Interferon regulatory factor 7 impairs cellular metabolism in aging adipose-derived stromal cells.

Dysregulated immunity and widespread metabolic dysfunctions are the most relevant hallmarks of the passing of time over the course of adult life, and their combination at midlife is strongly related to increased vulnerability to diseases; however, the causal connection between them remains largely unclear. By combining multi-omics and functional analyses of adipose-derived stromal cells established from young (1 month) and midlife (12 months) mice, we show that an increase in expression of interferon regulatory factor 7 (IRF7) during adult life drives major metabolic changes, which include impaired mitochondrial function, altered amino acid biogenesis and reduced expression of genes involved in branched-chain amino acid (BCAA) degradation. Our results draw a new paradigm of aging as the 'sterile' activation of a cell-autonomous pathway of self-defense and identify a crucial mediator of this pathway, IRF7, as driver of metabolic dysfunction with age.

In Vitro Characterization of Adipose Stem Cells Non-Enzymatically Extracted from the Thigh and Abdomen.

Autologous fat grafting is a surgical technique in which adipose tissue is transferred from one area of the body to another, in order to reconstruct or regenerate damaged or injured tissues. Before reinjection, adipose tissue needs to be purified from blood and cellular debris to avoid inflammation and preserve the graft viability. To perform this purification, different enzymatic and mechanical methods can be used. In this study, we characterized in vitro the product of a closed automatic device based on mechanical disaggregation, named Rigenera®, focusing on two sites of adipose tissue harvesting. At first, we optimized the Rigenera® operating timing, demonstrating that 60 s of treatment allows a higher cellular yield, in terms of the cell number and growth rate. This result optimizes the mechanical disaggregation and it can increase the clinical efficiency of the final product. When comparing the extracted adipose samples from the thigh and abdomen, our results showed that the thigh provides a higher number of mesenchymal-like cells, with a faster replication rate and a higher ability to form colonies. We can conclude that by collecting adipose tissue from the thigh and treating it with the Rigenera® device for 60 s, it is possible to obtain the most efficient product.

A Non-Enzymatic Method to Obtain a Fat Tissue Derivative Highly Enriched in Adipose Stem Cells (ASCs) from Human Lipoaspirates: Preliminary Results.

Adipose tissue possesses phenotypic gene expression characteristics that are similar to human mesenchymal stem cells (hMSCs). Nevertheless, the multilineage potential may be inhibited, and cells may not expand adequately to satisfy the requirements of Good Manufacturing Practice (cGMP). An autologous hMSC-enriched fat product would fulfil the void from a biomedical and clinical perspective. In this study, we suggest a novel mechanism using a closed system without enzymes, additives or other modifications, which will produce non-expanded, accessible material. This decentralized fat product, unlike unprocessed lipoaspirates, adequately encloses the vascular stroma with adipocytes and stromal stalks along with their vascular channels and lumina. This fat product contained hASCs and fewer hematopoietic elements such as lipoaspirates, which were digested enzymatically according to flow cytometric investigations, and molecular analysis also showed significant hASC uniformity within the cells of the stromal vascular tissue. Moreover, the fat product produced a higher quantity of hASCs similar to hMSCs in isolation with the typical characteristics of an osteogenic, chondrogenic and adipogenic lineage. Interestingly, these properties were evident in the non-enzymatic derived adipose tissue, as opposed to hASCs in isolation from the enzymatically digested lipoaspirates, suggesting that the aforementioned procedure may be an adequate alternative to regenerate and engineer tissue for the treatment of various medical conditions and promote efficient patient recovery.

Quantum dots labelling allows detection of the homing of mesenchymal stem cells administered as immunomodulatory therapy in an experimental model of pancreatic islets transplantation.

Cell transplantation is considered a promising therapeutic approach in several pathologies but still needs innovative and non-invasive imaging technologies to be validated. The use of mesenchymal stem cells (MSCs) attracts major interest in clinical transplantation thanks to their regenerative properties, low immunogenicity and ability to regulate immune responses. In several animal models, MSCs are used in co-transplantation with pancreatic islets (PIs) for the treatment of type I diabetes, supporting graft survival and prolonging normal glycaemia levels. In this study we investigated the homing of systemically administered MSCs in a rat model of pancreatic portal vein transplantation. MSCs labelled with quantum dots (Qdots) were systemically injected by tail vein and monitored by optical fluorescence imaging. The fluorescence signal of the liver in animals co-transplanted with MSCs and PIs was significantly higher than in control animals in which MSCs alone were transplanted. By using magnetic labelling of PIs, the homing of PIs into liver was independently confirmed. These results demonstrate that MSCs injected in peripheral blood vessels preferentially accumulate into liver when PIs are transplanted in the same organ. Moreover, we prove that bimodal MRI-fluorescence imaging allows specific monitoring of the fate of two types of cells.

