Publications by authors named "Tiziana Petrachi"

25 Publications

  • Page 1 of 1

GD2 CAR T cells against human glioblastoma.

NPJ Precis Oncol 2021 Oct 27;5(1):93. Epub 2021 Oct 27.

Laboratory of Cellular Therapy, Division of Oncology, Department of Medical and Surgical Sciences for Children & Adults, University of Modena and Reggio Emilia, Modena, Italy.

Glioblastoma is the most malignant primary brain tumor and is still in need of effective medical treatment. We isolated patient-derived glioblastoma cells showing high GD2 antigen expression representing a potential target for CAR T strategy. Data highlighted a robust GD2 CAR antitumor potential in 2D and 3D glioblastoma models associated with a significant and CAR T-restricted increase of selected cytokines. Interestingly, immunosuppressant TGF β1, expressed in all co-cultures, did not influence antitumor activity. The orthotopic NOD/SCID models using primary glioblastoma cells reproduced human histopathological features. Considering still-conflicting data on the delivery route for targeting brain tumors, we compared intracerebral versus intravenous CAR T injections. We report that the intracerebral route significantly increased the length of survival time in a dose-dependent manner, without any side effects. Collectively, the proposed anti-GD2 CAR can counteract human glioblastoma potentially opening a new therapeutic option for a still incurable cancer.
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http://dx.doi.org/10.1038/s41698-021-00233-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8551169PMC
October 2021

TRAIL receptors are expressed in both malignant and stromal cells in pancreatic ductal adenocarcinoma.

Am J Cancer Res 2021 15;11(9):4500-4514. Epub 2021 Sep 15.

Division of Oncology, Laboratory of Cellular Therapy, Department of Medical and Surgical Sciences of Children & Adults, University of Modena and Reggio Emilia Modena 41124, Italy.

This study assesses the expression of all TNF-related apoptosis-inducing ligand (TRAIL) receptors in pancreatic ductal adenocarcinoma (PDAC) tumor tissue. We aimed to include TRAIL receptor expression as an inclusion parameter in a future clinical study using a TRAIL-based therapy approach for PDAC patients. Considering the emerging influence of PDAC desmoplastic stroma on the efficacy of anti-PDAC therapies, this analysis was extended to tumor stromal cells. Additionally, we performed PDAC stroma characterization. Our retrospective cohort study (N=50) included patients with histologically confirmed PDAC who underwent surgery. The expression of TRAIL receptors (DR4, DR5, DcR1, DcR2, and OPG) in tumor and stromal cells was evaluated by immunohistochemistry (IHC). The amount of tumor stroma was assessed by anti-vimentin IHC and Mallory's trichrome staining. The prognostic impact was determined by the univariate Cox proportional hazards regression model. An extensive expression of functional receptors DR4 and DR5 and a variable expression of decoy receptors were detected in PDAC tumor and stromal cells. Functional receptors were detected also in metastatic tumor and stromal cells. A poor prognosis was associated with low or absent expression of decoy receptors in tumor cells of primary PDAC. After assessing that almost 80% of tumor mass was composed of stroma, we correlated a cellular-dense stroma in primary PDAC with reduced relapse-free survival. We demonstrated that TRAIL functional receptors are widely expressed in PDAC, representing a promising target for TRAIL-based therapies. Further, we demonstrated that a low expression of DcR1 and the absence of OPG in tumor cells, as well as a cellular-dense tumor stroma, could negatively impact the prognosis of PDAC patients.
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http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8493377PMC
September 2021

Microfragmented adipose tissue is associated with improved ex vivo performance linked to HOXB7 and b-FGF expression.

Stem Cell Res Ther 2021 08 28;12(1):481. Epub 2021 Aug 28.

Department of Medical and Surgical Sciences for Children and Adults, University-Hospital of Modena and Reggio Emilia, Modena, Italy.

Introduction: Adipose tissue (AT) has become a source of mesenchymal stromal/stem cells (MSC) for regenerative medicine applications, in particular skeletal disorders. Several enzymatic or mechanical procedures have been proposed to process AT with the aim to isolate cells that can be locally implanted. How AT is processed may impact its properties. Thus, we compared AT processed by centrifugation (C-AT) to microfragmentation (MF-AT). Focusing on MF-AT, we subsequently assessed the impact of synovial fluid (SF) alone on both MF-AT and isolated AT-MSC to better understand their cartilage repair mechanisms.

Materials And Methods: MF-AT and C-AT from the same donors were compared by histology and qRT-PCR immediately after isolation or as ex vivo cultures using a micro-tissue pellet system. The in vitro impact of SF on MF-AT and AT-MSC was assessed by histological staining and molecular analysis.

