Antitumour effects of SFX-01 molecule in combination with ionizing radiation in preclinical and in vivo models of rhabdomyosarcoma.

BACKGROUND: Despite a multimodal approach including surgery, chemo- and radiotherapy, the 5-year event-free survival rate for rhabdomyosarcoma (RMS), the most common soft tissue sarcoma in childhood, remains very poor for metastatic patients, mainly due to the selection and proliferation of tumour cells driving resistance mechanisms. Personalised medicine-based protocols using new drugs or targeted therapies in combination with conventional treatments have the potential to enhance the therapeutic effects, while minimizing damage to healthy tissues in a wide range of human malignancies, with several clinical trials being started. In this study, we analysed, for the first time, the antitumour activity of SFX-01, a complex of synthetic d, l-sulforaphane stabilised in alpha-cyclodextrin (Evgen Pharma plc, UK), used as single agent and in combination with irradiation, in four preclinical models of alveolar and embryonal RMS. Indeed, SFX-01 has shown promise in preclinical studies for its ability to modulate cellular pathways involved in inflammation and oxidative stress that are essential to be controlled in cancer treatment. METHODS: RH30, RH4 (alveolar RMS), RD and JR1 (embryonal RMS) cell lines as well as mouse xenograft models of RMS were used to evaluate the biological and molecular effects induced by SFX-01 treatment. Flow cytometry and the modulation of key markers analysed by q-PCR and Western blot were used to assess cell proliferation, apoptosis, autophagy and production of intracellular reactive oxygen species (ROS) in RMS cells exposed to SFX-01. The ability to migrate and invade was also investigated with specific assays. The possible synergistic effects between SFX-01 and ionising radiation (IR) was studied in both the in vitro and in vivo studies. Student's t-test or two-way ANOVA were used to test the statistical significance of two or more comparisons, respectively. RESULTS: SFX-01 treatment exhibited cytostatic and cytotoxic effects, mediated by G2 cell cycle arrest, apoptosis induction and suppression of autophagy. Moreover, SFX-01 was able to inhibit the formation and the proliferation of 3D tumorspheres as monotherapy and in combination with IR. Finally, SFX-01, when orally administered as single agent, displayed a pattern of efficacy at reducing the growth of tumour masses in RMS xenograft mouse models; when combined with a radiotherapy regime, it was observed to act synergistically, resulting in a more positive outcome than would be expected by adding each exposure alone. CONCLUSIONS: In summary, our results provide evidence for the antitumour properties of SFX-01 in preclinical models of RMS tumours, both as a standalone treatment and in combination with irradiation. These forthcoming findings are crucial for deeper investigations of SFX-01 molecular mechanisms against RMS and for setting up clinical trials in RMS patients in order to use the SFX-01/IR co-treatment as a promising therapeutic approach, particularly in the clinical management of aggressive RMS disease.

Assessing the Impact of Polyethylene Nano/Microplastic Exposure on Human Vaginal Keratinocytes.

The global rise of single-use throw-away plastic products has elicited a massive increase in the nano/microplastics (N/MPLs) exposure burden in humans. Recently, it has been demonstrated that disposable period products may release N/MPLs with usage, which represents a potential threat to women's health which has not been scientifically addressed yet. By using polyethyl ene (PE) particles (200 nm to 9 µm), we showed that acute exposure to a high concentration of N/MPLs induced cell toxicity in vaginal keratinocytes after effective cellular uptake, as viability and apoptosis data suggest, along with transmission electron microscopy (TEM) observations. The internalised N/MPLs altered the expression of junctional and adherence proteins and the organisation of the actin cortex, influencing the level of genes involved in oxidative stress signalling pathways and that of miRNAs related to epithelial barrier function. When the exposure to PE N/MPLs was discontinued or became chronic, cells were able to recover from the negative effects on viability and differentiation/proliferation gene expression in a few days. However, in all cases, PE N/MPL exposure prompted a sustained alteration of DNA methyltransferase and DNA demethylase expression, which might impact epigenetic regulation processes, leading to accelerated cell ageing and inflammation, or the occurrence of malignant transformation.