The transcriptional profile of adipose-derived stromal cells (ASC) mirrors the whitening of adipose tissue with age.

Multipotent stem cells persist within the stromal vascular fraction (SVF) of adipose tissue during adulthood. These cells, commonly referred to as adipose-derived stromal cells (ASC), have been extensively investigated over the past years as a promising therapeutic tool based on their regenerative and immunomodulatory properties. However, how ASC might mirror the age-related alteration of the fat they reside in remains unclear. Herein, we show that inguinal adipose tissue in mice turns from brown/beige- to white-like with age and resident ASC readily mirror these changes both at mRNA and microRNA transcriptional level. Mechanistically, our data suggest that these brown/age-related changes in ASC transcription rely on changes in the activity of E2F1 and NFkB transcription factors.

Improving the Cellular Uptake of Biomimetic Magnetic Nanoparticles.

Magnetococcus marinus magnetosome-associated protein MamC, expressed as recombinant, has been proven to mediate the formation of novel biomimetic magnetic nanoparticles (BMNPs) that are successful drug nanocarriers for targeted chemotherapy and hyperthermia agents. These BMNPs present several advantages over inorganic magnetic nanoparticles, such as larger sizes that allow the former to have larger magnetic moment per particle, and an isoelectric point at acidic pH values, which allows both the stable functionalization of BMNPs at physiological pH value and the molecule release at acidic (tumor) environments, simply based on electrostatic interactions. However, difficulties for BMNPs cell internalization still hold back the efficiency of these nanoparticles as drug nanocarriers and hyperthermia agents. In the present study we explore the enhanced BMNPs internalization following upon their encapsulation by poly (lactic-co-glycolic) acid (PLGA), a Food and Drug Administration (FDA) approved molecule. Internalization is further optimized by the functionalization of the nanoformulation with the cell-penetrating TAT peptide (TATp). Our results evidence that cells treated with the nanoformulation [TAT-PLGA(BMNPs)] show up to 80% more iron internalized (after 72 h) compared to that of cells treated with BMNPs (40%), without any significant decrease in cell viability. This nanoformulation showing optimal internalization is further characterized. In particular, the present manuscript demonstrates that neither its magnetic properties nor its performance as a hyperthermia agent are significantly altered due to the encapsulation. In vitro experiments demonstrate that, following upon the application of an alternating magnetic field on U87MG cells treated with BMNPs and TAT-PLGA(BMNPs), the cytotoxic effect of BMNPs was not affected by the TAT-PLGA enveloping. Based on that, difficulties shown in previous studies related to poor cell uptake of BMNPs can be overcome by the novel nanoassembly described here.

Comparative technical analysis of lipoaspirate mechanical processing devices.

Fat grafting is a well-established procedure in reconstructive, aesthetic, and regenerative medicine, in particular due to the presence in the adipose tissue of a high concentration of mesenchymal stem cells. The need to reduce fat processing times, for an immediate clinical use and regulatory restrictions on the degree of manipulation of human tissues, has led to the development of numerous devices for the mechanical, nonenzymatic processing of adipose tissue. The aim of this study is to describe the state of the art of mechanical devices used for fat processing, performing a technical analysis of the currently commercially available devices. This should facilitate the development of new devices that improve therapeutic results.

Simple and Rapid Non-Enzymatic Procedure Allows the Isolation of Structurally Preserved Connective Tissue Micro-Fragments Enriched with SVF.