Results: The main AT histological features (i.e., increased extracellular matrix and cellularity) of the freshly isolated or ex vivo-cultured MF-AT persisted compared to C-AT, which rapidly deteriorated during culture. Based on our previous studies of HOX genes in MSC, we investigated the involvement of Homeobox Protein HOX-B7 (HOXB7) and its target basic Fibroblast Growth Factor (bFGF) in the molecular mechanism underlying the improved performance of MF-AT. Indeed, both these biomarkers were more prominent in freshly isolated MF-AT compared to C-AT. SF alone preserved the AT histological features of MF-AT, together with HOXB7 and bFGF expression. Increased cell performance was also observed in isolated AT-MSC after SF treatment concomitant with enhanced HOXB7 expression, although there was no apparent association with bFGF.

Conclusions: Our findings show that MF has a positive effect on the maintenance of AT histology and may trigger the expression of trophic factors that improve tissue repair by processed AT.
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http://dx.doi.org/10.1186/s13287-021-02540-1DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8399787PMC
August 2021

Assessing Biocompatibility of Face Mask Materials during COVID-19 Pandemic by a Rapid Multi-Assays Strategy.

Int J Environ Res Public Health 2021 05 18;18(10). Epub 2021 May 18.

Technopole "Mario Veronesi", via 29 Maggio, 6, 41037 Mirandola, Italy.

During the coronavirus disease 2019 (COVID-19) pandemic, scientific authorities strongly suggested the use of face masks (FMs). FM materials (FMMs) have to satisfy the medical device biocompatibility requirements as indicated in the technical standard EN ISO 10993-1:2018. The biologic evaluation must be confirmed by in vivo tests to verify cytotoxicity, sensitisation, and skin irritation. Some of these tests require an extensive period of time for their execution, which is incompatible with an emergency situation. In this study, we propose to verify the safety of FMMs combining the assessment of 3-[4,5-dimethylthiazolyl-2]-2,5-diphenyltetrazolium bromide (MTT) with quantification of nitric oxide (NO) and interleukin-6 (IL-6), as predictive markers of skin sensitisation or irritation based on human primary fibroblasts. Two hundred and forty-two FMMs were collected and classified according to spectrometer IR in polypropylene, paper, cotton, polyester, polyethylene terephthalate, 3-dimensional printing, and viscose. Of all FMMs tested, 50.8% passed all the assays, 48% failed at least one, and only 1.2% failed all. By a low cost, rapid and highly sensitive multi assays strategy tested on human skin fibroblasts against a large variety of FMMs, we propose a strategy to promptly evaluate biocompatibility in wearable materials.
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http://dx.doi.org/10.3390/ijerph18105387DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8158333PMC
May 2021

Microscopic and chemical characterization of PVC tube used for dialysis lines: A new approach.

Int J Artif Organs 2021 Feb 17;44(2):75-84. Epub 2020 Jun 17.

Science and Technology Park for Medicine, Tecnopolo di Mirandola "Mario Veronesi", Mirandola, Italy.

Polyvinylchloride is universally agreed upon to be the material of choice for tubings and for containers for medical application. Many alterations of the chemical/physical surface conditions, mainly due to an altered extrusion process, could influence its biocompatibility by promoting platelet aggregation. Biocompatibility and safety of the medical device must be preserved, also monitoring the migration of additives within polyvinylchloride during the diffusion process. A large variety of methods are used to verify the correct composition and extrusion of polyvinylchloride but, generally, they need long experimental time and are expensive. The aim of the study is to propose a simple, economic and rapid approach based on Fourier transform-infrared spectroscopy and Coomassie Blue staining. The method has been used to detect chemical and morphological defects caused by an altered extrusion process on 20/75 polyvinylchloride tubings in a blind test. This approach positively identified altered samples in 80% of the cases. The suggested approach represents a reliable and versatile method to detect and monitor surface defects by an easy, inexpensive and reproducible method.
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http://dx.doi.org/10.1177/0391398820932178DOI Listing
February 2021

mTOR Pathway Expression as Potential Predictive Biomarker in Patients with Advanced Neuroendocrine Tumors Treated with Everolimus.

Cancers (Basel) 2020 May 10;12(5). Epub 2020 May 10.

Department of Oncology and Hematology, University Hospital of Modena, 41124 Modena, Italy.

Background: Everolimus (Eve), which is a mammalian target of Rapamicin (mTOR) inhibitor, is part of the therapeutic armamentarium of neuroendocrine tumors (NETs). Currently, there are no validated biomarkers predicting Eve efficacy in NETs. In this study, we explore whether the expression of phosphorilated (p)-mTOR and p70S6-kinase (S6K), a downstream effector of mTOR, correlates with the outcome of patients with NET that were treated with Eve.

Methods: Tissue specimens that were derived from NETs treated with Eve at our Institution were examined for the expression levels of p-mTOR and p-S6K by immunohistochemistry. Response rate (RR), progression-free survival (PFS), and overall survival (OS) were analyzed in two groups: p-mTOR/p-S6K positive (group 1) and p-mTOR/p-S6K negative (group 2). Univariate and multivariate Cox regression analysis were performed.

