Interaction between extracellular cancer matrix and stromal breast cells.

INTRODUCTION: Stromal-epithelial interactions are fundamental for normal organ development and there is a multitude of evidence that the different components of the microenvironment are also necessary for the maintenance and promotion of the "tumor organ". Deregulated tumor associated extracellular matrix (tECM) is a hallmark of cancer, causing an alteration in the amount and composition of the different components (i.e. proteins, proteoglycans, glycoproteins and polysaccharids) of the ECM. As epithelial-stromal interactions are reciprocal, it is possible that tECM itself is able to initiate tumor development. We therefore established a mouse model to examine the influence of tECM of murine breast cancer on developing breast tissue in mice. MATERIALS AND METHODS: Breast cancer was established in 5 BALB/c mice by subcutaneous injection of 1×106 4T1 cells in 100µl PBS into the left mammary fat pad. The mammary fat pad including the primary tumor was excised after two weeks, decellularised and labelled as tumor extracellular matrix (tECM). Tumor ECM of 4T1 tumors was implanted into the 4th inguinal mammary fat pad of BALB/c mice (n = 5) aged 5 days. After 12 weeks the fourth mammary fat pad including the primary tumor was excised. Tissue was used for paraffin embedding and mouse breast cancer PCR array. Murine breast cancer tissue (BCT) and normal murine breast tissue (BT) served as control. RESULTS: Gene array analysis of 84 breast cancer-specific transcripts revealed that the mammary gland cells which were exposed to tumor ECM (tECM-BT) showed a similarly high overexpression for 22 genes as apparent for breast cancer tissue (BCT). The corresponding scatter plot showed a high agreement in the expression of the examined genes between the mammary gland cells which were exposed to tumor ECM and the breast cancer tissue. DISCUSSION: Our results clearly demonstrate that the tECM is able to shift the gene expression pattern of murine mammary epithelial cells towards that of carcinoma, indicating a role in breast cancer initiation. These data underlines that the acellular component of the tumor (ECM) can lead to a transformation of mammary gland tissue cells. These data show for the first time that the interaction of normal breast tissue cells with tumor ECM leads to an exchange of information and a consecutive overexpression of tumor-specific genes.

PDGF regulated migration of mesenchymal stem cells towards malignancy acts via the PI3K signaling pathway.

INTRODUCTION: Mesenchymal stem cells (MSCs) have been described in breast cancer models to migrate towards carcinoma and integrate into tumor associated stroma supporting tumor growth, increasing their metastatic potency and contributing to tumor-angiogenesis. Platelet-derived growth factor (PDGF) isoforms (AA, BB, CC) stimulate growth, survival and motility of MSCs and certain other cell types. Noteworthy, breast carcinomas are known to express PDGF. We aim to further shed light on i) the relevance of the different PDGF isoforms on adipose tissue derived stem cells (ASCs) migration and ii) the underlying pathway dependent on PDGF stimulation. MATERIALS AND METHODS: Breast cancer cell lines were purchased and ASC's were isolated from murine subcutaneous adipose tissue. The transmigration of ASC's towards the PDGF-isoforms was assessed by using recombinant human PDGF-AA, PDGF-BB and PDGF-CC in a trans-well culture dish system. Transmigrated ASC's were quantified in 5 randomly selected fields per condition using fluorescence microscopy after calcein-staining. PDGF-BB depended transmigration of ASC's was verified by downregulation and overexpression of PDGF-BB in breast cancer cell line using lentiviral vectors. In addition, a PI3-kinase inhibitor (LY294002) and a MAP-kinase inhibitor (PD98059) were used to identify the pathway involved in the PDGF-BB mediated migration of ASC's towards tumor. RESULTS: ASC's transmigration significantly increased towards PDGF AA at 50 ng and only showed further increase by 500 ng which was similar to cell behavior when exposed to PDGF CC. In comparison, PDGF-BB significantly increased ASC's transmigration already at a low level of 5 ng with further significant increase for 20 ng and 40 ng. Cell transmigration was blocked with PDGFR-α antibodies but only for PDGF-AA and PDGF-CC whereas PDGFR-ß blockage showed a significant effect on transmigration for PDGF-BB and PDGF-CC but not for PDGF-AA. Neutralizing antibodies in combination with PDGF receptor blockage confirmed findings. In addition, only PI3-kinase inhibitor but not the MEK-1 selective inhibitor caused a significant decrease of transmigration for ASCs towards breast cancer cells. DISCUSSION: The transmigration of ASC's is most significantly enhanced by PDGF-BB via the PI3-kinase pathway. This data support that PI3-kinase is an important key player for MSC migration towards malignancy which need further research to prevent tumor progression in early disease stage.

Transcription Profile in Sporadic Multiple Symmetric Lipomatosis Reveals Differential Expression at the Level of Adipose Tissue-Derived Stem Cells.

