Notch-Mediated Tumor-Stroma-Inflammation Networks Promote Invasive Properties and CXCL8 Expression in Triple-Negative Breast Cancer. Liubomirski Yulia,Lerrer Shalom,Meshel Tsipi,Morein Dina,Rubinstein-Achiasaf Linor,Sprinzak David,Wiemann Stefan,Körner Cindy,Ehrlich Marcelo,Ben-Baruch Adit Frontiers in immunology Stromal cells and pro-inflammatory cytokines play key roles in promoting the aggressiveness of triple-negative breast cancers (TNBC; Basal/Basal-like). In our previous study we demonstrated that stimulation of TNBC and mesenchymal stem cells (MSCs) co-cultures by the pro-inflammatory cytokine tumor necrosis factor α (TNFα) has led to increased metastasis-related properties and . In this context, elevated release of the pro-metastatic chemokines CXCL8 (IL-8) and CCL5 (RANTES) was noted in TNFα- and interleukin-1β (IL-1β)-stimulated TNBC:MSC co-cultures; the process was partly (CXCL8) and entirely (CCL5) dependent on physical contacts between the two cell types. Here, we demonstrate that DAPT, inhibitor of γ-secretase that participates in activation of Notch receptors, inhibited the migration and invasion of TNBC cells that were grown in "Contact" co-cultures with MSCs or with patient-derived cancer-associated fibroblasts (CAFs), in the presence of TNFα. DAPT also inhibited the contact-dependent induction of CXCL8, but not of CCL5, in TNFα- and IL-1β-stimulated TNBC:MSC/CAF co-cultures; some level of heterogeneity between the responses of different TNBC cell lines was noted, with MDA-MB-231:MSC/CAF co-cultures being the most sensitive to DAPT. Patient dataset studies comparing basal tumors to luminal-A tumors, and mRNA analyses of Notch receptors in TNBC and luminal-A cells pointed at Notch1 as possible mediator of CXCL8 increase in TNFα-stimulated TNBC:stroma "Contact" co-cultures. Accordingly, down-regulation of Notch1 in TNBC cells by siRNA has substantially reduced the contact-dependent elevation in CXCL8 in TNFα- and also in IL-1β-stimulated TNBC:MSC "Contact" co-cultures. Then, studies in which CXCL8 or p65 (NF-κB pathway) were down-regulated (siRNAs; CRISPR/Cas9) in TNBC cells and/or MSCs, indicated that upon TNFα stimulation of "Contact" co-cultures, p65 was activated and led to CXCL8 production mainly in TNBC cells. Moreover, our findings indicated that when tumor cells interacted with stromal cells in the presence of pro-inflammatory stimuli, TNFα-induced p65 activation has led to elevated Notch1 expression and activation, which then gave rise to elevated production of CXCL8. Overall, tumor:stroma interactions set the stage for Notch1 activation by pro-inflammatory signals, leading to CXCL8 induction and consequently to pro-metastatic activities. These observations may have important clinical implications in designing novel therapy combinations in TNBC. 10.3389/fimmu.2019.00804
    Downregulation of CXCL12 in mesenchymal stromal cells by TGFβ promotes breast cancer metastasis. Yu P F,Huang Y,Xu C L,Lin L Y,Han Y Y,Sun W H,Hu G H,Rabson A B,Wang Y,Shi Y F Oncogene Mesenchymal stromal cells (MSCs) are one of major components of the tumour microenvironment. Recent studies have shown that MSC tumour residence and their close interactions with inflammatory factors are important factors that affect tumour progression. Among tumour-associated inflammatory factors, transforming growth factor β (TGFβ) is regarded as a key determinant of malignancy. By employing a lung metastasis model of a murine breast cancer, we show here that the prometastatic effect of MSCs was dependent on their response to TGFβ. Interestingly, we found that MSC-produced CXCL12, an important chemokine in tumour metastasis, was markedly inhibited by TGFβ. Furthermore, silencing of CXCL12 in TGFβ-unresponsive MSCs restored their ability to promote tumour