Transcription Factor Activation Profiles (TFAP) identify compounds promoting differentiation of Acute Myeloid Leukemia cell lines.

Repurposing of drugs for new therapeutic use has received considerable attention for its potential to limit time and cost of drug development. Here we present a new strategy to identify chemicals that are likely to promote a desired phenotype. We used data from the Connectivity Map (CMap) to produce a ranked list of drugs according to their potential to activate transcription factors that mediate myeloid differentiation of leukemic progenitor cells. To validate our strategy, we tested the in vitro differentiation potential of candidate compounds using the HL-60 human cell line as a myeloid differentiation model. Ten out of 22 compounds, which were ranked high in the inferred list, were confirmed to promote significant differentiation of HL-60. These compounds may be considered candidate for differentiation therapy. The method that we have developed is versatile and it can be adapted to different drug repurposing projects.

Skeletal Muscle Subpopulation Rearrangements upon Rhabdomyosarcoma Development through Single-Cell Mass Cytometry.

The embryonal rhabdomyosarcoma (eRMS) is a soft tissue sarcoma commonly affecting the head and neck, the extremities and the genitourinary tract. To contribute to revealing the cell types that may originate this tumor, we exploited mass cytometry, a single-cell technique that, by using heavy-metal-tagged antibodies, allows the accurate monitoring of the changes occurring in the mononuclear cell composition of skeletal muscle tissue during tumor development. To this end, we compared cell populations of healthy muscles with those from spatiotemporal-induced eRMS tumors in a mouse model (LSL-KrasG12D/+;Tp53Fl/Fl) that can be used to develop rhabdomyosarcoma by means of infection with an adenovirus vector expressing Cre (Ad-Cre) recombinase. By monitoring different time points after tumor induction, we were able to analyze tumor progression and composition, identifying fibro/adipogenic progenitors (FAPs) as the cell type that, in this model system, had a pivotal role in tumor development. In vitro studies highlighted that both FAPs and satellite cells (SCs), upon infection with the Ad-Cre, acquired the potential to develop rhabdomyosarcomas when transplanted into immunocompromised mice. However, only infected FAPs had an antigen profile that was similar to embryonal rhabdomyosarcoma cells. Overall, our analysis supports the involvement of FAPs in eRMS development.

SCA-1 micro-heterogeneity in the fate decision of dystrophic fibro/adipogenic progenitors.

The term micro-heterogeneity refers to non-genetic cell to cell variability observed in a bell-shaped distribution of the expression of a trait within a population. The contribution of micro-heterogeneity to physiology and pathology remains largely uncharacterised. To address such an issue, we investigated the impact of heterogeneity in skeletal muscle fibro/adipogenic progenitors (FAPs) isolated from an animal model of Duchenne muscular dystrophy (DMD), the mdx mouse. FAPs play an essential role in muscle homoeostasis. However, in pathological conditions or ageing, they are the source of intramuscular infiltrations of fibrotic or adipose tissue. By applying a multiplex flow cytometry assay, we characterised and purified from mdx muscles two FAP cell states expressing different levels of SCA-1. The two cell states are morphologically identical and repopulate each other after several growth cycles. However, they differ in their in vitro behaviour. Cells expressing higher levels of SCA-1 (SCA1-High-FAPs) differentiate more readily into adipocytes while, when exposed to a fibrogenic stimulation, increase the expression of Col1a1 and Timp1 mRNA. A transcriptomic analysis confirmed the adipogenic propensity of SCA1-High-FAPs. In addition, SCA1-High-FAPs proliferate more extensively ex vivo and display more proliferating cells in dystrophic muscles in comparison to SCA1-Low-FAPs. Adipogenesis of both FAP cell states is inhibited in vitro by leucocytes from young dystrophic mice, while leucocytes isolated from aged dystrophic mice are less effective in limiting the adipogenesis of SCA1-High-FAPs suggesting a differential regulatory effect of the microenvironment on micro-heterogeneity. Our data suggest that FAP micro-heterogeneity is modulated in pathological conditions and that this heterogeneity in turn may impact on the behaviour of interstitial mesenchymal cells in genetic diseases.

Ionizing Radiation-Induced Extracellular Vesicle Release Promotes AKT-Associated Survival Response in SH-SY5Y Neuroblastoma Cells.