Results: Twenty-four patients with advanced NETs that were treated with Eve were included in the analysis. Eight out 24 (33.3%) patients were both p-mTOR and p-S6K positive. A better median PFS and OS in group 1 (18.2 and 39.9 months) as compared to group 2 (13 and 32.4 months) was depicted, with a toxicity profile that was comparable with data literature.

Conclusions: Our study suggests that the activation of mTOR pathway can predict better outcomes in patients with NET treated with Eve. However, these results warrant further confirmation in a prospective setting.
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http://dx.doi.org/10.3390/cancers12051201DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7281483PMC
May 2020

Human Mesenchymal Stem Cell Combined with a New Strontium-Enriched Bioactive Glass: An Model for Bone Regeneration.

Materials (Basel) 2019 Nov 5;12(21). Epub 2019 Nov 5.

Department of Engineering "Enzo Ferrari", University of Modena and Reggio Emilia, Via P. Vivarelli 10, 41125 Modena, Italy.

A 3D cellular model that mimics the potential clinical application of a biomaterial is here applied for the first time to a bioactive glass, in order to assess its biological potential. A recently developed bioactive glass (BGMS10), whose composition contained strontium and magnesium, was produced in the form of granules and fully investigated in terms of biocompatibility in vitro. Apart from standard biological characterization (Simulated Body Fluid (SBF) testing and biocompatibility as per ISO10993), human bone marrow mesenchymal stromal/stem cells (BM-MSCs) were used to investigate the performance of the bioactive glass granules in an innovative 3D cellular model. The results showed that BGMS10 supported human BM-MSCs adhesion, colonization, and bone differentiation. Thus, bioactive glass granules seem to drive osteogenic differentiation and thus look particularly promising for orthopedic applications, bone tissue engineering and regenerative medicine.
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http://dx.doi.org/10.3390/ma12213633DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6862168PMC
November 2019

A Novel 3D In Vitro Platform for Pre-Clinical Investigations in Drug Testing, Gene Therapy, and Immuno-oncology.

Sci Rep 2019 05 9;9(1):7154. Epub 2019 May 9.

Rigenerand srl, Medolla, Modena, Italy.

Tumors develop within complex cell-to-cell interactions, with accessory cells playing a relevant role starting in the early phases of cancer progression. This event occurs in a three-dimensional (3D) environment, which to date, has been difficult to reproduce in vitro due to its complexity. While bi-dimensional cultures have generated substantial data, there is a progressive awareness that 3D culture strategies may rapidly increase the understanding of tumor development and be used in anti-cancer compound screening and for predicting response to new drugs utilizing personalized approaches. However, simple systems capable of rapidly rebuilding cancer tissues ex-vivo in 3D are needed and could be used for a variety of applications. Therefore, we developed a flat, handheld and versatile 3D cell culture bioreactor that can be loaded with tumor and/or normal cells in combination which can be monitored using a variety of read-outs. This biocompatible device sustained 3D growth of tumor cell lines representative of various cancers, such as pancreatic and breast adenocarcinoma, sarcoma, and glioblastoma. The cells repopulated the thin matrix which was completely separated from the outer space by two gas-permeable membranes and was monitored in real-time using both microscopy and luminometry, even after transportation. The device was tested in 3D cytotoxicity assays to investigate the anti-cancer potential of chemotherapy, biologic agents, and cell-based therapy in co-cultures. The addition of luciferase in target cancer cells is suitable for comparative studies that may also involve parallel in vivo investigations. Notably, the system was challenged using primary tumor cells harvested from lung cancer patients as an innovative predictive functional assay for cancer responsiveness to checkpoint inhibitors, such as nivolumab. This bioreactor has several novel features in the 3D-culture field of research, representing a valid tool useful for cancer investigations, drug screenings, and other toxicology approaches.
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http://dx.doi.org/10.1038/s41598-019-43613-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6509120PMC
May 2019

MSC-Delivered Soluble TRAIL and Paclitaxel as Novel Combinatory Treatment for Pancreatic Adenocarcinoma.

Theranostics 2019 1;9(2):436-448. Epub 2019 Jan 1.

Division of Oncology, Department of Medical and Surgical Sciences for Children & Adults, University-Hospital of Modena and Reggio Emilia, Modena, Italy.