metastasis. We found that 4T1 breast cancer cells expressed high levels of CXCR7, but not of CXCR4, both of which are CXCL12 receptors. In presence of CXCL12, CXCR7 expression on tumour cells was decreased. Indeed, when CXCR7 was silenced in breast cancer cells, their metastatic ability was inhibited. Therefore, our data demonstrated that sustained expression of CXCL12 by MSCs in the primary tumour site inhibits metastasis through reduction of CXCR7, while, in the presence of TGFβ, this CXCL12 effect of MSCs on tumour cells is relieved. Importantly, elevated CXCR7 and depressed CXCL12 expression levels were prominent features of clinical breast cancer lesions and were related significantly with poor survival. Our findings reveal a novel mechanism of MSC effects on malignant cells through which crosstalk between MSCs and TGFβ regulates tumour metastasis. 10.1038/onc.2016.252
    Mesenchymal Stem/Stromal Cell Engulfment Reveals Metastatic Advantage in Breast Cancer. Chen Yu-Chih,Gonzalez Maria E,Burman Boris,Zhao Xintao,Anwar Talha,Tran Mai,Medhora Natasha,Hiziroglu Ayse B,Lee Woncheol,Cheng Yu-Heng,Choi Yehyun,Yoon Euisik,Kleer Celina G Cell reports Twenty percent of breast cancer (BC) patients develop distant metastasis for which there is no cure. Mesenchymal stem/stromal cells (MSCs) in the tumor microenvironment were shown to stimulate metastasis, but the mechanisms are unclear. Here, we identified and quantified cancer cells engulfing stromal cells in clinical samples of BC metastasis by dual immunostaining for EZH2 and ALDH1 expression. Using flow cytometry and a microfluidic single-cell paring and retrieval platform, we show that MSC engulfment capacity is associated with BC cell metastatic potential and generates cells with mesenchymal-like, invasion, and stem cell traits. Whole-transcriptome analyses of selectively retrieved engulfing BC cells identify a gene signature of MSC engulfment consisting of WNT5A, MSR1, ELMO1, IL1RL2, ZPLD1, and SIRPB1. These results delineate a mechanism by which MSCs in the tumor microenvironment promote metastasis and provide a microfluidic platform with the potential to predict BC metastasis in clinical samples. 10.1016/j.celrep.2019.05.084
    Mesenchymal Stem Cell-Secreted Extracellular Vesicles Instruct Stepwise Dedifferentiation of Breast Cancer Cells into Dormancy at the Bone Marrow Perivascular Region. Sandiford Oleta A,Donnelly Robert J,El-Far Markos H,Burgmeyer Lisa M,Sinha Garima,Pamarthi Sri Harika,Sherman Lauren S,Ferrer Alejandra I,DeVore Dariana E,Patel Shyam A,Naaldijk Yahaira,Alonso Sara,Barak Pradeep,Bryan Margarette,Ponzio Nicholas M,Narayanan Ramaswamy,Etchegaray Jean-Pierre,Kumar Rakesh,Rameshwar Pranela Cancer research In the bone marrow (BM), breast cancer cells (BCC) can survive in dormancy for decades as cancer stem cells (CSC), resurging as tertiary metastasis. The endosteal region where BCCs exist as CSCs poses a challenge to target them, mostly due to the coexistence of endogenous hematopoietic stem cells. This study addresses the early period of dormancy when BCCs enter BM at the perivascular region to begin the transition into CSCs, which we propose as the final step in dormancy. A two-step process comprises the Wnt-β-catenin pathway mediating BCC dedifferentiation into CSCs at the BM perivascular niche. At this site, BCCs responded to two types of mesenchymal stem cell (MSC)-released extracellular vesicles (EV) that may include exosomes. Early released EVs began the transition into cycling quiescence, DNA repair, and reorganization into distinct BCC subsets. After contact with breast cancer, the content of EVs changed (primed) to complete dedifferentiation into a more homogeneous population with CSC properties. BCC progenitors (Oct4alo), which are distant from CSCs in