Adipose stem cells on the basis of tumor transformation

Mesenchymal stem cells thanks to their differentiated multipotent ability are considered the most promising candidate for tissue engineering and regenerative medicine. The source of mesenchymal stem cells up to about 20 years was represented by the bone marrow, but due to their limited amount of cells together with a very invasive and painfull surgical treatment, poorly accepted by patients, has led the scientific community to investigate an alternative mesenchymal stem cells source with similar properties. In particular, the adipose tissue has attracted the greatest interest thanks to its relative abundance, the easy of availability and the large amounts of mesenchymal stem cells which are obtenible. So this adipose stem cells subpopulation, once verified the same differentiation potential of them derived from bone marrow, represent the ideal type of adult mesenchymal stem cells useful for numerous regenerative and tissue engineering applications. In particular the bone regeneration, obtained after the lipotransfer of a specific stem cells subpopulation isolated from adipose tissue, should be considered one of the most successful applications in the field of regenerative medicine and tissue engineering. (Chapter 1). A further widespread use of mesenchymal stem cells is to reconstructive medicine. In particular, most patients with breast cancer, whenever possible, based on the type and stage cancer disease (Chapter 2), first make use of a demolitive surgery, in order to completely remove the tumor mass presence, and then resort to a reconstructive plastic surgery procedure. In breast cancer patients it is often used the adipose tissue as autologous filler. Even though the surgical treatment does not represent a potential danger for the patient, many scientific works have highlighted how the use of these self transplantations constitute a great risk for a possible cancer relapse despite the absence of a minimal residual disease. The presence of a mature adipocytes population infact, is responsible of a specific microenvironment, composed by pro-inflammatory cytokines and paracrine signals, that induce a generalized inflammatory state and which stimulate a proliferative return of those few quiescent cancer cells that still remain in the implantation site. In particular we have seen that the action of adipokines and interleukins generated by adipose autologous filler works both on differentiated tumor cells, just in a active proliferation cell phase but also on the cancer stem cell population that instead reside in a quiescent cell cycle phase. (Chapter 3). Therefore it is essential develop a more accurate and specific lipofilling procedure, especially going to select an appropriate autologous filler, in which a particular adipose stem cell subpopulation, with great regenerating properties has to be chosen among the adipose populations already present in conventional fillers. (Chapters 4). So the goal of this study is to enable the development of new selective therapies useful to remove the cancer stem cells, real responsible for tumor relapse. One of the most promising cancer therapies among those currently in the testing phase could be the differentiative therapy, and specifically one that uses the negative regulatory properties of the miRNAs (Chapter 5)

The miRNA Contribution in Adipocyte Maturation

Mesenchymal stem cells, due to their multipotent ability, are considered one of the best candidates to be used in regenerative medicine. To date, the most used source is represented by the bone marrow, despite the limited number of cells and the painful/invasive procedure for collection. Therefore, the scientific community has investigated many alternative sources for the collection of mesenchymal stem cells, with the adipose tissue representing the best option, given the abundance of mesenchymal stem cells and the easy access. Although adipose mesenchymal stem cells have recently been investigated for their multipotency, the molecular mechanisms underlying their adipogenic potential are still unclear. In this scenario, this communication is aimed at defining the role of miRNAs in adipogenic potential of adipose-derived mesenchymal stem cells via real-time PCR. Even if preliminary, our data show that cell culture conditions affect the expression of specific miRNA involved in the adipogenic potential of mesenchymal stem cells. The in vitro/in vivo validation of these results could pave the way for novel therapeutic strategies in the field of regenerative medicine. In conclusion, our research highlights how specific cell culture conditions can modulate the adipogenic potential of adipose mesenchymal stem cells through the regulation of specific miRNAs

The biological interplay between air pollutants and miRNAs regulation in cancer

Air pollution, especially fine particulate matter (PM2.5, with an aerodynamic diameter of less than 2.5 μm), represents a risk factor for human health. Many studies, regarding cancer onset and progression, correlated with the short and/or long exposition to PM2.5. This is mainly mediated by the ability of PM2.5 to reach the pulmonary alveoli by penetrating into the blood circulation. This review recapitulates the methodologies used to study PM2.5 in cellular models and the downstream effects on the main molecular pathways implicated in cancer. We report a set of data from the literature, that describe the involvement of miRNAs or long noncoding RNAs on the main biological processes involved in oxidative stress, inflammation, autophagy (PI3K), cell proliferation (NFkB, STAT3), and EMT (Notch, AKT, Wnt/β-catenin) pathways. microRNAs, as well as gene expression profile, responds to air pollution environment modulating some key genes involved in epigenetic modification or in key mediators of the biological processes described below. In this review, we provide some scientific evidences about the thigh correlation between miRNAs dysregulation, PM2.5 exposition, and gene pathways involved in cancer progression

By promoting cell differentiation, miR-100 sensitizes basal-like breast cancer stem cells to hormonal therapy