Pancreatic cancer is the fourth leading cause of cancer death in western countries with more than 100,000 new cases per year in Europe and a mortality rate higher than 90%. In this scenario, advanced therapies based on gene therapies are emerging, thanks to a better understanding of tumour architecture and cancer cell alterations. We have demonstrated the efficacy of an innovative approach for pancreatic cancer based on mesenchymal stromal cells (MSC) genetically engineered to produce TNF-related Apoptosis Inducing Ligand (TRAIL). Here we investigated the combination of this MSC-based approach with the administration of a paclitaxel (PTX)-based chemotherapy to improve the potential of the treatment, also accounting for a possible resistance onset. Starting from the BXPC3 cell line, we generated and profiled a TRAIL-resistant model of pancreatic cancer, testing the impact of the combined treatment with specific cytotoxicity and metabolic assays. We then challenged the rationale in a subcutaneous mouse model of pancreatic cancer, assessing its effect on tumour size accounting stromal and parenchymal organization. PTX was able to restore pancreatic cancer sensitivity to MSC-delivered TRAIL by reverting its pro-survival gene expression profile. The two compounds cooperate both and and the combined treatment resulted in an improved cytotoxicity on tumour cells. In summary, this study uncovers the potential of a combinatory approach between MSC-delivered TRAIL and PTX, supporting the combination of cell-based products and conventional chemotherapeutics as a tool to improve the efficacy of the treatments, also addressing possible mechanisms of resistance.
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http://dx.doi.org/10.7150/thno.27576DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6376176PMC
December 2019

Soluble TRAIL Armed Human MSC As Gene Therapy For Pancreatic Cancer.

Sci Rep 2019 02 11;9(1):1788. Epub 2019 Feb 11.

Division of Oncology, Department of Medical and Surgical Sciences for Children & Adults, University-Hospital of Modena and Reggio Emilia, Modena, Italy.

Pancreatic ductal adenocarcinoma (PDAC) is still one of the most aggressive adult cancers with an unacceptable prognosis. For this reason novel therapies accounting for PDAC peculiarities, such as the relevant stromal reaction, are urgently needed. Here adipose mesenchymal stromal/stem cells (AD-MSC) have been armed to constantly release a soluble trimeric and multimeric variant of the known anti-cancer TNF-related apoptosis-inducing ligand (sTRAIL). This cancer gene therapy strategy was in vitro challenged demonstrating that sTRAIL was thermally stable and able to induce apoptosis in the PDAC lines BxPC-3, MIA PaCa-2 and against primary PDAC cells. sTRAIL released by AD-MSC relocated into the tumor stroma was able to significantly counteract tumor growth in vivo with a significant reduction in tumor size, in cytokeratin-7+ cells and by an anti-angiogenic effect. In parallel, histology on PDAC specimens form patients (n = 19) was performed to investigate the levels of TRAIL DR4, DR5 and OPG receptors generating promising insights on the possible clinical translation of our approach. These results indicate that adipose MSC can very efficiently vehicle a novel TRAIL variant opening unexplored opportunities for PDAC treatment.
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http://dx.doi.org/10.1038/s41598-018-37433-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6370785PMC
February 2019

Label-free toxicology screening of primary human mesenchymal cells and iPS-derived neurons.

PLoS One 2018 4;13(9):e0201671. Epub 2018 Sep 4.

Science & Technology Park for Medicine (TPM), Mirandola, Italy.

The high-throughput, label-free Corning Epic assay has applications in drug discovery, pharmacogenomics, cell receptor signaling, cell migration, and viral titration. The utility of Epic technology for biocompatibility testing has not been well established. In manufacturing of medical devices, in vitro and in vivo biocompatibility assessments are mandatory, according to ISO 10993. The new medical device regulation MDR 745/2017 specifies that ex vivo assays that can closely recapitulate in vivo scenarios are needed to better evaluate biomedical devices. We propose herein that Epic technology-which enables detection of variations in cell mass distribution-is suitable for biocompatibility screening of compounds. In this study, we challenged primary human osteoblasts, endothelial cells, and neurons derived from induced pluripotent stem cells with specific concentrations of methyl methacrylate (MMA). Polymeric MMA has long been applied in cranioplasty, where it makes contact with multiple cell types. Application of Epic technology yielded real-time cytotoxicity profiles for all considered cell types. The results were compared with those from microscopic observation of the same culture plate used in the Epic analyses. The Epic assay should be further examined for its utility for cell biology, genomics, and proteomics companion assays. Our results suggest that Epic technology can be applied to biocompatibility evaluation of human cells in medical device development.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0201671PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6122932PMC
February 2019

Soluble Fas Ligand Is Essential for Blister Formation in Pemphigus.

Front Immunol 2018 26;9:370. Epub 2018 Feb 26.

Laboratory of Cutaneous Biology, Department of Surgical, Medical, Dental and Morphological Sciences, University of Modena and Reggio Emilia, Modena, Italy.