a hierarchical stratification, were sensitive to MSC EVs. Despite CSC function, Oct4alo BCCs expressed multipotent pathways similar to CSCs. Oct4alo BCCs dedifferentiated and colocalized with MSCs (murine and human BM) . Overall, these findings elucidate a mechanism of early dormancy at the BM perivascular region and provide evidence of epigenome reorganization as a potential new therapy for breast cancer. SIGNIFICANCE: These findings describe how the initial process of dormancy and dedifferentiation of breast cancer cells at the bone marrow perivascular niche requires mesenchymal stem cell-derived exosomes, indicating a potential target for therapeutic intervention. 10.1158/0008-5472.CAN-20-2434
    Biomimetic nanovesicles made from iPS cell-derived mesenchymal stem cells for targeted therapy of triple-negative breast cancer. Zhao Qingguo,Hai Bo,Zhang Xiao,Xu Jing,Koehler Brian,Liu Fei Nanomedicine : nanotechnology, biology, and medicine Nanoparticles made from membrane of mesenchymal stem cells (MSCs) showed active targeting capacities to prostate and lung cancers, but further studies are hindered by limited expandability and donor variations of tissue-derived MSCs. We have derived MSCs with an unlimited supply and uniform homing capacity to triple-negative breast cancer (TNBC) from human induced pluripotent stem cells (iPSCs). By breaking down intact iPSC-MSCs, we efficiently developed nanovesicles that selectively accumulated in primary and metastatic TNBC after systemic infusion in mouse models. When loaded with a chemotherapeutic drug doxorubicin, iPSC-MSC nanovesicles showed superior cytotoxic effects on doxorubicin-resistant TNBC cells, and significantly decreased the incidence and burden of metastases in mouse models of spontaneous and experimental metastatic TNBC compared with free or liposomal doxorubicin. These nanovesicles showed no detectable immunogenicity or toxicity, and are stable after storage. Our data indicate that iPSC-MSC nanovesicles are promising to improve TNBC treatment as a standardized targeting platform. 10.1016/j.nano.2019.102146
    Tumor-Stroma-Inflammation Networks Promote Pro-metastatic Chemokines and Aggressiveness Characteristics in Triple-Negative Breast Cancer. Liubomirski Yulia,Lerrer Shalom,Meshel Tsipi,Rubinstein-Achiasaf Linor,Morein Dina,Wiemann Stefan,Körner Cindy,Ben-Baruch Adit Frontiers in immunology The tumor microenvironment (TME) plays key roles in promoting disease progression in the aggressive triple-negative subtype of breast cancer (TNBC; Basal/Basal-like). Here, we took an integrative approach and determined the impact of tumor-stroma-inflammation networks on pro-metastatic phenotypes in TNBC. With the TCGA dataset we found that the pro-inflammatory cytokines tumor necrosis factor α (TNFα) and interleukin 1β (IL-1β), as well as their target pro-metastatic chemokines CXCL8 (IL-8), CCL2 (MCP-1), and CCL5 (RANTES) were expressed at significantly higher levels in basal patients than luminal-A patients. Then, we found that TNFα- or IL-1β-stimulated co-cultures of TNBC cells (MDA-MB-231, MDA-MB-468, BT-549) with mesenchymal stem cells (MSCs) expressed significantly higher levels of CXCL8 compared to non-stimulated co-cultures or each cell type alone, with or without cytokine stimulation. CXCL8 was also up-regulated in TNBC co-cultures with breast cancer-associated fibroblasts (CAFs) derived from patients. CCL2 and CCL5 also reached the highest expression levels in TNFα/IL-1β-stimulated TNBC:MSC/CAF co-cultures. The elevations in CXCL8 and CCL2 expression partly depended on direct physical contacts between the tumor cells and the MSCs/CAFs, whereas CCL5 up-regulation was entirely dependent on cell-to-cell contacts. Supernatants of TNFα-stimulated TNBC:MSC "Contact" co-cultures induced