Identification of Human Breast Adipose Tissue Progenitors Displaying Distinct Differentiation Potentials and Interactions with Cancer Cells

Breast adipose tissue (AT) participates in the physiological evolution and remodeling of the mammary gland due to its high plasticity. It is also a favorable microenvironment for breast cancer progression. However, information on the properties of human breast adipose progenitor cells (APCs) involved in breast physiology or pathology is scant. We performed differential enzymatic dissociation of human breast AT lobules. We isolated and characterized two populations of APCs. Here we report that these distinct breast APC populations selectively expressed markers suitable for characterization. The population preferentially expressing ALPL (MSCA1) showed higher adipogenic potential. The population expressing higher levels of INHBA and CD142 acquired myofibroblast characteristics upon TGF-β treatment and a myo-cancer-associated fibroblast profile in the presence of breast cancer cells. This population expressed the immune checkpoint CD274 (PD-L1) and facilitated the expansion of breast cancer mammospheres compared with the adipogenic population. Indeed, the breast, as with other fat depots, contains distinct types of APCs with differences in their ability to specialize. This indicates that they were differentially involved in breast remodeling. Their interactions with breast cancer cells revealed differences in the potential for tumor dissemination and estrogen receptor expression, and these differences might be relevant to improve therapies targeting the tumor microenvironment

Impact of thermogenesis induced by chronic β3-adrenergic receptor agonist treatment on inflammatory and infectious response during bacteremia in mice

International audienceWhite adipocytes store energy differently than brown and brite adipocytes which dissipate energy under the form of heat. Studies have shown that adipocytes are able to respond to bacteria thanks to the presence of Toll-like receptors at their surface. Despite this, little is known about the involvement of each class of adipocytes in the infectious response. We treated mice for one week with a β3-adrenergic receptor agonist to induce activation of brown adipose tissue and brite adipocytes within white adipose tissue. Mice were then injected intraperitoneally with E . coli to generate acute infection. The metabolic, infectious and inflammatory parameters of the mice were analysed during 48 hours after infection. Our results shown that in response to bacteria, thermogenic activity promoted a discrete and local anti-inflammatory environment in white adipose tissue characterized by the increase of the IL-1RA secretion. More generally, activation of brown and brite adipocytes did not modify the host response to infection including no additive effect with fever and an equivalent bacteria clearance and inflammatory response. In conclusion, these results suggest an IL-1RA-mediated immunomodulatory activity of thermogenic adipocytes in response to acute bacterial infection and open a way to characterize their effect along more chronic infection as septicaemia

Regulation of Adipose Progenitor Cell Expansion in a Novel Micro-Physiological Model of Human Adipose Tissue Mimicking Fibrotic and Pro-Inflammatory Microenvironments

The expansion of adipose progenitor cells (APCs) plays an important role in the regeneration of the adipose tissue in physiological and pathological situations. The major role of CD26-expressing APCs in the generation of adipocytes has recently been highlighted, revealing that the CD26 APC subtype displays features of multipotent stem cells, giving rise to CD54- and CD142-expressing preadipocytes. However, a relevant human in vitro model to explore the regulation of the APC subpopulation expansion in lean and obese adipose tissue microenvironments is still lacking. In this work, we describe a novel adipose tissue model, named ExAdEx, that can be obtained from cosmetic surgery wastes. ExAdEx products are adipose tissue units maintaining the characteristics and organization of adipose tissue as it presents in vivo. The model was viable and metabolically active for up to two months and could adopt a pathological-like phenotype. The results revealed that inflammatory and fibrotic microenvironments differentially regulated the expansion of the CD26 APC subpopulation and its CD54 and CD142 APC progenies. The approach used significantly improves the method of generating adipose tissue models, and ExAdEx constitutes a relevant model that could be used to identify pathways promoting the expansion of APCs in physiological and pathological microenvironments