Pemphigus is a blistering disease characterized by pemphigus autoantibodies (PVIgG) directed mostly against desmogleins (Dsgs), resulting in the loss of keratinocyte adhesion (acantholysis). Yet, the mechanisms underlying blister formation remain to be clarified. We have shown previously that anti-Fas ligand (FasL) antibody (Ab) prevents PVIgG-induced caspase-8 activation and Dsg cleavage in human keratinocytes, and that sera from pemphigus patients contain abnormally increased levels of FasL. Here, we demonstrate that recombinant FasL induces the activation of caspases prior to Dsg degradation, and anti-FasL Ab prevents acantholysis in cultured keratinocytes. Moreover, the silencing of FasL reduces PVIgG-induced caspase-8 activation and Dsg3 cleavage. Following injection of PVIgG into mice, FasL is upregulated at 1-3 h and is followed by caspase-8-mediated keratinocyte apoptosis, before blister formation. The administration of anti-FasL Ab after PVIgG injection blocks blister formation in mice. Furthermore, we injected PVIgG into two different gene-targeted mutant mice that selectively lack either secreted soluble FasL (sFasL), mice, or the membrane-bound form of FasL (mFasL), mice. After PVIgG treatment, blisters are only visible in animals, lacking mFasL, but still producing sFasL, similar to wild-type (C57BL/6) animals. By contrast, a significant decrease in the relative acantholytic area is observed in the animals. These results demonstrate that soluble FasL plays a crucial role in the mechanisms of blister formation, and blockade of FasL could be an effective therapeutic approach for pemphigus.
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http://dx.doi.org/10.3389/fimmu.2018.00370DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5834757PMC
April 2019

An Alternative Approach to Investigate Biofilm in Medical Devices: A Feasibility Study.

Int J Environ Res Public Health 2017 12 18;14(12). Epub 2017 Dec 18.

Science and Technology Park for Medicine, via 29 Maggio 6, 41037 Mirandola, Modena, Italy.

Biofilms are assemblages of bacterial cells irreversibly associated with a surface where moisture is present. In particular, they retain a relevant impact on public health since through biofilms bacteria are able to survive and populate biomedical devices causing severe nosocomial infections that are generally resistant to antimicrobial agents. Therefore, controlling biofilm formation is a mandatory feature during medical device manufacturing and during their use. In this study, combining a crystal violet staining together with advanced stereomicroscopy, we report an alternative rapid protocol for both qualitative and semi-quantitative biofilm determination having high specificity, high repeatability, and low variability. The suggested approach represents a reliable and versatile method to detect, monitor, and measure biofilm colonization by an easy, more affordable, and reproducible method.
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http://dx.doi.org/10.3390/ijerph14121587DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5751004PMC
December 2017

GD2 expression in breast cancer.

Oncotarget 2017 May;8(19):31592-31600

Department of Medical and Surgical Sciences for Children and Adults, University-Hospital of Modena and Reggio Emilia, 71-41124 Modena, Italy.

Breast cancer (BC) is a heterogeneous disease, including different subtypes having diverse incidence, drug-sensitivity and survival rates. In particular, claudin-low and basal-like BC have mesenchymal features with a dismal prognosis. Disialoganglioside GD2 is a typical neuroectodermal antigen expressed in a variety of cancers. Despite its potential relevance in cancer diagnostics and therapeutics, the presence and role of GD2 require further investigation, especially in BC. Therefore, we evaluated GD2 expression in a cohort of BC patients and its correlation with clinical-pathological features.Sixty-three patients with BC who underwent surgery without prior chemo- and/or radiotherapy between 2001 and 2014 were considered. Cancer specimens were analyzed by immunohistochemistry and GD2-staining was expressed according to the percentage of positive cells and by a semi-quantitative scoring system.Patient characteristics were heterogeneous by age at diagnosis, histotype, grading, tumor size, Ki-67 and receptor-status. GD2 staining revealed positive cancer cells in 59% of patients. Among them, 26 cases (41%) were labeled with score 1+ and 11 (18%) with score 2+. Notably, the majority of metaplastic carcinoma specimens stained positive for GD2. The univariate regression logistic analysis revealed a significant association of GD2 with triple-receptor negative phenotype and older age (> 78) at diagnosis.We demonstrate for the first time that GD2 is highly prevalent in a cohort of BC patients clustering on very aggressive BC subtypes, such as triple-negative and metaplastic variants.
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http://dx.doi.org/10.18632/oncotarget.16363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5458232PMC
May 2017

Therapeutic potential of the metabolic modulator phenformin in targeting the stem cell compartment in melanoma.

Oncotarget 2017 Jan;8(4):6914-6928

Laboratory of Translational Research, Department of Scientific Direction, Arcispedale S. Maria Nuova-IRCCS, Reggio Emilia, Italy.