BACKGROUND: The cause of the rare fat distribution disorder multiple symmetric lipomatosis is unknown. Independent reports suggest a higher proliferative activity, hormone resistance, and involvement of mitochondrial function in the disease. METHODS: The authors performed morphologic comparison of affected and unaffected tissues in five unrelated patients and generated adipose-derived stem cell cultures from the tissue samples and characterized them as a possible cellular model of multiple symmetric lipomatosis evolution. The authors investigated proliferative activity and the expression of genes relevant to disease processes. RESULTS: There was no difference in the morphologic appearance and the surface marker profile. Stem cells from lipomatous tissue showed significantly higher proliferative activity. Polymerase chain reaction arrays showed marked changes in genes associated with proliferation, hormonal regulation, and mitochondria. The authors show that multiple symmetric lipomatosis tissue is morphologically and histologically different from regular subcutaneous fat. CONCLUSIONS: This study indicates an involvement of mesenchymal stem cells in the pathogenesis of multiple symmetric lipomatosis and that the evolution of multiple symmetric lipomatosis tissue is a process driven by an inherent defect of the respective cell clone(s). Further molecular genetics and functional analysis will be required to unravel the pathogenetic mechanism underlying the derailment in fat cell metabolism and proliferation. Here, the authors show for the first time that adipose-derived stem cells exhibit many characteristics previously described for native multiple symmetric lipomatosis fat tissue and propose that they are therefore an excellent tool for further functional investigations in multiple symmetric lipomatosis and other disorders of the fat tissue. CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, V.

Adipose tissue-derived stem cell secreted IGF-1 protects myoblasts from the negative effect of myostatin.

Myostatin, a TGF-ß family member, is associated with inhibition of muscle growth and differentiation and might interact with the IGF-1 signaling pathway. Since IGF-1 is secreted at a bioactive level by adipose tissue-derived mesenchymal stem cells (ASCs), these cells (ASCs) provide a therapeutic option for Duchenne Muscular Dystrophy (DMD). But the protective effect of stem cell secreted IGF-1 on myoblast under high level of myostatin remains unclear. In the present study murine myoblasts were exposed to myostatin under presence of ASCs conditioned medium and investigated for proliferation and apoptosis. The protective effect of IGF-1 was further examined by using IGF-1 neutralizing and receptor antibodies as well as gene silencing RNAi technology. MyoD expression was detected to identify impact of IGF-1 on myoblasts differentiation when exposed to myostatin. IGF-1 was accountable for 43.6% of the antiapoptotic impact and 48.8% for the proliferative effect of ASCs conditioned medium. Furthermore, IGF-1 restored mRNA and protein MyoD expression of myoblasts under risk. Beside fusion and transdifferentiation the beneficial effect of ASCs is mediated by paracrine secreted cytokines, particularly IGF-1. The present study underlines the potential of ASCs as a therapeutic option for Duchenne muscular dystrophy and other dystrophic muscle diseases.

Angiogenesis: the role of PDGF-BB on adipose-tissue derived stem cells (ASCs).

Recently, it was shown that mesenchymal stem cells (MSCs) are capable of differentiating into endothelial cells which highlights the potential role of MSCs in neovascularization. In the present study, we investigated the paracrine factors responsible for tube formation in human adipose-tissue derived stem cells (ASCs). Moreover, we analyzed ASC's migration towards PDGF-BB and altered levels of proteins involved in different pathways. Freshly isolated human adipose tissue-derived stem cells were seeded onto wells coated with Matrigel and cultured in endothelial growth medium. Capillary-like tube formation was observed after 18 hours culture. Tube formation was significantly reduced in the presence of antibodies against platelet-derived growth factor receptor beta (PDGF) or basic fibroblast growth factor (bFGF). Reverse phase proteomic assay (RPPA) was used to interrogate the expression of 139 phosphorylated or native proteins after incubation with PDGF-BB protein for 24 hours. The present data suggest, that freshly isolated ASCs contain a subpopuplation of stem cells that can form capillary like tubes which is dependent on PDGF and bFGF signaling pathway. Furthermore, Migration of human ASCs significantly increased in response to increased concentrations of PDGF-BB. In addition, incubation of ASCs with PDGF-BB altered phosphorylation of several transcription proteins that are widely expressed throughout the hematopoietic system, targeting genes that have been associated with proliferation, anti-apoptosis or differentiation.

Fibroblasts share mesenchymal phenotypes with stem cells, but lack their differentiation and colony-forming potential.