The stromal vascular fraction (SVF) consists of a heterogeneous population of stem and stromal cells, generally obtained from adipose tissue by enzymatic digestion. For human cell-based therapies, mechanical process methods to obtain SVF represent an advantageous approach because they have fewer regulatory restrictions for their clinical use. The aim of this study was to characterize a novel commercial system for obtaining SVF from adipose tissue by a mechanical approach without substantial manipulations. Lipoaspirate samples collected from 27 informed patients were processed by a simple and fast mechanical system (by means of Hy-Tissue SVF). The Hy-Tissue SVF product contained a free cell fraction and micro-fragments of stromal connective tissue. The enzymatic digestion of the micro-fragments increased the yield of free cells (3.2 times) and CFU-F (2.4 times). Additionally, 10% of free cells from SVF were positive for CD34+, suggesting the presence of endothelial cells, pericytes, and potential adipose-derived stem cells (ADSC). Moreover, the SVF cells were able to proliferate and differentiate in vitro toward adipocytes, osteocytes, and chondrocytes. The immunophenotypic analysis of expanded cells showed positivity for typical mesenchymal stem cell markers. The Hy-Tissue SVF system allows the isolation of stromal vascular fraction, making this product of potential interest in regenerative medicine.

Artificial dermal substitutes for tissue regeneration: comparison of the clinical outcomes and histological findings of two templates.

OBJECTIVE: Artificial dermal substitutes (DSs) are fundamental in physiological wound healing to ensure consistent and enduring wound closure and provide a suitable scaffold to repair tissue. We compared the clinical and histological features of two DSs, Pelnac and Integra, in the treatment of traumatic and iatrogenic skin defects. METHODS: This prospective observational study involved 71 randomly selected patients from our hospital. Wound healing was analyzed using the Wound Surface Area Assessment, the Vancouver Scar Scale, and a visual analog scale. Histological and immunohistochemical evaluations were also performed. RESULTS: At 2 weeks, greater regeneration with respect to proliferation of the epidermis and renewal of the dermis was observed with Pelnac than with Integra. At 4 weeks, the dermis had regenerated with both DSs. Both templates induced renewed collagen and revascularization. Differences in the Vancouver Scar Scale score were statistically significant at 4 weeks and 1 year. Pelnac produced a significant increase in contraction at 2 weeks with increasing effectiveness at 4 weeks. Integra produced a higher percentage reduction in the wound surface area and a shorter healing time than Pelnac for wounds >1.5 cm deep. CONCLUSION: Our observational data indicate that both DSs are effective and applicable in different clinical contexts.

A Correlative Imaging Study of in vivo and ex vivo Biodistribution of Solid Lipid Nanoparticles.

PURPOSE: Solid lipid nanoparticles are largely used in biomedical research and are characterized by high stability and biocompatibility and are also able to improve the stability of various loaded molecules. In vitro studies demonstrated that these nanoparticles are low cytotoxic, while in vivo studies proved their efficiency as nanocarriers for molecules characterized by a low bioavailability. However, to our knowledge, no data on the systemic biodistribution and organ accumulation of solid lipid nanoparticles in itself are presently available. METHODS: In this view, we investigated the solid lipid nanoparticles biodistribution by a multimodal imaging approach correlating in vivo and ex vivo analyses. We loaded solid lipid nanoparticles with two different fluorophores (cardiogreen and rhodamine) to observe them with an optical imager in the whole organism and in the excised organs, and with fluorescence microscopy in tissue sections. Light and transmission electron microscopy analyses were also performed to evaluate possible structural modification or damage due to nanoparticle administration. RESULTS: Solid lipid nanoparticles loaded with the two fluorochromes showed good optic characteristics and stable polydispersity. After in vivo administration, they were clearly detectable in the organism. Four  hours after the injection, the fluorescent signal occurred in anatomical districts corresponding to the liver and this was confirmed by the ex vivo acquisitions of excised organs. Brightfield, fluorescence and transmission electron microscopy confirmed solid lipid nanoparticles accumulation in hepatocytes without structural damage. CONCLUSION: Our results support the systemic biocompatibility of solid lipid nanoparticles and demonstrate their detailed biodistribution from the whole organism to organs until the cells.

Colloidal polymer-coated Zn-doped iron oxide nanoparticles with high relaxivity and specific absorption rate for efficient magnetic resonance imaging and magnetic hyperthermia.