Melanoma is the most dangerous and treatment-resistant skin cancer. Tumor resistance and recurrence are due to the persistence in the patient of aggressive cells with stem cell features, the cancer stem cells (CSC). Recent evidences have shown that CSC display a distinct metabolic profile as compared to tumor bulk population: a promising anti-tumor strategy is therefore to target specific metabolic pathways driving CSC behavior. Biguanides (metformin and phenformin) are anti-diabetic drugs able to perturb cellular metabolism and displaying anti-cancer activity. However, their ability to target the CSC compartment in melanoma is not known. Here we show that phenformin, but not metformin, strongly reduces melanoma cell viability, growth and invasion in both 2D and 3D (spheroids) models. While phenformin decreases melanoma CSC markers expression and the levels of the pro-survival factor MITF, MITF overexpression fails to prevent phenformin effects. Phenformin significantly reduces cell viability in melanoma by targeting both CSC (ALDHhigh) and non-CSC cells and by significantly reducing the number of viable cells in ALDHhigh and ALDHlow-derived spheroids. Consistently, phenformin reduces melanoma cell viability and growth independently from SOX2 levels. Our results show that phenformin is able to affect both CSC and non-CSC melanoma cell viability and growth and suggests its potential use as anti-cancer therapy in melanoma.
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http://dx.doi.org/10.18632/oncotarget.14321DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5351679PMC
January 2017

Survivin Modulates Squamous Cell Carcinoma-Derived Stem-Like Cell Proliferation, Viability and Tumor Formation in Vivo.

Int J Mol Sci 2016 Jan 12;17(1). Epub 2016 Jan 12.

Laboratory of Cutaneous Biology, Department of Surgical, Medical, Dental and Morphological Sciences, University of Modena and Reggio Emilia, via del Pozzo 71, 41124 Modena, Italy.

Squamous Cell Carcinoma-derived Stem-like Cells (SCC-SC) originate from alterations in keratinocyte stem cells (KSC) gene expression and sustain tumor development, invasion and recurrence. Since survivin, a KSC marker, is highly expressed in SCC-SC, we evaluate its role in SCC-SC cell growth and SCC models. Survivin silencing by siRNA decreases clonal growth of SCC keratinocytes and viability of total, rapidly adhering (RAD) and non-RAD (NRAD) cells from primary SCC. Similarly, survivin silencing reduces the expression of stem cell markers (OCT4, NOTCH1, CD133, β₁-integrin), while it increases the level of differentiation markers (K10, involucrin). Moreover, survivin silencing improves the malignant phenotype of SCC 3D-reconstruct, as demonstrated by reduced epidermal thickness, lower Ki-67 positive cell number, and decreased expression of MMP9 and psoriasin. Furthermore, survivin depletion by siRNA in Ras(G12V)-IκBα-derived tumors leads to smaller tumor formation characterized by lower mitotic index and reduced expression of the tumor-associated marker HIF1α, VEGF and CD51. Therefore, our results indicate survivin as a key gene in regulating SCC cancer stem cell formation and cSCC development.
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http://dx.doi.org/10.3390/ijms17010089DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4730332PMC
January 2016

A novel anti-GD2/4-1BB chimeric antigen receptor triggers neuroblastoma cell killing.

Oncotarget 2015 Sep;6(28):24884-94

Department of Medical and Surgical Sciences for Children & Adults, Division of Oncology, University-Hospital of Modena and Reggio Emilia, Modena, Italy.

Chimeric antigen receptor (CAR)-expressing T cells are a promising therapeutic option for patients with cancer. We developed a new CAR directed against the disialoganglioside GD2, a surface molecule expressed in neuroblastoma and in other neuroectoderm-derived neoplasms. The anti-GD2 single-chain variable fragment (scFv) derived from a murine antibody of IgM class was linked, via a human CD8α hinge-transmembrane domain, to the signaling domains of the costimulatory molecules 4-1BB (CD137) and CD3-ζ. The receptor was expressed in T lymphocytes by retroviral transduction and anti-tumor activities were assessed by targeting GD2-positive neuroblastoma cells using in vitro cytotoxicity assays and a xenograft model. Transduced T cells expressed high levels of anti-GD2 CAR and exerted a robust and specific anti-tumor activity in 4- and 48-hour cultures with neuroblastoma cells. Cytotoxicity was associated with the release of pro-apoptotic molecules such as TRAIL and IFN-γ. These results were confirmed in a xenograft model, where anti-GD2 CAR T cells infiltrating tumors and persisting into blood circulation induced massive apoptosis of neuroblastoma cells and completely abrogated tumor growth. This anti-GD2 CAR represents a powerful new tool to redirect T cells against GD2. The preclinical results of this study warrant clinical testing of this approach in neuroblastoma and other GD2-positive malignancies.
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http://dx.doi.org/10.18632/oncotarget.4670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4694800PMC
September 2015

Organ culture and Reflectance Confocal Microscopy as new integrated tools for barrier rescue studies in inflammatory skin diseases.

Exp Dermatol 2015 Dec 2;24(12):980-2. Epub 2015 Oct 2.

Laboratory of Cutaneous Biology, Department of Surgical, Medical, Dental and Morphological Sciences, University of Modena and Reggio Emilia, Modena, Italy.

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http://dx.doi.org/10.1111/exd.12823DOI Listing
December 2015

Mesenchymal progenitors expressing TRAIL induce apoptosis in sarcomas.

