, we speculate on the different aspects of their influence on normal mammary gland development, function, and carcinogenesis.

Loss of EphB4 receptor tyrosine kinase protein expression during carcinogenesis of the human breast.

Members of the Eph family of receptor tyrosine kinase have been implicated in cell-cell communication and tissue integrity during embryogenesis. We have previously demonstrated cell type specific and hormone dependent EphB4 expression in the mouse mammary parenchyma suggesting involvement in the homeostasis of this organ. Since disruption of tissue organization is crucial for metastatic dissemination, we have investigated the expression of EphB4 during carcinogenesis of the human breast. Immunohistochemical analysis of 24 normal human breast samples and 124 consecutive breast carcinomas was correlated with tumor characteristics (stage, histology, grade, lymph node involvement) and the expression of ER, PR, Ki-67, p53 and HER2. In normal breast tissue, the EphB4 protein was expressed exclusively in parenchymal cells. Strikingly, a drastic reduction in the number of EphB4 protein expressing cells was observed in almost all invasive carcinomas analyzed, irrespective of the tumor type (p<0.0001). Furthermore, we found a highly significant correlation between EphB4 positivity and low histological grading of the tumor cells (p=0.002) suggesting that in breast cancer, EphB4 expression is not compatible with tumor progression. This raises the possibility that EphB4 could represent a potent tool for therapeutic intervention.

Tumor cell specific expression of MMP-2 correlates with tumor vascularisation in breast cancer.

The metastatic potential of tumors is dependent on the ability of tumor cells to degrade extracellular matrix components by the expression of matrix metalloproteinases (MMPs) and to induce vascularisation of the tumor tissue. Thus, expression of MMPs and the number of blood vessel in tumor tissue may serve as prognostic markers of aggressive and metastasizing tumor growth. We have determined the vascularisation and the expression of MMP-2 by immuno-histochemical staining of 19 benign and 75 malignant breast tissue specimens with CD31- and MMP-2 specific antisera. The degree of vascularisation was expressed by intratumoral microvascular density (IMD), which takes into account all vessels present in a hot spot irrespective of their size. In addition, we have introduced a novel parameter, vascular grading (VG), which describes the percentage of small microvessels of <20 microm in diameter in the total number of blood vessels. IMD tended to indicate an elevated risk for metastasis formation and disease recurrence, while VG did not correlate with metastasis formation. Similarly, MMP-2 expression neither correlated with the clinical outcome of the disease nor with the classical histo-pathological parameters such as stage, grade, lymph node involvement and estrogen receptor status. Tumor cell-specific MMP-2 expression, however, showed a highly significant correlation with VG but not with IMD. These results indicate that MMP-2 expression is rather involved in the formation of small capillaries than in vessel maturation and tumor cell invasion. Thus, MMP-2 expression by tumor cells may serve as indicator of strong angiogenic induction potential of breast tumor cells.

Altered mammary epithelial development, pattern formation and involution in transgenic mice expressing the EphB4 receptor tyrosine kinase.

We have previously documented the cell-type-specific and hormone-dependent expression of the EphB4 receptor in the mouse mammary gland. To investigate its role in the biology of the mammary gland, we have established transgenic mice bearing the EphB4 receptor under the control of the MMTV-LTR promoter, which represents the first transgenic mouse model to investigate the effect(s) of unscheduled expression of EphB4 in adult organisms. Transgene expression in the mammary epithelium was induced at puberty, increased during pregnancy, culminated at early lactation and persisted until day three of post-lactational involution. In contrast, expression of the endogenous EphB4 gene is downregulated during pregnancy, is essentially absent during lactation and is re-induced after day three of post-lactational involution. The unscheduled expression of EphB4 led to a delayed development of the mammary epithelium at puberty and during pregnancy. During pregnancy, less lobules were formed, these however exhibited more numerous but smaller alveolar units. Transgenic mammary glands were characterized by a fragile, irregular morphology at lactation; however, sufficient functionality was maintained to nourish the young. Transgenic mammary glands exhibited untimely epithelial apoptotic cell death during pregnancy and abnormal epithelial DNA synthesis at early post-lactational involution, indicating a disturbed response to proliferative/apoptotic signals. Mammary tumours were not observed in the EphB4 transgenic animals; however, in double transgenic animals expressing both EphB4 and the neuT genes, tumour appearance was significantly accelerated and, in contrast to neuT-only animals, metastases were observed in the lung. These results implicate EphB4 in the regulation of tissue architecture, cellular growth response and establishment of the invasive phenotype in the adult mammary gland.