Basal-like breast cancer is an aggressive tumor subtype with a poor response to conventional therapies. Tumor formation and relapse are sustained by a cell subset of Breast Cancer Stem Cells (BrCSCs). Here we show that miR-100 inhibits maintenance and expansion of BrCSCs in basal-like cancer through Polo-like kinase1 (Plk1) down-regulation. Moreover, miR-100 favors BrCSC differentiation, converting a basal like phenotype into luminal. It induces the expression of a functional estrogen receptor (ER) and renders basal-like BrCSCs responsive to hormonal therapy. The key role played by miR-100 in breast cancer free-survival is confirmed by the analysis of a cohort of patients' tumors, which shows that low expression of miR-100 is a negative prognostic factor and is associated with gene signatures of high grade undifferentiated tumors. Our findings indicate a new possible therapeutic strategy, which could make aggressive breast cancers responsive to standard treatments

TRAM (Transcriptome Mapper): database-driven creation and analysis of transcriptome maps from multiple sources

<p>Abstract</p> <p>Background</p> <p>Several tools have been developed to perform global gene expression profile data analysis, to search for specific chromosomal regions whose features meet defined criteria as well as to study neighbouring gene expression. However, most of these tools are tailored for a specific use in a particular context (e.g. they are species-specific, or limited to a particular data format) and they typically accept only gene lists as input.</p> <p>Results</p> <p>TRAM (Transcriptome Mapper) is a new general tool that allows the simple generation and analysis of quantitative transcriptome maps, starting from any source listing gene expression values for a given gene set (e.g. expression microarrays), implemented as a relational database. It includes a parser able to assign univocal and updated gene symbols to gene identifiers from different data sources. Moreover, TRAM is able to perform intra-sample and inter-sample data normalization, including an original variant of quantile normalization (scaled quantile), useful to normalize data from platforms with highly different numbers of investigated genes. When in 'Map' mode, the software generates a quantitative representation of the transcriptome of a sample (or of a pool of samples) and identifies if segments of defined lengths are over/under-expressed compared to the desired threshold. When in 'Cluster' mode, the software searches for a set of over/under-expressed consecutive genes. Statistical significance for all results is calculated with respect to genes localized on the same chromosome or to all genome genes. Transcriptome maps, showing differential expression between two sample groups, relative to two different biological conditions, may be easily generated. We present the results of a biological model test, based on a meta-analysis comparison between a sample pool of human CD34+ hematopoietic progenitor cells and a sample pool of megakaryocytic cells. Biologically relevant chromosomal segments and gene clusters with differential expression during the differentiation toward megakaryocyte were identified.</p> <p>Conclusions</p> <p>TRAM is designed to create, and statistically analyze, quantitative transcriptome maps, based on gene expression data from multiple sources. The release includes FileMaker Pro database management runtime application and it is freely available at <url>http://apollo11.isto.unibo.it/software/</url>, along with preconfigured implementations for mapping of human, mouse and zebrafish transcriptomes.</p

Emerging Roles of Hedgehog Signaling in Cancer Immunity

Hedgehog–GLI (HH) signaling plays an essential role in embryogenesis and tissue homeostasis. Aberrant activation of the pathway through mutations or other mechanisms is involved in the development and progression of numerous types of cancer, including basal cell carcinoma, medulloblastoma, melanoma, breast, prostate, hepatocellular and pancreatic carcinomas. Activation of HH signaling sustains proliferation, suppresses cell death signals, enhances invasion and metastasis, deregulates cellular metabolism and promotes angiogenesis and tumor inflammation. Targeted inhibition of the HH pathway has therefore emerged as an attractive therapeutic strategy for the treatment of a wide range of cancers. Currently, the Smoothened (SMO) receptor and the downstream GLI transcriptional factors have been investigated for the development of targeted drugs. Recent studies have revealed that the HH signaling is also involved in tumor immune evasion and poor responses to cancer immunotherapy. Here we focus on the effects of HH signaling on the major cellular components of the adaptive and innate immune systems, and we present recent discoveries elucidating how the immunosuppressive function of the HH pathway is engaged by cancer cells to prevent immune surveillance. In addition, we discuss the future prospect of therapeutic options combining the HH pathway and immune checkpoint inhibitors

Injectable in situ forming hydrogels based on natural and synthetic polymers for potential application in cartilage repair

In this work we prepared two new hyaluronic acid (HA) based in situ forming hydrogels for the potential treatment of articular cartilage damages. In particular the amino derivative of HA (HA-EDA) and its graft copolymer with α-elastin (HA-EDA-g-α-elastin) were crosslinked, in mild physiological conditions via Michael-type addition, with α,β-poly(N-2-hydroxyethyl)-dl-aspartamide (PHEA) derivatized with divinylsulfone (DV). The swelling and degradation profile of the obtained hydrogels as well as the metabolic activity of incorporated bovine articular chondrocytes were investigated. Histological analysis and scanning electron microscopy (SEM) were performed to analyze the morphology of cells after scheduled times of incubation. Moreover the influence of the α-elastin on the elastic modulus of cell free and cell containing hydrogels has been studied through atomic force microscopy (AFM). This journal i