Clues to CD2-associated Protein Involvement in Cytokinesis

Cytokinesis requires membrane trafficking coupled to actin remodeling and involves a number of trafficking molecules. CD2-associated protein (CD2AP) has been implicated in dynamic actin remodeling and membrane trafficking that occurs during endocytosis leading to the degradative pathway. In this study, we present several arguments for its implication in cytokinesis. First, endogenous CD2AP was found concentrated in the narrow region of the midzone microtubules during anaphase and in the midbody during late telophase. Moreover, we found that CD2AP is a membrane- and not a microtubule-associated protein. Second, the overexpression of the first two Src homology 3 domains of CD2AP, which are responsible for this localization, led to a significant increase in the rate of cell multinucleation. Third, the CD2AP small interfering RNA interfered with the cell separation, indicating that CD2AP is required for HeLa cells cytokinesis. Fourth, using the yeast two-hybrid system, we found that CD2AP interacted with anillin, a specific cleavage furrow component, and the two proteins colocalized at the midbody. Both CD2AP and anillin were found phosphorylated early in mitosis and also CD2AP phosphorylation was coupled to its delocalization from membrane to cytosol. All these observations led us to propose CD2AP as a new player in cytokinesis

Diet Supplementation in ω3 Polyunsaturated Fatty Acid Favors an Anti-Inflammatory Basal Environment in Mouse Adipose Tissue

Oxylipins are metabolized from dietary ω3 and ω6 polyunsaturated fatty acids and are involved in an inflammatory response. Adipose tissue inflammatory background is a key factor of metabolic disorders and it is accepted that dietary fatty acids, in terms of quality and quantity, modulate oxylipin synthesis in this tissue. Moreover, it has been reported that diet supplementation in ω3 polyunsaturated fatty acids resolves some inflammatory situations. Thus, it is crucial to assess the influence of dietary polyunsaturated fatty acids on oxylipin synthesis and their impact on adipose tissue inflammation. To this end, mice fed an ω6- or ω3-enriched standard diet (ω6/ω3 ratio of 30 and 3.75, respectively) were analyzed for inflammatory phenotype and adipose tissue oxylipin content. Diet enrichment with an ω3 polyunsaturated fatty acid induced an increase in the oxylipins derived from ω6 linoleic acid, ω3 eicosapentaenoic, and ω3 docosahexaenoic acids in brown and white adipose tissues. Among these, the level of pro-resolving mediator intermediates, as well as anti-inflammatory metabolites, were augmented. Concomitantly, expressions of M2 macrophage markers were increased without affecting inflammatory cytokine contents. In vitro, these metabolites did not activate macrophages but participated in macrophage polarization by inflammatory stimuli. In conclusion, we demonstrated that an ω3-enriched diet, in non-obesogenic non-inflammatory conditions, induced synthesis of oxylipins which were involved in an anti-inflammatory response as well as enhancement of the M2 macrophage molecular signature, without affecting inflammatory cytokine secretion

Isolation of head and neck squamous carcinoma cancer stem-like cells in a syngeneic mouse model and analysis of hypoxia effect.

International audienceThe incidence of oral tumors is increasing around the world and despite recent advances in early detection and diagnosis, current treatments are still unsatisfactory. Recent data suggest that tumor persistence and recurrence could be due to the presence of a rare cell population called cancer stem cells (CSCs), which are generally spared by traditional treatments. Therefore, identification and characterization of CSCs are extremely important to develop novel and effective treatment strategies for cancer. The aim of this study was to identify and isolate CSCs in an established murine head and neck squamous cell carcinoma (HNSCC) cell line and to investigate the influence of hypoxic conditions on the isolated cell popul-ation. Using the expression of the aldehyde dehydrogenase 1 (ALDH1) enzymatic activity, which is now recognized as a CSC marker in various tumors, we isolated a cell population expressing high levels of ALDH1 (ALDH1high) representing 1±0.6% in the murine SCC-VII cell line. These cells were injected subcutaneously in syngeneic animals to evaluate their tumorigenic properties. For the lowest injected cell dose (250 injected cells), tumor occurrence and median tumor size were higher in ALDH1high injected mice than in ALDH1low injected mice. Following an in vivo passage and culture in serum-free medium, the percentage of ALDH1high cells increased by 3‑fold in SCC-VII CSCs (oral spheres) compared to the SCC-VII cell line. This percentage was further increased when oral spheres were cultured under hypoxic conditions. In conclusion, this study reports for the first time the isolation of HNSCC CSCs in a syngeneic mouse model and the use of hypoxia as a method to further enrich the ALDH1high cell population

Dynamic Characterization of the Molecular Events During In Vitro Epidermal Wound Healing