Microgravity Modifies the Phenotype of Fibroblast and Promotes Remodeling of the Fibroblast-Keratinocyte Interaction in a 3D Co-Culture Model.

Microgravity impairs tissue organization and critical pathways involved in the cell-microenvironment interplay, where fibroblasts have a critical role. We exposed dermal fibroblasts to simulated microgravity by means of a Random Positioning Machine (RPM), a device that reproduces conditions of weightlessness. Molecular and structural changes were analyzed and compared to control samples growing in a normal gravity field. Simulated microgravity impairs fibroblast conversion into myofibroblast and inhibits their migratory properties. Consequently, the normal interplay between fibroblasts and keratinocytes were remarkably altered in 3D co-culture experiments, giving rise to several ultra-structural abnormalities. Such phenotypic changes are associated with down-regulation of α-SMA that translocate in the nucleoplasm, altogether with the concomitant modification of the actin-vinculin apparatus. Noticeably, the stress associated with weightlessness induced oxidative damage, which seemed to concur with such modifications. These findings disclose new opportunities to establish antioxidant strategies that counteract the microgravity-induced disruptive effects on fibroblasts and tissue organization.

Gene expression profiles of oral soft tissue-derived fibroblast from healing wounds: correlation with clinical outcome, autophagy activation and fibrotic markers expression.

AIM: Our aim was to evaluate gene expression profiling of fibroblasts from human alveolar mucosa (M), buccal attached gingiva (G) and palatal (P) tissues during early wound healing, correlating it with clinical response. MATERIALS AND METHODS: M, G and P biopsies were harvested from six patients at baseline and 24 hr after surgery. Clinical response was evaluated through Early wound Healing Score (EHS). Fibrotic markers expression and autophagy were assessed on fibroblasts isolated from those tissues by Western blot and qRT-PCR. Fibroblasts from two patients were subjected to RT2 profiler array, followed by network analysis of the differentially expressed genes. The expression of key genes was validated with qRT-PCR on all patients. RESULTS: At 24 hr after surgery, EHS was higher in P and G than in M. In line with our clinical results, no autophagy and myofibroblast differentiation were observed in G and P. We observed significant variations in mRNA expression of key genes: RAC1, SERPINE1 and TIMP1, involved in scar formation; CDH1, ITGA4 and ITGB5, contributing to myofibroblast differentiation; and IL6 and CXCL1, involved in inflammation. CONCLUSIONS: We identified some genes involved in periodontal soft tissue clinical outcome, providing novel insights into the molecular mechanisms of oral repair (ClinicalTrial.gov-NCT04202822).

Autophagy activation is required for myofibroblast differentiation during healing of oral mucosa.

AIM: It is known that periodontal tissues heal faster that skin, and gingiva in particular heal without scar formation. The mechanisms regulating this behaviour are still unclear. The aim of our work was to compare wound healing in oral mucosa and gingiva, investigating the role of α-smooth muscle actin (αSMA)-expressing myofibroblasts and autophagy. MATERIALS AND METHODS: Biopsies were obtained from seven patients immediately before and 24 hr after vertical releasing incision in oral mucosa and attached gingiva. Both whole biopsies and primary cultures of fibroblasts derived from the same tissues were subjected to immunofluorescence, Western blot and quantitative real-time PCR analyses. RESULTS: We demonstrated that in oral mucosa, characterized by partially fibrotic outcome during repair, the activation of autophagy determined an increase in αSMA and collagen 1a1 production. Conversely, wound healing did not stimulate autophagy in attached gingiva, and subsequently, no increase in myofibroblast differentiation and collagen deposition could be seen, thus justifying its scarless outcome. CONCLUSIONS: The elucidation of the differential regulation of autophagy in periodontal tissues and its correlation with myofibroblast differentiation and fibrotic outcome could allow the identification of new molecules involved in periodontal healing and the development of new surgical approaches for periodontal treatment that could improve the outcome of postoperative wounds.

Design and synthesis of selective, dual fatty acid binding protein 4 and 5 inhibitors.