Colloidally stable nanoparticles-based magnetic agents endowed with very high relaxivity and specific absorption rate are extremely desirable for efficient magnetic resonance imaging and magnetic hyperthermia, respectively. Here, we report a water dispersible magnetic agent consisting of zinc-doped superparamagnetic iron oxide nanoparticles (i.e., Zn-SPIONs) of 15 nm size with high saturation magnetization coated with an amphiphilic polymer for effective magnetic resonance imaging and magnetic hyperthermia of glioblastoma cells. These biocompatible polymer-coated Zn-SPIONs had 24 nm hydrodynamic diameter and exhibited high colloidal stability in various aqueous media, very high transverse relaxivity of 471 mM-1 s-1, and specific absorption rate up to 743.8 W g-1, which perform better than most iron oxide nanoparticles reported in the literature, including commercially available agents. Therefore, using these polymer-coated Zn-SPIONs even at low concentrations, T2-weighted magnetic resonance imaging and moderate magnetic hyperthermia of glioblastoma cells under clinically relevant magnetic field were successfully implemented. In addition, the results of this in vitro study suggest the superior potential of Zn-SPIONs as a theranostic nanosystem for brain cancer treatment, simultaneously acting as a contrast agent for magnetic resonance imaging and a heat mediator for localized magnetic hyperthermia.

Easy formulation of liposomal doxorubicin modified with a bombesin peptide analogue for selective targeting of GRP receptors overexpressed by cancer cells.

The article concerns the obtainment of liposomal doxorubicin (Dox) in which liposomes are externally modified with a targeting peptide able to drive the formulation in a selective way on membrane receptors overexpressed in tumors. We developed a kit composed by three different vials: (A) a vial containing a sterile, translucent, red dispersion of the liposomal doxorubicin drug (Doxil®), (B) a vial filled with a lyophilized powder of a modified phospholipid with a reactive function (DSPE-Peg-maleimide), and (C) a vial containing a 1-9 bombesin peptide analogue (Cys-BN-AA1) chemically modified to react in stoichiometric ratio respect to DSPE-Peg-maleimide. The chosen peptide is a stable analogue antagonist of the wild-type 1-9 bombesin peptide; it is very stable in serum; maintains high specificity, with nanomolar affinity, towards gastrin release peptide receptors (GRPRs indicated also as BB2); and is overexpressed in some cancer cells. Results on animal studies clearly indicate that in mice treated with the kit product (i.e., pegylated liposomal Dox modified with the bombesin analogue, Doxil-BN-AA1), tumor growth is reduced, with an improved efficacy respect to mice treated with non-modified pegylated liposomal Dox or with saline solution.

Light Entrains Diurnal Changes in Insulin Sensitivity of Skeletal Muscle via Ventromedial Hypothalamic Neurons.

Loss of synchrony between geophysical time and insulin action predisposes to metabolic diseases. Yet the brain and peripheral pathways linking proper insulin effect to diurnal changes in light-dark and feeding-fasting inputs are poorly understood. Here, we show that the insulin sensitivity of several metabolically relevant tissues fluctuates during the 24 h period. For example, in mice, the insulin sensitivity of skeletal muscle, liver, and adipose tissue is lowest during the light period. Mechanistically, by performing loss- and gain-of-light-action and food-restriction experiments, we demonstrate that SIRT1 in steroidogenic factor 1 (SF1) neurons of the ventromedial hypothalamic nucleus (VMH) convey photic inputs to entrain the biochemical and metabolic action of insulin in skeletal muscle. These findings uncover a critical light-SF1-neuron-skeletal-muscle axis that acts to finely tune diurnal changes in insulin sensitivity and reveal a light regulatory mechanism of skeletal muscle function.

Mild ozonisation activates antioxidant cell response by the Keap1/Nrf2 dependent pathway.

Treatment with low-dose ozone is successfully exploited as an adjuvant therapy in the treatment of several disorders. Although the list of medical applications of ozone therapy is increasing, molecular mechanisms underlying its beneficial effects are still partially known. Clinical and experimental evidence suggests that the therapeutic effects of ozone treatment may rely on its capability to mount a beneficial antioxidant response through activation of the nuclear factor erythroid-derived-like 2 (Nrf2) pathway. However, a conclusive mechanistic demonstration is still lacking. Here, we bridge this gap of knowledge by providing evidence that treatment with a low concentration of ozone in cultured cells promotes nuclear translocation of Nrf2 at the chromatin sites of active transcription and increases the expression of antioxidant response element (ARE)-driven genes. Importantly, we show that ozone-induced ARE activation can be reverted by the ectopic expression of the Nrf2 specific inhibitor Kelch-like ECH associated protein (Keap1), thus proving the role of the Nrf2 pathway in the antioxidant response induced by mild ozonisation.

Magnetosomes Extracted from Magnetospirillum gryphiswaldense as Theranostic Agents in an Experimental Model of Glioblastoma.