robust endothelial cell migration and sprouting. TNBC cells co-cultured with MSCs and TNFα gained migration-related morphology and potent migratory properties; they also became more invasive when co-cultured with MSCs/CAFs in the presence of TNFα. Using siRNA to CXCL8, we found that CXCL8 was significantly involved in mediating the pro-metastatic activities gained by TNFα-stimulated TNBC:MSC "Contact" co-cultures: angiogenesis, migration-related morphology of the tumor cells, as well as cancer cell migration and invasion. Importantly, TNFα stimulation of TNBC:MSC "Contact" co-cultures has increased the aggressiveness of the tumor cells , leading to higher incidence of mice with lung metastases than non-stimulated TNBC:MSC co-cultures. Similar tumor-stromal-inflammation networks established in-culture with luminal-A cells demonstrated less effective or differently-active pro-metastatic functions than those of TNBC cells. Overall, our studies identify novel tumor-stroma-inflammation networks that may promote TNBC aggressiveness by increasing the pro-malignancy potential of the TME and of the tumor cells themselves, and reveal key roles for CXCL8 in mediating these metastasis-promoting activities. 10.3389/fimmu.2019.00757
    MSC-regulated microRNAs converge on the transcription factor FOXP2 and promote breast cancer metastasis. Cuiffo Benjamin G,Campagne Antoine,Bell George W,Lembo Antonio,Orso Francesca,Lien Evan C,Bhasin Manoj K,Raimo Monica,Hanson Summer E,Marusyk Andriy,El-Ashry Dorraya,Hematti Peiman,Polyak Kornelia,Mechta-Grigoriou Fatima,Mariani Odette,Volinia Stefano,Vincent-Salomon Anne,Taverna Daniela,Karnoub Antoine E Cell stem cell Mesenchymal stem/stromal cells (MSCs) are progenitor cells shown to participate in breast tumor stroma formation and to promote metastasis. Despite expanding knowledge of their contributions to breast malignancy, the underlying molecular responses of breast cancer cells (BCCs) to MSC influences remain incompletely understood. Here, we show that MSCs cause aberrant expression of microRNAs, which, led by microRNA-199a, provide BCCs with enhanced cancer stem cell (CSC) properties. We demonstrate that such MSC-deregulated microRNAs constitute a network that converges on and represses the expression of FOXP2, a forkhead transcription factor tightly associated with speech and language development. FOXP2 knockdown in BCCs was sufficient in promoting CSC propagation, tumor initiation, and metastasis. Importantly, elevated microRNA-199a and depressed FOXP2 expression levels are prominent features of malignant clinical breast cancer and are associated significantly with poor survival. Our results identify molecular determinants of cancer progression of potential utility in the prognosis and therapy of breast cancer. 10.1016/j.stem.2014.10.001
    MSCs inhibit tumor progression and enhance radiosensitivity of breast cancer cells by down-regulating Stat3 signaling pathway. He Ningning,Kong Yangyang,Lei Xudan,Liu Yang,Wang Jinhan,Xu Chang,Wang Yan,Du Liqing,Ji Kaihua,Wang Qin,Li Zongjin,Liu Qiang Cell death & disease The acquisition of radioresistance by breast cancer cells during radiotherapy may lead to cancer recurrence and poor survival. Signal transducer and activator of transcription 3 (Stat3) is activated in breast cancer cells and, therefore, may be an effective target for overcoming therapeutic resistance. Mesenchymal stem cells (MSCs) have been investigated for use in cancer treatment. Here, we investigated the potential of MSC conditioned medium (MSC-CM) in sensitizing breast cancer to radiotherapy. It was found that MSC-CM could inhibit the level of activated Stat3, suppress cancer growth, and exhibit synergetic effects with radiation treatment in vitro and in vivo. Furthermore, MSC-CM reduced the ALDH-positive cancer stem cells (CSCs) population, modulated several potential stem cell markers, and decreased tumor migration, as well as metastasis. These results demonstrate that MSC-CM suppresses breast cancer cells growth and sensitizes cancer cells to radiotherapy through inhibition of the Stat3 signaling pathway, thus, providing a novel strategy for breast cancer therapy by overcoming radioresistance. 10.1038/s41419-018-0949-3