Dual inhibition of fatty acid binding proteins 4 and 5 (FABP4 and FABP5) is expected to provide beneficial effects on a number of metabolic parameters such as insulin sensitivity and blood glucose levels and should protect against atherosclerosis. Starting from a FABP4 selective focused screening hit, biostructure information was used to modulate the selectivity profile in the desired way and to design potent dual FABP4/5 inhibitors with good selectivity against FABP3. With very good pharmacokinetic properties and no major safety alerts, compound 12 was identified as a suitable tool compound for further in vivo investigations.

Autologous in vitro cultured vaginal tissue for vaginoplasty in women with Mayer-Rokitansky-Küster-Hauser syndrome: anatomic and functional results.

STUDY OBJECTIVE: To present the procedure and the results of a technique in which in vitro autologous cell cultures were used for the canal lining in patients with Mayer-Rokitansky-Küster-Hauser syndrome (MRKHS) subjected to vaginoplasty with a modified Abbè-McIndoe technique. MRKHS is a rare anomaly characterized by vaginal agenesis with variable müllerian duct abnormalities. The Abbè-McIndoe procedure is 1 of the most frequent surgical treatments adopted in these women. In the last decades, several modifications have been introduced by different authors, mostly changing the lining material, but no consensus has been reached on what material should be used for the neovagina canal wall lining. DESIGN: A pilot study (Canadian Task Force classification II-1). SETTING: Policlinico Umberto I, "Sapienza" University of Rome. PATIENTS: A consecutive series of 23 women with MRKHS underwent neovaginoplasty with autologous vaginal tissue as the graft material between 2006 and 2013. INTERVENTIONS: Each patient with MRKHS was subjected to a full-thickness mucosal biopsy from the vaginal vestibule. After enzymatic dissociation, cells were inoculated onto collagen IV-coated plates and cultured for 2 to 3 weeks. The patients were subjected to vaginoplasty with a modified Abbè-McIndoe technique with autologous in vitro cultured vaginal tissue. Patients underwent clinical follow-up visits at 1, 3, 6, and 12 months after surgery and every year thereafter. Anatomic, functional, and sexual results were assessed. MEASUREMENTS AND MAIN RESULTS: In all cases, the vagina appeared normal in length and depth. A vaginal cytology and a vaginal biopsy obtained at the 3-month follow-up visit revealed physiological vaginal tissue. All 23 patients completed the Female Sexual Function Index questionnaire at 12 months after surgery. The results showed a total score of 27.2. These results indicate a satisfactory quality of sexual life. CONCLUSION: The modified Abbè-McIndoe technique with autologous vaginal tissue appears to be safe and feasible. This technique allows normal and satisfying sexual intercourse. Larger series with longer follow-ups will be necessary to confirm if this technique represents the ideal procedure for vaginal agenesis.

Topical KGF treatment as a therapeutic strategy for vaginal atrophy in a model of ovariectomized mice.

One of the most frequent complaints for post-menopausal women is vaginal atrophy, because of reduction in circulating oestrogens. Treatments based on local oestrogen administration have been questioned as topic oestrogens can reach the bloodstream, thus leading to consider their safety as controversial, especially for patients with a history of breast or endometrial cancers. Recently, growth factors have been shown to interact with the oestrogen pathway, but the mechanisms still need to be fully clarified. In this study, we investigated the effect of keratinocyte growth factor (KGF), a known mitogen for epithelial cells, on human vaginal mucosa cells, and its potential crosstalk with oestrogen pathways. We also tested the in vivo efficacy of KGF local administration on vaginal atrophy in a murine model. We demonstrated that KGF is able to induce proliferation of vaginal mucosa, and we gained insight on its mechanism of action by highlighting its contribution to switch ERα signalling towards non-genomic pathway. Moreover, we demonstrated that KGF restores vaginal trophism in vivo similarly to intravaginal oestrogenic preparations, without systemic effects. Therefore, we suggest a possible alternative therapy for vaginal atrophy devoid of the risks related to oestrogen-based treatments, and a patent (no. RM2012A000404) has been applied for this study.