BACKGROUND INFORMATION: Although MSCs (mesenchymal stem cells) and fibroblasts have been well studied, differences between these two cell types are not fully understood. We therefore comparatively analysed antigen and gene profiles, colony-forming ability and differentiation potential of four human cell types in vitro: commercially available skin-derived fibroblasts [hSDFs (human skin-derived fibroblasts)], adipose tissue-derived stem cells [hASCs (human adipose tissue-derived stem cells)], embryonic lung fibroblasts (WI38) and dermal microvascular endothelial cells [hECs (human dermal microvascular endothelial cells)]. RESULTS: hSDFs, hASCs and WI38 exhibited a similar spindle-like morphology and expressed same antigen profiles: positive for MSC markers (CD44, CD73 and CD105) and fibroblastic markers [collagen I, HSP47 (heat shock protein 47), vimentin, FSP (fibroblast surface protein) and αSMA (α smooth muscle actin)], and negative for endothelial cell marker CD31 and haemopoietic lineage markers (CD14 and CD45). We further analysed 90 stem cell-associated gene expressions by performing real-time PCR and found a more similar gene expression pattern between hASCs and hSDFs than between hSDFs and WI38. The expression of embryonic stem cell markers [OCT4, KLF4, NANOG, LIN28, FGF4 (fibroblast growth factor 4) and REST] in hASCs and hSDFs was observed to differ more than 2.5-fold as compared with WI38. In addition, hSDFs and hASCs were able to form colonies and differentiate into adipocytes, osteoblasts and chondrocytes in vitro, but not WI38. Moreover, single cell-derived hSDFs and hASCs obtained by clonal expansion were able to differentiate into adipocytes and osteoblasts. However, CD31 positive hECs did not show differentiation potential. CONCLUSIONS: These findings suggest that (i) so-called commercially available fibroblast preparations from skin (hSDFs) consist of a significant number of cells with differentiation potential apart from terminally differentiated fibroblasts; (ii) colony-forming capacity and differentiation potential are specific important properties that discriminate MSCs from fibroblasts (WI38), while conventional stem cell properties such as plastic adherence and the expression of CD44, CD90 and CD105 are unspecific for stem cells.

Limitation of in vivo models investigating angiogenesis in breast cancer.

MSCs reside within their niche and pathologic conditions such as hypoxia and inflammation can lead to mobilization and migration of Mesenchymal Stem Cells (MSCs). Xenograft animal models using immundeficient mouse demonstrated that MSCs migrated to and distributed throughout the tumors and were found to engraft into tumor stroma and vasculature. In contrast, MSCs primarily incorporated within tumor-capsula and did not invade the tumor using immuncompetent tumor allograft models. Here we hypothesize that MSCs migrate primarily towards an inflammatory milieu independent of the underlying biological process causing the inflammation. Murine MSCs (mASCs) were isolated from subcutaneous fat tissues and transduced at passage 0 with lentiviral vector encoding green fluorescent protein (GFP) and luciferase reporter. Breast cancer was established in BALB/c mice by subcutaneous injection of 4T1 cells into the left mammary fat pad. E. coli were injected subcutaneously in the right 4th mammary fat pad. After 24 h luciferase labeled mASCs were administered intraperitoneal (i.p.) and monitored with IVIS Bioluminescence camera for 72 hours. Control group received either tumor implantation or E. coli injection. MSCs significantly migrated towards tumor when compared to control mice without tumor or inflammatory process. However, mASCs injected in 4T1 bearing mice with E. coli only migrated towards the bacterial inflammatory focus. Our results substantiate the notation the MSCs response predominantly to the inflammatory milieu created by bacteria or tumor rather than specifically to the tumor. Thus, it is suggested that the migration of MSCs in immunodeficient mice depends on cancer secreted cytokines due to the lack of the inflammatory response by the immune system. Therefore, in vivo studies investigating the role of MSCs in tumor angiogenesis have shown controversy results and should be interpreted with caution in terms of tumor secreted cytokine dependent stem cell migration.

Breast cancer cells attract the migration of adipose tissue-derived stem cells via the PDGF-BB/PDGFR-beta signaling pathway.

The origin of vascular cells in tumors is unknown, but it is believed that tumors use cells from the host to build new vessels. To determine whether adipose tissue stem cells (ASCs) could be attracted by cancer cells, we performed migration assays in which ASCs were seeded on a transwell migration system top chamber and tumor-conditioned medium was placed in the bottom chamber. Our data showed that a significant number of ASCs migrated toward the tumor-conditioned medium (p<0.0001), and migration of human ASCs significantly (p<0.0001) increased in response to increased concentrations of recombinant PDGF-BB. In addition, neutralizing antibodies to PDGF receptor (PDGFR)-beta decreased migration of ASCs toward a breast cancer-conditioned medium to the level of serum-free control. These data suggest that tumor cell-derived PDGF-BB is an important factor in governing the microenvironment interaction between tumor cells and local tissue-resident stem cells.

PDGF and bFGF modulate tube formation in adipose tissue-derived stem cells.

Recent evidence indicates that mesenchymal stem cells contribute to tumor angiogenesis through yet undefined mechanisms. In the present study, we investigated the angiogenic properties of human adipose tissue-derived stem cells and the mechanisms involved. Freshly isolated human adipose tissue-derived stem cells were seeded onto wells coated with Matrigel and cultured in endothelial growth medium. Capillary-like tube formation was observed after 18 hours culture. Tube formation was significantly reduced in the presence of antibodies against platelet-derived growth factor receptor beta or basic fibroblast growth factor. Collectively, these data suggest that freshly isolated adipose tissue-derived stem cells have the capacity to differentiate into capillary structures and platelet-derived growth factor and bFGF plays a critical role in this process.