Radiation therapy is one of the most effective methods of tumor eradication; however, in some forms of neuroblastoma, radiation can increase the risk of secondary neoplasms, due to the ability of irradiated cells to transmit pro-survival signals to non-irradiated cells through vesicle secretion. The aims of this study were to characterize the vesicles released by the human neuroblastoma cell line SH-SY5Y following X-ray radiations and their ability to increase invasiveness in non-irradiated SH-SY5Y cells. We first purified the extracellular vesicles released by the SH-SY5Y cells following X-rays, and then determined their total amount, dimensions, membrane protein composition, and cellular uptake. We also examined the effects of these extracellular vesicles on viability, migration, and DNA damage in recipient SH-SY5Y cells. We found that exposure to X-rays increased the release of extracellular vesicles and altered their protein composition. These vesicles were readily uptaken by non-irradiated cells, inducing an increase in viability, migration, and radio-resistance. The same results were obtained in an MYCN-amplified SK-N-BE cell line. Our study demonstrates that vesicles released from irradiated neuroblastoma cells stimulate proliferation and invasiveness that correlate with the epithelial to mesenchymal transition in non-irradiated cells. Moreover, our results suggest that, at least in neuroblastomas, targeting the extracellular vesicles may represent a novel therapeutic approach to counteract the side effects associated with radiotherapy.

Effect of microvesicles from Moringa oleifera containing miRNA on proliferation and apoptosis in tumor cell lines.

Human microvesicles are key mediators of cell-cell communication. Exosomes function as microRNA transporters, playing a crucial role in physiological and pathological processes. Plant microvesicles (MVs) display similar features to mammalian exosomes, and these MVs might enhance plant microRNA delivery in mammals. Considering that plant microRNAs have been newly identified as bioactive constituents in medicinal plants, and that their potential role as regulators in mammals has been underlined, in this study, we characterized MVs purified from Moringa oleifera seeds aqueous extract (MOES MVs) and used flow cytometry methods to quantify the ability to deliver their content to host cells. The microRNAs present in MOES MVs were characterized, and through a bioinformatic analysis, specific human apoptosis-related target genes of plant miRNAs were identified. In tumor cell lines, MOES MVs treatment reduced viability, increased apoptosis levels associated with a decrease in B-cell lymphoma 2 protein expression and reduced mitochondrial membrane potential. Interestingly, the effects observed with MOES MVs treatment were comparable to those observed with MOES treatment and transfection with the pool of small RNAs isolated from MOES, used as a control. These results highlight the role of microRNAs transported by MOES MVs as natural bioactive plant compounds that counteract tumorigenesis.

Janus effect of glucocorticoids on differentiation of muscle fibro/adipogenic progenitors.

Muscle resident fibro-adipogenic progenitors (FAPs), support muscle regeneration by releasing cytokines that stimulate the differentiation of myogenic stem cells. However, in non-physiological contexts (myopathies, atrophy, aging) FAPs cause fibrotic and fat infiltrations that impair muscle function. We set out to perform a fluorescence microscopy-based screening to identify compounds that perturb the differentiation trajectories of these multipotent stem cells. From a primary screen of 1,120 FDA/EMA approved drugs, we identified 34 compounds as potential inhibitors of adipogenic differentiation of FAPs isolated from the murine model (mdx) of Duchenne muscular dystrophy (DMD). The hit list from this screen was surprisingly enriched with compounds from the glucocorticoid (GCs) chemical class, drugs that are known to promote adipogenesis in vitro and in vivo. To shed light on these data, three GCs identified in our screening efforts were characterized by different approaches. We found that like dexamethasone, budesonide inhibits adipogenesis induced by insulin in sub-confluent FAPs. However, both drugs have a pro-adipogenic impact when the adipogenic mix contains factors that increase the concentration of cAMP. Gene expression analysis demonstrated that treatment with glucocorticoids induces the transcription of Gilz/Tsc22d3, an inhibitor of the adipogenic master regulator PPARγ, only in anti-adipogenic conditions. Additionally, alongside their anti-adipogenic effect, GCs are shown to promote terminal differentiation of satellite cells. Both the anti-adipogenic and pro-myogenic effects are mediated by the glucocorticoid receptor and are not observed in the presence of receptor inhibitors. Steroid administration currently represents the standard treatment for DMD patients, the rationale being based on their anti-inflammatory effects. The findings presented here offer new insights on additional glucocorticoid effects on muscle stem cells that may affect muscle homeostasis and physiology.

Fibro-adipogenic progenitors of dystrophic mice are insensitive to NOTCH regulation of adipogenesis.

Fibro-adipogenic progenitors (FAPs) promote satellite cell differentiation in adult skeletal muscle regeneration. However, in pathological conditions, FAPs are responsible for fibrosis and fatty infiltrations. Here we show that the NOTCH pathway negatively modulates FAP differentiation both in vitro and in vivo. However, FAPs isolated from young dystrophin-deficient mdx mice are insensitive to this control mechanism. An unbiased mass spectrometry-based proteomic analysis of FAPs from muscles of wild-type and mdx mice suggested that the synergistic cooperation between NOTCH and inflammatory signals controls FAP differentiation. Remarkably, we demonstrated that factors released by hematopoietic cells restore the sensitivity to NOTCH adipogenic inhibition in mdx FAPs. These results offer a basis for rationalizing pathological ectopic fat infiltrations in skeletal muscle and may suggest new therapeutic strategies to mitigate the detrimental effects of fat depositions in muscles of dystrophic patients.