Characterization of Wnt/ß-catenin signaling in rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in children and accounts for about 5% of all malignant paediatric tumours. ß-Catenin, a multifunctional nuclear transcription factor in the canonical Wnt signaling pathway, is active in myogenesis and embryonal somite patterning. Dysregulation of Wnt signaling facilitates tumour invasion and metastasis. This study characterizes Wnt/ß-catenin signaling and functional activity in paediatric embryonal and alveolar RMS. Immunohistochemical assessment of paraffin-embedded tissues from 44 RMS showed ß-catenin expression in 26 cases with cytoplasmic/membranous expression in 9/14 cases of alveolar RMS, and 15/30 cases of embryonal RMS, whereas nuclear expression was only seen in 2 cases of embryonal RMS. The potential functional significance of ß-catenin expression was tested in four RMS cell lines, two derived from embryonal (RD and RD18) RMS and two from alveolar (Rh4 and Rh30) RMS. Western blot analysis demonstrated the expression of Wnt-associated proteins including ß-catenin, glycogen synthase kinase-3ß, disheveled, axin-1, naked, LRP-6 and cadherins in all cell lines. Cell fractionation and immunofluorescence studies of the cell lines (after stimulation by human recombinant Wnt3a) showed reduced phosphorylation of ß-catenin, stabilization of the active cytosolic form and nuclear translocation of ß-catenin. Reporter gene assay demonstrated a T-cell factor/lymphoid-enhancing factor-mediated transactivation in these cells. In response to human recombinant Wnt3a, the alveolar RMS cells showed a significant decrease in proliferation rate and induction of myogenic differentiation (myogenin, MyoD1 and myf5). These data indicate that the central regulatory components of canonical Wnt/ß-catenin signaling are expressed and that this pathway is functionally active in a significant subset of RMS tumours and might represent a novel therapeutic target.

Effect of Chlorhexidine Digluconate in Early Wound Healing of Human Gingival Tissues. A Histological, Immunohistochemical and Biomolecular Analysis.

Chlorhexidine digluconate (CHX) is considered the gold standard for oral cavity antiseptic treatment. Nevertheless, several in vitro studies have reported detrimental effects in oral tissue repair. The aim of the present study was to evaluate the in vivo effect of post-surgical CHX mouth rinse on gingival tissue (G) 24 h after injury. G biopsies were obtained in three patients 24 h after surgery with the indication of post-surgical 0.12% CHX use and were compared with those obtained from the same patients without any antiseptic use. Changes in collagen production, cell proliferation, and apoptosis were examined by histological and Ki-67/P53 immunohistochemical analysis. Fibrotic markers (COL1A1, αSMA), proapoptotic protein (BAX) expression, and wound healing-related gene modulation (RAC1, SERPINE1, TIMP1) were analyzed by quantitative real-time PCR analysis. CHX was able to reduce cellular proliferation and increase collagen deposition, proapoptotic molecule and fibrotic marker expression, and myofibroblast differentiation, reduce expression of RAC1 and trigger expression of SERPINE1 and TIMP1, showing "scar wound healing response" pattern. This study assessed for the first time the in vivo effects of CHX on gingival tissue. The demonstration of a CHX-induced fibrotic transformation, leading to scar repair, supports the need for new post-surgical clinical protocols based on a strategic and personalized use of CHX.

Sex-Related Factors in Cardiovascular Complications Associated to COVID-19.

Coronavirus disease 2019 (COVID-19), the pandemic infection caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), presents with an extremely heterogeneous spectrum of symptoms and signs. The clinical manifestations seem to be correlated with disease severity. COVID-19 susceptibility and mortality show a significant sex imbalance, with men being more prone to infection and showing a higher rate of hospitalization and mortality compared to women. Such variability can be ascribed to both sex-related biological factors and gender-related behavioral cues. This review will discuss the potential mechanisms accounting for sex/gender influence in vulnerability to COVID-19. Cardiovascular diseases play a central role in determining COVID-19 outcome, whether they are pre-existent or arose upon infection. We will pay particular attention to the impact of sex and gender on cardiovascular manifestations related to COVID-19. Finally, we will discuss the sex-dependent variability in some biomarkers for the evaluation of COVID-19 infection and prognosis. The aim of this work is to highlight the significance of gendered medicine in setting up personalized programs for COVID-19 prevention, clinical evaluation and treatment.

Protein-protein interaction network analysis applied to DNA copy number profiling suggests new perspectives on the aetiology of Mayer-Rokitansky-Küster-Hauser syndrome.

Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is a rare disease, characterised by the aplasia of vagina and uterus in women with a 46,XX karyotype. Most cases are sporadic, but familial recurrence has also been described. Herein, we investigated an Italian cohort of 36 unrelated MRKH patients to explore the presence of pathogenic copy number variations (CNVs) by array-CGH and MLPA assays. On the whole, aberrations were found in 9/36 (25%) patients. Interestingly, one patient showed a novel heterozygous microduplication at Xp22.33, not yet described in MRKH patients, containing the PRKX gene. Moreover, a novel duplication of a specific SHOX enhancer was highlighted by MLPA. To predict the potential significance of CNVs in MRKH pathogenesis, we provided a network analysis for protein-coding genes found in the altered genomic regions. Although not all of these genes taken individually showed a clear clinical significance, their combination in a computational network highlighted that the most relevant biological connections are related to the anatomical structure development. In conclusion, the results described in the present study identified novel genetic alterations and interactions that may be likely involved in MRKH phenotype determination, so adding new insights into the complex puzzle of MRKH disease.

Altered Expression of Candidate Genes in Mayer-Rokitansky-Küster-Hauser Syndrome May Influence Vaginal Keratinocytes Biology: A Focus on Protein Kinase X.

Mayer-Rokitansky-Küster-Hauser (MRKH) syndrome is a rare and complex disease defined by congenital aplasia of the vagina and uterus in 46,XX women, often associated with kidney and urinary tract anomalies. The aetiopathogenesis of MRKH syndrome is still largely unknown. Herein, we investigated the role of selected candidate genes in the aetiopathogenesis of MRKH syndrome, with a focus on PRKX, which encodes for protein kinase X. Through RT-qPCR analyses performed on vaginal dimple samples from patients, and principal component analysis (PCA), we highlighted a phenotype-related expression pattern of PRKX, MUC1, HOXC8 and GREB1L in MRKH patients. By using an in vitro approach, we proved that PRKX ectopic overexpression in a cell model of vaginal keratinocytes promotes cell motility through epithelial-to-mesenchymal transition (EMT) activation, a fundamental process in urogenital tract morphogenesis. Moreover, our findings showed that PRKX upregulation in vaginal keratinocytes is able to affect transcriptional levels of HOX genes, implicated in urinary and genital tract development. Our study identified the dysregulation of PRKX expression as a possible molecular cause for MRKH syndrome. Moreover, we propose the specific role of PRKX in vaginal keratinocyte biology as one of the possible mechanisms underlying this complex disease.

Calcineurin Gamma Catalytic Subunit PPP3CC Inhibition by miR-200c-3p Affects Apoptosis in Epithelial Ovarian Cancer.

Epithelial ovarian cancer (EOC) outpaces all the other forms of the female reproductive system malignancies. MicroRNAs have emerged as promising predictive biomarkers to therapeutic treatments as their expression might characterize the tumor stage or grade. In EOC, miR-200c is considered a master regulator of oncogenes or tumor suppressors. To investigate novel miR-200c-3p target genes involved in EOC tumorigenesis, we evaluated the association between this miRNA and the mRNA expression of several potential target genes by RNA-seq data of both 46 EOC cell lines from Cancer Cell line Encyclopedia (CCLE) and 456 EOC patient bio-specimens from The Cancer Genome Atlas (TCGA). Both analyses showed a significant anticorrelation between miR-200c-3p and the protein phosphatase 3 catalytic subunit γ of calcineurin (PPP3CC) levels involved in the apoptosis pathway. Quantitative mRNA expression analysis in patient biopsies confirmed the inverse correlation between miR-200c-3p and PPP3CC levels. In vitro regulation of PPP3CC expression through miR-200c-3p and RNA interference technology led to a concomitant modulation of BCL2- and p-AKT-related pathways, suggesting the tumor suppressive role of PPP3CC in EOC. Our results suggest that inhibition of high expression of miR-200c-3p in EOC might lead to overexpression of the tumor suppressor PPP3CC and subsequent induction of apoptosis in EOC patients.

When Viruses Cross Developmental Pathways.