Stem Cells 2015 Mar;33(3):859-69

Division of Oncology, Department of Medical and Surgical Sciences for Children & Adults, University-Hospital of Modena and Reggio Emilia, Modena, Italy.

Sarcomas are frequent tumors in children and young adults that, despite a relative chemo-sensitivity, show high relapse rates with up to 80% of metastatic patients dying in 5 years from diagnosis. The real ontogeny of sarcomas is still debated and evidences suggest they may derive from precursors identified within mesenchymal stromal/stem cells (MSC) fractions. Recent studies on sarcoma microenvironment additionally indicated that MSC could take active part in generation of a supportive stroma. Based on this knowledge, we conceived to use modified MSC to deliver tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) targeting different sarcoma histotypes. Gene modified MSC expressing TRAIL were cocultured with different osteosarcoma, rhabdomyosarcoma, and Ewing's Sarcoma (ES) cell lines assessing viability and caspase-8 activation. An in vivo model focused on ES was then implemented considering the impact of MSC-TRAIL on tumor size, apoptosis, and angiogenesis. MSC expressing TRAIL induced significantly high apoptosis in all tested lines. Sarcoma death was specifically associated with caspase-8 activation starting from 8 hours of coculture with MSC-TRAIL. When injected into pre-established ES xenotransplants, MSC-TRAIL persisted within its stroma, causing significant tumor apoptosis versus control groups. Additional histological and in vitro studies reveal that MSC-TRAIL could also exert potent antiangiogenic functions. Our results suggest that MSC as TRAIL vehicles could open novel therapeutic opportunities for sarcoma by multiple mechanisms.
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http://dx.doi.org/10.1002/stem.1903DOI Listing
March 2015

CD271 mediates stem cells to early progeny transition in human epidermis.

J Invest Dermatol 2015 Mar 20;135(3):786-795. Epub 2014 Oct 20.

Laboratory of Cutaneous Biology, Department of Surgical, Medical, Dental and Morphological Sciences, University of Modena and Reggio Emilia, Modena, Italy.

CD271 is the low-affinity neurotrophin (p75NTR) receptor that belongs to the tumor necrosis factor receptor superfamily. Because in human epidermis, CD271 is predominantly expressed in transit-amplifying (TA) cells, we evaluated the role of this receptor in keratinocyte differentiation and in the transition from keratinocyte stem cells (KSCs) to progeny. Calcium induced an upregulation of CD271 in subconfluent keratinocytes, which was prevented by CD271 small interfering RNA. Furthermore, CD271 overexpression provoked the switch of KSCs to TA cells, whereas silencing CD271 induced TA cells to revert to a KSC phenotype, as shown by the expression of β1-integrin and by the increased clonogenic ability. CD271(+) keratinocytes sorted from freshly isolated TA cells expressed more survivin and keratin 15 (K15) compared with CD271(-) cells and displayed a higher proliferative capacity. Early differentiation markers and K15 were more expressed in the skin equivalent generated from CD271(+) TA than from those derived from CD271(-) TA cells. By contrast, late differentiation markers were more expressed in skin equivalents from CD271(-) than in reconstructs from CD271(+) TA cells. Finally, skin equivalents originated from CD271(-) TA cells displayed a psoriatic phenotype. These results indicate that CD271 is critical for keratinocyte differentiation and regulates the transition from KSCs to TA cells.
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http://dx.doi.org/10.1038/jid.2014.454DOI Listing
March 2015

Proteomic analysis of PTCH1+/- fibroblast lysate and conditioned culture media isolated from the skin of healthy subjects and nevoid basal cell carcinoma syndrome patients.

Biomed Res Int 2013 4;2013:794028. Epub 2013 Dec 4.

Department of Diagnostic and Clinical Medicine and Public Health, University of Modena and Reggio Emilia, Via del Pozzo, 41100 Modena, Italy.

Background: The pathogenesis underlying the increased predisposition to the development of basal cell carcinomas (BCCs) in the context of Gorlin-Goltz syndrome is linked to molecular mechanisms that differ from sporadic BCCs. Patients with Gorlin syndrome tend to develop multiple BCCs at an early age and present with tumors of non-sun-exposed skin. The aim of this study was to compare the proteomic profile of cultured fibroblast and fibroblast conditioned culture media of PTCH1+ and nonmutated fibroblasts.

Results: Proteomic analysis was performed using Surface-Enhanced Laser Desorption/Ionization Time-of-Flight mass spectrometry in PTCH1+ fibroblast conditioned media isolated from not affected sun-protected skin areas of Gorlin patients and from healthy subjects. 12 protein cluster peaks, >5 kDa, had significant differences in their peak intensities between PTCH1+ and PTCH1- subject groups. We detected a strongly MMP1 overexpression in PTCH1+ fibroblasts obtained from NBCCS patients with respect to healthy donors.