The aim of this study was to characterize some of the molecular events stimulated in vitro in response to injury within a confluent culture of normal epidermal keratinocytes as a model to understand the mechanisms of wound healing. To this end, an original device was developed specifically designed to perform calibrated injuries of great lengths within mono-stratified or pluri-stratified keratinocyte cultures. The experiments performed in this study validate this device as an appropriate tool for studying epidermal wound healing; this is because it performs mechanical injuries that stimulate the expression of multiple healing markers also known to be upregulated during wound healing in vivo (growth factors, cytokines, proteinases, extracellular matrix proteins). Using this device, it was demonstrated in human keratinocytes: mechanical injuries (i) immediately stimulate the tyrosine phosphory lation of numerous cellular proteins; (ii) induce molecular cascades leading to the activation of p21ras, mitogen-activated protein kinases, extracellular signal-regulated kinases 1/2, c-Jun NH2 terminal kinase, and p38 mitogen-activated protein kinase; and (iii) increase the phosphorylation of their respective substrates, c-jun and activator transcription factor 1. Wounding of these cells also results in increases in the DNA binding activities of several jun/fos activator protein-1 transcription factor complexes. It is important to note that the development of an appropriate wounding system was essential for performing this study, as use of a classical wounding procedure did not enable the detection of the biologic parameters reported above. In conclusion, these data indicate that using the appropriate system, it is possible to identify the signaling pathways activated in normal human keratinocyte cells after injury. In this study, it was shown that the mitogen-activated protein kinase pathways and activator protein-1 are stimulated in response to physical injury, and may be involved in regulating the expression of healing markers

The antidiabetic drug metformin exerts an antitumoral effect in vitro and in vivo through a decrease of cyclin D1 level.

International audienceMetformin is a widely used antidiabetic agent, which regulates glucose homeostasis through inhibition of liver glucose production and an increase in muscle glucose uptake. Recent studies suggest that metformin may reduce the risk of cancer, but its mode of action in cancer remains not elucidated. We investigated the effect of metformin on human prostate cancer cell proliferation in vitro and in vivo. Metformin inhibited the proliferation of DU145, PC-3 and LNCaP cancer cells with a 50% decrease of cell viability and had a modest effect on normal prostate epithelial cell line P69. Metformin did not induce apoptosis but blocked cell cycle in G(0)/G(1). This blockade was accompanied by a strong decrease of cyclin D1 protein level, pRb phosphorylation and an increase in p27(kip) protein expression. Metformin activated the AMP kinase pathway, a fuel sensor signaling pathway. However, inhibition of the AMPK pathway using siRNA against the two catalytic subunits of AMPK did not prevent the antiproliferative effect of metformin in prostate cancer cells. Importantly, oral and intraperitoneal treatment with metformin led to a 50 and 35% reduction of tumor growth, respectively, in mice bearing xenografts of LNCaP. Similar, to the in vitro study, metformin led to a strong reduction of cyclin D1 protein level in tumors providing evidence for a mechanism that may contribute to the antineoplastic effects of metformin suggested by recent epidemiological studies

Arrest of WNT/β-catenin signaling enables the transition from pluripotent to differentiated germ cells in mouse ovaries

International audienceGerm cells form the basis for sexual reproduction by producing gametes. In ovaries, primordial germ cells exit the cell cycle and the pluripotency-associated state, differentiate into oogonia, and initiate meiosis. Despite the importance of germ cell differentiation for sexual reproduction, signaling pathways regulating their fate remain largely unknown. Here, we show in mouse embryonic ovaries that germ cell–intrinsic β-catenin activity maintains pluripotency and that its repression is essential to allow differentiation and meiosis entry in a timely manner. Accordingly, in β-catenin loss-of-function and gain-of-function mouse models, the germ cells precociously enter meiosis or remain in the pluripotent state, respectively. We further show that interaction of β-catenin and the pluripotent-associated factor POU5F1 in the nucleus is associated with germ cell pluripotency. The exit of this complex from the nucleus correlates with germ cell differentiation, a process promoted by the up-regulation of Znrf3 , a negative regulator of WNT/β-catenin signaling. Together, these data identify the molecular basis of the transition from primordial germ cells to oogonia and demonstrate that β-catenin is a central gatekeeper in ovarian differentiation and gametogenesis