Aberrant regulation of developmental pathways plays a key role in tumorigenesis. Tumor cells differ from normal cells in their sustained proliferation, replicative immortality, resistance to cell death and growth inhibition, angiogenesis, and metastatic behavior. Often they acquire these features as a consequence of dysregulated Hedgehog, Notch, or WNT signaling pathways. Human tumor viruses affect the cancer cell hallmarks by encoding oncogenic proteins, and/or by modifying the microenvironment, as well as by conveying genomic instability to accelerate cancer development. In addition, viral immune evasion mechanisms may compromise developmental pathways to accelerate tumor growth. Viruses achieve this by influencing both coding and non-coding gene regulatory pathways. Elucidating how oncogenic viruses intersect with and modulate developmental pathways is crucial to understanding viral tumorigenesis. Many currently available antiviral therapies target viral lytic cycle replication but with low efficacy and severe side effects. A greater understanding of the cross-signaling between oncogenic viruses and developmental pathways will improve the efficacy of next-generation inhibitors and pave the way to more targeted antiviral therapies.

OTX015 Epi-Drug Exerts Antitumor Effects in Ovarian Cancer Cells by Blocking GNL3-Mediated Radioresistance Mechanisms: Cellular, Molecular and Computational Evidence.

Ovarian cancer (OC) is the most aggressive gynecological tumor worldwide and, notwithstanding the increment in conventional treatments, many resistance mechanisms arise, this leading to cure failure and patient death. So, the use of novel adjuvant drugs able to counteract these pathways is urgently needed to improve patient overall survival. A growing interest is focused on epigenetic drugs for cancer therapy, such as Bromodomain and Extra-Terminal motif inhibitors (BETi). Here, we investigate the antitumor effects of OTX015, a novel BETi, as a single agent or in combination with ionizing radiation (IR) in OC cellular models. OTX015 treatment significantly reduced tumor cell proliferation by triggering cell cycle arrest and apoptosis that were linked to nucleolar stress and DNA damage. OTX015 impaired migration capacity and potentiated IR effects by reducing the expression of different drivers of cancer resistance mechanisms, including GNL3 gene, whose expression was found to be significantly higher in OC biopsies than in normal ovarian tissues. Gene specific knocking down and computational network analysis confirmed the centrality of GNL3 in OTX015-mediated OC antitumor effects. Altogether, our findings suggest OTX015 as an effective option to improve therapeutic strategies and overcome the development of resistant cancer cells in patients with OC.

DNMT3A and DNMT3B Targeting as an Effective Radiosensitizing Strategy in Embryonal Rhabdomyosarcoma.

Rhabdomyosarcoma (RMS) is the most common soft tissue sarcoma in childhood. Recently, we demonstrated the overexpression of both DNA methyltransferase 3A (DNMT3A) and 3B (DNMT3B) in RMS tumour biopsies and cell lines compared to normal skeletal muscle. Radiotherapy may often fail due to the abnormal expression of some molecules able to drive resistance mechanisms. The aim of this study was to analyse the involvement of DNMT3A and DNMT3B in radioresistance in RMS. RNA interference experiments against DNMT3A/3B were performed in embryonal RMS cells, upon ionizing radiation (IR) exposure and the effects of the combined treatment on RMS cells were analysed. DNMT3A and DNMT3B knocking down increased the sensitivity of RMS cells to IR, as indicated by the drastic decrease of colony formation ability. Interestingly, DNMT3A/3B act in two different ways: DNMT3A silencing triggers the cellular senescence program by up-regulating p16 and p21, whilst DNMT3B depletion induces significant DNA damage and impairs the DNA repair machinery (ATM, DNA-PKcs and Rad51 reduction). Our findings demonstrate for the first time that DNMT3A and DNMT3B overexpression may contribute to radiotherapy failure, and their inhibition might be a promising radiosensitizing strategy, mainly in the treatment of patients with metastatic or recurrent RMS tumours.

MiR-200c-3p maintains stemness and proliferative potential in adipose-derived stem cells by counteracting senescence mechanisms.