IN SITU FORMING HYDROGELS OF HYALURONIC ACID AND INULIN DERIVATIVES FOR CARTILAGE REGENERATION

An in situ forming hydrogel obtained by crosslinking of amino functionalized hyaluronic acid derivativeswith divinylsulfone functionalized inulin (INU-DV) has been here designed and characterized. In par-ticular two hyaluronic acid derivatives bearing respectively a pendant ethylenediamino (EDA) portion(HA-EDA) and both EDA and octadecyl pendant groups (HA-EDA-C18) were crosslinked through an azo-Michael reaction with INU-DV. Gelation time and consumption of DV portions have been evaluated onhydrogel obtained using HA-EDA and HA-EDA-C18derivatives with a concentration of 3% w/v and a ratio80/20 w/w respect to the crosslinker INU-DV. The presence of pendant C18chains improves mechanicalperformances of hydrogels and decreases the susceptibility to hyaluronidase hydrolysis. Bovine chon-drocytes, encapsulated during crosslinking, sufficiently survive and efficiently proliferate until 28 daysof analysis

Hepatocyte-targeted fluorescent nanoparticles based on a polyaspartamide for potential theranostic applications

Here, the synthesis of a galactosylated amphiphilic copolymer bearing rhodamine (RhB) moieties and its use for the preparation of polymeric fluorescent nanoparticles for potential applications in therapy and diagnosis are described. To do this, firstly, a fluorescent derivative of alpha,beta-poly(N-2-hydroxyethyl)-D,L-aspartamide (PHEA) was synthesized by chemical reaction with RhB, and with polylactic acid (PLA), to obtain PHEA-RhB-PLA. Then, the derivatization of PHEA-RhB-PLA with GAL-PEG-NH2 allows obtaining PHEA-RhB-PLA-PEGGAL copolymer, with derivatization degrees in -PLA and -PEG-GAL equal to 1.9 mol% and 4.5 mol%, respectively. Starting from this copolymer, liver-targeted fluorescent nanoparticles were prepared by high pressure homogenizationesolvent evaporation method, and showed nanoscaled size, slightly negative zeta potential and spherical shape. Chemical and enzymatic stability of fluorescent dye covalently linked to the copolymer backbone by ester linkage was demonstrated until 4 days of incubation. Finally, thanks to the covalently-linked fluorescent RhB, it was demonstrated that these galactosylated nanoparticles interact with HepG2 cells that are positive for the asialoglycoprotein receptor (ASGPR), while these do not interact with HeLa cells that are negative for the same receptor, demonstrating the contributor of ASGPR to the internalization process

POLYMERIC FLUORESCENT NANOPARTICLES BASED ON A POLYASPARTAMIDE FOR IMAGING APPLICATIONS: EVALUATION OF GALACTOSE TARGETING ON HEPG2 CELL INTERNALIZATION

In the field of nanomedicine, the use of polymeric fluorescent nanoparticles for in vitro and in vivo imaging is a promising application in order to evaluate passive as well as active targeting strategies [1]. In this work, the synthesis of an amphiphilic galactosylated polylactide-polyaminoacid copolymer bearing rhodamine (RhB) moieties and its use for the preparation of polymeric fluorescent nanoparticles for potential in vitro and in vivo imaging applications are described. To do this, firstly, a fluorescent derivative of α,β-poly(N-2-hydroxyethyl)-D,L-aspartamide (PHEA) was obtained by chemical reaction of PHEA with RhB. Then, a galactosylated derivative of polyethylene glycol, the O-(2-aminoethyl)-O’-galactosyl polyethylene glycol (GAL-PEG-NH2) was obtained by a reductive amination of lactose with primary amine function of poly(ethylene glycol)bis(amine) (H2N-PEG-NH2) in the presence of sodium cyanoborohydride. The fluorescent galactosylated polylactide-polyaminoacid conjugate was obtained by chemical reaction of PHEA-RhB with polylactic acid (PLA), and subsequent reaction with GAL-PEG-NH2, obtaining PHEA-RhB-PLA-PEG-GAL copolymer [2]. Starting from this copolymer, liver-targeted fluorescent nanoparticles, were successfully prepared by high pressure homogenization– solvent evaporation method [2]. Fluorescenti nanoparticles have nanoscaled size and spherical shape as showed by Dinamic Light Scattering (DLS) and Transmission Electron Microscopy (TEM) analyses. Moreover, thanks to the fluorescence given by covalently-linked RhB, by confocal microscopy studies it was demonstrated that these nanoparticles bearing GAL moieties interact with HepG2 cells that are positive for the asialoglycoprotein receptor (ASGP-R), while these do not interact with HeLa cells that are negative for the same receptor, demonstrating the contributor of ASGPR to the internalization process. [1] Vollrath A, S. Schubert, U.S. Schubert, Fluorescence imaging of cancer tissue based on metal-free polimeric nanopaticles - A review, J Mat Chem. 1, 1994 (2013). [2] E.F. Craparo, G. Teresi, M.C. Ognibene, M.P. Casaletto, M.L. Bondì, G. Cavallaro. J Nanoparticle Res. 12, 2629 (2010)