Magnetic fluid hyperthermia (MFH) with chemically synthesized nanoparticles is currently used in clinical trials as it destroys tumor cells with an extremely localized deposition of thermal energy. In this paper, we investigated an MFH protocol based on magnetic nanoparticles naturally produced by magnetotactic bacteria: magnetosomes. The efficacy of such protocol is tested in a xenograft model of glioblastoma. Mice receive a single intratumoral injection of magnetosomes, and they are exposed three times in a week to an alternating magnetic field with concurrent temperature measurements. MRI is used to visualize the nanoparticles and to monitor tumor size before and after the treatment. Statistically significant inhibition of the tumor growth is detected in subjects exposed to the alternating magnetic field compared to control groups. Moreover, thanks to magnetosomes high transversal relaxivity, their effective delivery to the tumor tissue is monitored by MRI. It is apparent that the efficacy of this protocol is limited by inhomogeneous delivery of magnetosomes to tumor tissue. These results suggest that naturally synthesized magnetosomes could be effectively considered as theranostic agent candidates for hyperthermia based on iron oxide nanoparticles.

Liposomes derivatized with multimeric copies of KCCYSL peptide as targeting agents for HER-2-overexpressing tumor cells.

Mixed liposomes, obtained by coaggregation of 1,2-dioleoyl-sn-glycero-3-phosphocholine and of the synthetic monomer containing a gadolinium complex ([C18]2DTPA[Gd]) have been prepared. Liposomes externally decorated with KCCYSL (P6.1 peptide) sequence in its monomeric, dimeric, and tetrameric forms are studied as target-selective delivery systems toward cancer cells overexpressing human epidermal growth factor receptor-2 (HER-2) receptors. Derivatization of liposomal surface with targeting peptides is achieved using the postmodification method: the alkyne-peptide derivative Pra-KCCYSL reacts, through click chemistry procedures, with a synthetic surfactant modified with 1, 2, or 4 azido moieties previously inserted in liposome formulation. Preliminary in vitro data on MDA-MB-231 and BT-474 cells indicated that liposomes functionalized with P6.1 peptide in its tetrameric form had better binding to and uptake into BT-474 cells compared to liposomes decorated with monomeric or dimeric versions of the P6.1 peptide. BT-474 cells treated with liposomes functionalized with the tetrameric form of P6.1 showed high degree of liposome uptake, which was comparable with the uptake of anti-HER-2 antibodies such as Herceptin. Moreover, magnetic MRI experiments have demonstrated the potential of liposomes to act as MRI contrast agents.

Nanoformulations for dimethyl fumarate: Physicochemical characterization and in vitro/in vivo behavior.

Dimethyl fumarate has been demonstrated useful in relapsing remitting multiple sclerosis treatment (Tecfidera®). Nevertheless, since Tecfidera® capsules induce flushing, gastro-intestinal events and other more serious drawbacks, in this investigation a nanoparticle based system to be administered by an alternative way is proposed. In particular this study describes the preparation and characterization of dimethyl fumarate-containing solid lipid nanoparticles (SLN). Namely SLN based on tristearin, tristearin SLN treated with polysorbate 80 and cationic SLN constituted of tristearin in mixture with dimethyldioctadecylammonium chloride were investigated. The effect of the presence of dimethyl fumarate, functionalization by polysorbate 80 and dimethyldioctadecylammonium chloride was studied on morphology and dimensional distribution of SLN, by photon correlation spectroscopy and cryogenic transmission electron microscopy. Dimethyl fumarate release from SLN, studied by Franz cell, evidenced a Fickian dissolutive type kinetic in the case of SLN treated by polysorbate 80. Moreover fluorescent SLN were produced and characterized in order to investigate their in vitro permeability and in vivo biodistribution in mice. An in vitro study of fluorescent SLN permeability performed through a model of mouse brain microvascular endothelial cells, indicated that cationic SLN displayed higher permeability values with respect to neutral SLN and SLN treated by polysorbate 80. Biodistribution of polysorbate 80 treated SLN was studied by fluorescent imaging after intraperitoneal or intranasal administration in mice. The in vivo images indicate that polysorbate 80 treated SLN were able to reach the brain, even if they prevalently accumulated in liver and spleen, especially by intraperitoneal route.

Innovative approach to safely induce controlled lipolysis by superparamagnetic iron oxide nanoparticles-mediated hyperthermic treatment.