Adipose-derived mesenchymal stem cells (ASCs) are promising therapeutic tools in regenerative medicine because they possess self-renewal, differentiation and immunomodulatory capacities. After isolation, ASCs are passaged multiple times in vitro passages to obtain a sufficient amount of cells for clinical applications. During this time-consuming procedure, ASCs become senescent and less proliferative, compromising their clinical efficacy. Here, we sought to investigate how in vitro passages impact ASC proliferation/senescence and expression of immune regulatory proteins. MicroRNAs are pivotal regulators of ASC physiology. Particularly, miR-200c is known to maintain pluripotency and targets the immune checkpoint Programmed death-ligand 1 (PD-L1). We therefore investigated its involvement in these critical characteristics of ASCs during in vitro passages. We found that when transiently expressed, miR-200c-3p promotes proliferation, maintains stemness, and contrasts senescence in late passaged ASCs. Additionally, this miRNA modulates PD-L1 and Indoleamine 2,3-Dioxygenase (IDO1) expression, thus most likely interfering with the immunoregulatory capacity of ASCs. Based on our results, we suggest that expression of miR-200c-3p may prime ASC towards a self-renewing phenotype by improving their in vitro expansion. Contrarily, its inhibition is associated with senescence, reduced proliferation and induction of immune regulators. Our data underline the potential use of miR-200c-3p as a switch for ASCs reprogramming and their clinical application.

MiR-200c-3p Contrasts PD-L1 Induction by Combinatorial Therapies and Slows Proliferation of Epithelial Ovarian Cancer through Downregulation of ß-Catenin and c-Myc.

Conventional/targeted chemotherapies and ionizing radiation (IR) are being used both as monotherapies and in combination for the treatment of epithelial ovarian cancer (EOC). Several studies show that these therapies might favor oncogenic signaling and impede anti-tumor responses. MiR-200c is considered a master regulator of EOC-related oncogenes. In this study, we sought to investigate if chemotherapy and IR could influence the expression of miR-200c-3p and its target genes, like the immune checkpoint PD-L1 and other oncogenes in a cohort of EOC patients' biopsies. Indeed, PD-L1 expression was induced, while miR-200c-3p was significantly reduced in these biopsies post-therapy. The effect of miR-200c-3p target genes was assessed in miR-200c transfected SKOV3 cells untreated and treated with olaparib and IR alone. Under all experimental conditions, miR-200c-3p concomitantly reduced PD-L1, c-Myc and ß-catenin expression and sensitized ovarian cancer cells to olaparib and irradiation. In silico analyses further confirmed the anti-correlation between miR-200c-3p with c-Myc and ß-catenin in 46 OC cell lines and showed that a higher miR-200c-3p expression associates with a less tumorigenic microenvironment. These findings provide new insights into how miR-200c-3p could be used to hold in check the adverse effects of conventional chemotherapy, targeted therapy and radiation therapy, and offer a novel therapeutic strategy for EOC.

Immunomodulatory Effect of Adipose-Derived Stem Cells: The Cutting Edge of Clinical Application.

Adipose-derived stem cells (ASCs) represent a promising tool for soft tissue engineering as well as for clinical treatment of inflammatory and autoimmune pathologies. The well-characterized multi-differentiation potential and self-renewal properties of ASCs are coupled with their immunomodulatory ability in providing therapeutic efficacy. Yet, their impact in immune or inflammatory disorders might rely both on cell contact-dependent mechanisms and paracrine effects, resulting in the release of various soluble factors that regulate immune cells functions. Despite the widespread use of ASCs in clinical trials addressing several pathologies, the pathophysiological mechanisms at the basis of their clinical use have been not yet fully investigated. In particular, a thorough analysis of ASC immunomodulatory potential is mandatory. Here we explore such molecular mechanisms involved in ASC immunomodulatory properties, emphasizing the relevance of the milieu composition. We review the potential clinical use of ASC secretome as a mediator for immunomodulation, with a focus on in vitro and in vivo environmental conditions affecting clinical outcome. We describe some potential strategies for optimization of ASCs immunomodulatory capacity in clinical settings, which act either on adult stem cells gene expression and local microenvironment. Finally, we discuss the limitations of both allogeneic and autologous ASC use, highlighting the issues to be fixed in order to significantly improve the efficacy of ASC-based cell therapy.