Conclusion: Protein profiles in the fibroblast conditioned media revealed statistically significant differences between two different types (missense versus nonsense) of PTCH1 mutations. These differences could be useful as signatures to identify PTCH1 gene carriers at high risk for the development of NBCCS-associated malignancies and to develop novel experimental molecular tailored therapies based on these druggable targets.
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http://dx.doi.org/10.1155/2013/794028DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3867831PMC
July 2014

Isolation and characterization of squamous cell carcinoma-derived stem-like cells: role in tumor formation.

Int J Mol Sci 2013 Sep 26;14(10):19540-55. Epub 2013 Sep 26.

Department of Dermatology, University of Modena and Reggio Emilia, Via del Pozzo 71, 41124 Modena, Italy.

In human epidermis, keratinocyte stem cells (KSC) are characterized by high levels of β1-integrin, resulting in the rapid adhesion to type IV collagen. Since epithelial tumors originate from KSC, we evaluated the features of rapidly adhering (RAD) keratinocytes derived from primary human squamous cell carcinoma of the skin (cSCC). RAD cells expressed higher levels of survivin, a KSC marker, as compared to non-rapidly adhering (NRAD) cells. Moreover, RAD cells proliferated to a greater extent and were more efficient in forming colonies than NRAD cells. RAD cells also migrated significantly better than NRAD cells. When seeded in a silicone chamber and grafted onto the back skin of NOD SCID mice, RAD cells formed tumors 2-4 fold bigger than those derived from NRAD cells. In tumors derived from RAD cells, the mitotic index was significantly higher than in those derived from NRAD cells, while Ki-67 and survivin expression were more pronounced in RAD tumors. This study suggests that SCC RAD stem cells play a critical role in the formation and development of epithelial tumors.
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http://dx.doi.org/10.3390/ijms141019540DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3821572PMC
September 2013

E-FABP induces differentiation in normal human keratinocytes and modulates the differentiation process in psoriatic keratinocytes in vitro.

Exp Dermatol 2013 Apr;22(4):255-61

Institute of Dermatology, School of Medicine, University of Modena and Reggio Emilia, Modena, Italy.

Epidermal fatty acid-binding protein (E-FABP) is a lipid carrier, originally discovered in human epidermis. We show that E-FABP is almost exclusively expressed in postmitotic (PM) keratinocytes, corresponding to its localization in the highest suprabasal layers, while it is barely expressed in keratinocyte stem cells (KSC) and transit amplifying (TA) keratinocytes. Transfection of normal human keratinocytes with recombinant (r) E-FABP induces overexpression of K10 and involucrin. On the other hand, E-FABP inhibition by siRNA downregulates K10 and involucrin expression in normal keratinocytes through NF-κB and JNK signalling pathways. E-FABP is highly expressed in psoriatic epidermis, and it is mainly localized in stratum spinosum. Psoriatic PM keratinocytes overexpress E-FABP as compared to the same population in normal epidermis. E-FABP inhibition in psoriatic keratinocytes markedly reduces differentiation, while it upregulates psoriatic markers such as survivin and K16. However, under high-calcium conditions, E-FABP silencing downregulates K10 and involucrin, while survivin and K16 expression is completely abolished. These data strongly indicate that E-FABP plays an important role in keratinocyte differentiation. Moreover, E-FABP modulates differentiation in psoriatic keratinocytes.
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http://dx.doi.org/10.1111/exd.12111DOI Listing
April 2013

Role of neurotrophins on dermal fibroblast survival and differentiation.

J Cell Physiol 2012 Mar;227(3):1017-25

Institute of Dermatology, School of Biosciences and Biotechnologies, University of Modena and Reggio Emilia, Modena, Italy.

Neurotrophins (NTs) belong to a family of growth factors that play a critical role in the control of skin homeostasis. NTs act through the low-affinity receptor p75NTR and the high-affinity receptors TrkA, TrkB, and TrkC. Here we show that dermal fibroblasts (DF) and myofibroblasts (DM) synthesize and secrete all NTs and express NT receptors. NTs induce differentiation of DF into DM, as shown by the expression of α-SMA protein. The Trk inhibitor K252a, TrkA/Fc, TrkB/Fc, or TrkC/Fc chimera prevents DF and DM proliferation. In addition, p75NTR siRNA inhibits DF proliferation, indicating that both NT receptors mediate DF proliferation induced by endogenous NTs. Autocrine NTs also induce DF migration through p75NTR and Trk, as either silencing of p75NTR or Trk/Fc chimeras prevent this effect, in absence of exogenous NTs. Finally, NGF or BDNF statistically increase the tensile strength in a dose dependent manner, as measured in a collagen gel through the GlaSbox device. Taken together, these results indicate that NTs exert a critical role on fibroblast and could be involved in tissue re-modeling and wound healing.
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http://dx.doi.org/10.1002/jcp.22811DOI Listing
March 2012
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