    MiR-375 inhibits the hepatocyte growth factor-elicited migration of mesenchymal stem cells by downregulating Akt signaling. He Lihong,Wang Xianyao,Kang Naixin,Xu Jianwei,Dai Nan,Xu Xiaojing,Zhang Huanxiang Cell and tissue research The migration of mesenchymal stem cells (MSCs) is critical for their use in cell-based therapies. Accumulating evidence suggests that microRNAs are important regulators of MSC migration. Here, we report that the expression of miR-375 was downregulated in MSCs treated with hepatocyte growth factor (HGF), which strongly stimulates the migration of these cells. Overexpression of miR-375 decreased the transfilter migration and the migration velocity of MSCs triggered by HGF. In our efforts to determine the mechanism by which miR-375 affects MSC migration, we found that miR-375 significantly inhibited the activation of Akt by downregulating its phosphorylation at T308 and S473, but had no effect on the activity of mitogen-activated protein kinases. Further, we showed that 3'phosphoinositide-dependent protein kinase-1 (PDK1), an upstream kinase necessary for full activation of Akt, was negatively regulated by miR-375 at the protein level. Moreover, miR-375 suppressed the phosphorylation of focal adhesion kinase (FAK) and paxillin, two important regulators of focal adhesion (FA) assembly and turnover, and decreased the number of FAs at cell periphery. Taken together, our results demonstrate that miR-375 inhibits HGF-elicited migration of MSCs through downregulating the expression of PDK1 and suppressing the activation of Akt, as well as influencing the tyrosine phosphorylation of FAK and paxillin and FA periphery distribution. 10.1007/s00441-017-2765-y
    Breast cancer carcinoma-associated fibroblasts differ from breast fibroblasts in immunological and extracellular matrix regulating pathways. Pasanen I,Lehtonen S,Sormunen R,Skarp S,Lehtilahti E,Pietilä M,Sequeiros R Blanco,Lehenkari P,Kuvaja P Experimental cell research Tumor stroma has been recently shown to play a crucial role in the development of breast cancer. Since the origin of the stromal cells in the tumor is unknown, we have examined differences and similarities between three stromal cell types of mesenchymal origin, namely carcinoma associated fibroblasts from breast tumor (CAFs), fibroblasts from normal breast area (NFs) and bone marrow derived mesenchymal stromal cells (MSCs). In a microarray analysis, immunological, developmental and extracellular matrix -related pathways were over-represented in CAFs when compared to NFs (p<0.001). Under hypoxic conditions, the expression levels of pyruvate dehydrogenase kinase-1 (PDK1) and pyruvate dehydrogenase kinase-4 (PDK4) were lower in CAFs when compared to NFs (fold changes 0.6 and 0.4, respectively). In normoxia, when compared to NFs, CAFs displayed increased expression of glucose transporter 1 (GLUT-1) and PDK1 (fold changes 1.5 and 1.3, respectively). With respect to the assessed surface markers, only CD105 was expressed differently in MSCs when compared to fibroblasts, being more often expressed on MSCs. Cells with myofibroblast features were present in both NF and CAF samples. We conclude, that CAFs differ distinctly from NFs at the gene expression level, this hypothesis was also tested in silico for other available gene expression data. 10.1016/j.yexcr.2016.04.016