During last years, evidence has been provided on the involvement of overweight and obesity in the pathogenesis and aggravation of several life-threatening diseases. Here, we demonstrate that, under appropriate administration conditions, polyhedral iron oxide nanoparticles are efficiently and safely taken up by 3T3 cell line-derived adipocytes (3T3 adipocytes) in vitro. Since these nanoparticles proved to effectively produce heat when subjected to alternating magnetic field, 3T3 adipocytes were submitted to superparamagnetic iron oxide nanoparticles-mediated hyperthermia treatment (SMHT), with the aim of modulating their lipid content. Notably, the treatment resulted in a significant delipidation persisting for at least 24h, and in the absence of cell death, damage or dedifferentiation. Interestingly, transcript expression of adipose triglyceride lipase (ATGL), a key gene involved in canonical lipolysis, was not modulated upon SMHT, suggesting the involvement of a novel/alternative mechanism in the effective lipolysis observed. By applying the same experimental conditions successfully used for 3T3 adipocytes, SMHT was able to induce delipidation also in primary cultures of human adipose-derived adult stem cells. The success of this pioneering approach in vitro opens promising perspectives for the application of SMHT in vivo as an innovative safe and physiologically mild strategy against obesity, potentially useful in association with balanced diet and healthy lifestyle.

Magnetic nanoparticles from Magnetospirillum gryphiswaldense increase the efficacy of thermotherapy in a model of colon carcinoma.

Magnetic nanoparticles (MNPs) are capable of generate heating power under the influence of alternating magnetic fields (AMF); this behaviour recently opened new scenarios for advanced biomedical applications, mainly as new promising tumor therapies. In this paper we have tested magnetic nanoparticles called magnetosomes (MNs): a class of MNPs naturally produced by magnetotactic bacteria. We extracted MNs from Magnetospirillum gryphiswaldense strain MSR-1 and tested the interaction with cellular elements and anti-neoplastic activity both in vitro and in vivo, with the aim of developing new therapeutic approaches for neoplastic diseases. In vitro experiments performed on Human Colon Carcinoma HT-29 cell cultures demonstrated a strong uptake of MNs with no evident signs of cytotoxicity and revealed three phases in the interaction: adherence, transport and accumulation in Golgi vesicles. In vivo studies were performed on subcutaneous tumors in mice; in this model MNs are administered by direct injection in the tumor volume, then a protocol consisting of three exposures to an AMF rated at 187 kHz and 23kA/m is carried out on alternate days, over a week. Tumors were monitored by Magnetic Resonance Imaging (MRI) to obtain information about MNs distribution and possible tissue modifications induced by hyperthermia. Histological analysis showed fibrous and necrotic areas close to MNs injection sites in mice subjected to a complete thermotherapy protocol. These results, although concerning a specific tumor model, could be useful to further investigate the feasibility and efficacy of protocols based on MFH. Magnetic nanoparticles naturally produced and extracted from bacteria seem to be promising candidates for theranostic applications in cancer therapy.

Mesenchymal/stromal gene expression signature relates to basal-like breast cancers, identifies bone metastasis and predicts resistance to therapies.

BACKGROUND: Mounting clinical and experimental evidence suggests that the shift of carcinomas towards a mesenchymal phenotype is a common paradigm for both resistance to therapy and tumor recurrence. However, the mesenchymalization of carcinomas has not yet entered clinical practice as a crucial diagnostic paradigm. METHODOLOGY/PRINCIPAL FINDINGS: By integrating in silico and in vitro studies with our epithelial and mesenchymal tumor models, we compare herein crucial molecular pathways of previously described carcinoma-derived mesenchymal tumor cells (A17) with that of both carcinomas and other mesenchymal phenotypes, such as mesenchymal stem cells (MSCs), breast stroma, and various types of sarcomas. We identified three mesenchymal/stromal-signatures which A17 cells shares with MSCs and breast stroma. By using a recently developed computational approach with publicly available microarray data, we show that these signatures: 1) significantly relates to basal-like breast cancer subtypes; 2) significantly relates to bone metastasis; 3) are up-regulated after hormonal treatment; 4) predict resistance to neoadjuvant therapies. CONCLUSIONS/SIGNIFICANCE: Our results demonstrate that mesenchymalization is an intrinsic property of the most aggressive tumors and it relates to therapy resistance as well as bone metastasis.