Publications by authors named "Carlotta Spano"

22 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41698-021-00233-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8551169PMC
October 2021

Anti-GD2 CAR MSCs against metastatic Ewing's sarcoma.

Transl Oncol 2022 Jan 12;15(1):101240. Epub 2021 Oct 12.

Division of Oncology, Department of Medical and Surgical Sciences for Children & Adults, University-Hospital of Modena and Reggio Emilia, Modena, Italy; Rigenerand Srl, Medolla, Modena, Italy. Electronic address:

Background: Ewing's sarcoma (ES) is an aggressive cancer affecting children and young adults. We pre-clinically demonstrated that mesenchymal stromal/stem cells (MSCs) can deliver tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) against primary ES after local injection. However, ES is often metastatic calling for approaches able to support MSC targeting to the ES multiple remote sites. Considering that the disialoganglioside GD2 is expressed by ES and to optimise MSC tumour affinity, bi-functional (BF) MSCs expressing both TRAIL and a truncated anti-GD2 chimeric antigen receptor (GD2 tCAR) were generated and challenged against ES.

Methods: The anti-GD2 BF MSCs delivering a soluble variant of TRAIL (sTRAIL) were tested in several in vitro ES models. Tumour targeting and killing by BF MSCs was further investigated by a novel immunodeficient ES metastatic model characterized by different metastatic sites, including lungs, liver and bone, mimicking the deadly clinical scenario.

Findings: In vitro data revealed both tumour affinity and killing of BF MSCs. In vivo, GD2 tCAR molecule ameliorated the tumour targeting and persistence of BF MSCs counteracting ES in lungs but not in liver.

Interpretation: We here generated data on the potential effects of BF MSCs within a complex ES metastatic in vivo model, exploring also the biodistribution of MSCs. Our BF MSC-based strategy promises to pave the way for potential improvements in the therapeutic delivery of TRAIL for the treatment of metastatic ES and other deadly GD2-positive malignancies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1016/j.tranon.2021.101240DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8517927PMC
January 2022

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41598-018-37433-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6370785PMC
February 2019

Targeting GD2-positive glioblastoma by chimeric antigen receptor empowered mesenchymal progenitors.

Cancer Gene Ther 2020 08 22;27(7-8):558-570. Epub 2018 Nov 22.

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

Tumor targeting by genetically modified mesenchymal stromal/stem cells (MSCs) carrying anti-cancer molecules represents a promising cell-based strategy. We previously showed that the pro-apoptotic agent tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can be successfully delivered by MSCs to cancer sites. While the interaction between TRAIL and its receptors is clear, more obscure is the way in which MSCs can selectively target tumors and their antigens. Several neuroectoderm-derived neoplasms, including glioblastoma (GBM), sarcomas, and neuroblastoma, express high levels of the tumor-associated antigen GD2. We have already challenged this cell surface disialoganglioside by a chimeric antigen receptor (CAR)-T cell approach against neuroblastoma. With the intent to maximize the therapeutic profile of MSCs delivering TRAIL, we here originally developed a bi-functional strategy where TRAIL is delivered by MSCs that are also gene modified with the truncated form of the anti-GD2 CAR (GD2 tCAR) to mediate an immunoselective recognition of GD2-positive tumors. These bi-functional MSCs expressed high levels of TRAIL and GD2 tCAR associated with a robust anti-tumor activity against GD2-positive GBM cells. Most importantly, the anti-cancer action was reinforced by the enhanced targeting potential of such bi-functional cells. Collectively, our results suggest that a truncated anti-GD2 CAR might be a powerful new tool to redirect MSCs carrying TRAIL against GD2-expressing tumors. This affinity-based dual targeting holds the promise to combine site-specific and prolonged retention of MSCs in GD2-expressing tumors, thereby providing a more effective delivery of TRAIL for still incurable cancers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41417-018-0062-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7445885PMC
August 2020

Targeting GD2-positive glioblastoma by chimeric antigen receptor empowered mesenchymal progenitors.

Cancer Gene Ther 2020 08 22;27(7-8):558-570. Epub 2018 Nov 22.

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

Tumor targeting by genetically modified mesenchymal stromal/stem cells (MSCs) carrying anti-cancer molecules represents a promising cell-based strategy. We previously showed that the pro-apoptotic agent tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can be successfully delivered by MSCs to cancer sites. While the interaction between TRAIL and its receptors is clear, more obscure is the way in which MSCs can selectively target tumors and their antigens. Several neuroectoderm-derived neoplasms, including glioblastoma (GBM), sarcomas, and neuroblastoma, express high levels of the tumor-associated antigen GD2. We have already challenged this cell surface disialoganglioside by a chimeric antigen receptor (CAR)-T cell approach against neuroblastoma. With the intent to maximize the therapeutic profile of MSCs delivering TRAIL, we here originally developed a bi-functional strategy where TRAIL is delivered by MSCs that are also gene modified with the truncated form of the anti-GD2 CAR (GD2 tCAR) to mediate an immunoselective recognition of GD2-positive tumors. These bi-functional MSCs expressed high levels of TRAIL and GD2 tCAR associated with a robust anti-tumor activity against GD2-positive GBM cells. Most importantly, the anti-cancer action was reinforced by the enhanced targeting potential of such bi-functional cells. Collectively, our results suggest that a truncated anti-GD2 CAR might be a powerful new tool to redirect MSCs carrying TRAIL against GD2-expressing tumors. This affinity-based dual targeting holds the promise to combine site-specific and prolonged retention of MSCs in GD2-expressing tumors, thereby providing a more effective delivery of TRAIL for still incurable cancers.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1038/s41417-018-0062-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7445885PMC
August 2020

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.16363DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5458232PMC
May 2017

Microglia are less pro-inflammatory than myeloid infiltrates in the hippocampus of mice exposed to status epilepticus.

Glia 2016 08 1;64(8):1350-62. Epub 2016 Jun 1.

Department of Biomedical, Metabolic and Neural Sciences, University of Modena and Reggio Emilia, via G. Campi, 287, Modena, Italy.

Activated microglia, astrogliosis, expression of pro-inflammatory cytokines, blood brain barrier (BBB) leakage and peripheral immune cell infiltration are features of mesial temporal lobe epilepsy. Numerous studies correlated the expression of pro-inflammatory cytokines with the activated morphology of microglia, attributing them a pro-epileptogenic role. However, microglia and myeloid cells such as macrophages have always been difficult to distinguish due to an overlap in expressed cell surface molecules. Thus, the detrimental role in epilepsy that is attributed to microglia might be shared with myeloid infiltrates. Here, we used a FACS-based approach to discriminate between microglia and myeloid infiltrates isolated from the hippocampus 24 h and 96 h after status epilepticus (SE) in pilocarpine-treated CD1 mice. We observed that microglia do not express MHCII whereas myeloid infiltrates express high levels of MHCII and CD40 96 h after SE. This antigen-presenting cell phenotype correlated with the presence of CD4(pos) T cells. Moreover, microglia only expressed TNFα 24 h after SE while myeloid infiltrates expressed high levels of IL-1β and TNFα. Immunofluorescence showed that astrocytes but not microglia expressed IL-1β. Myeloid infiltrates also expressed matrix metalloproteinase (MMP)-9 and 12 while microglia only expressed MMP-12, suggesting the involvement of both cell types in the BBB leakage that follows SE. Finally, both cell types expressed the phagocytosis receptor Axl, pointing to phagocytosis of apoptotic cells as one of the main functions of microglia. Our data suggests that, during early epileptogenesis, microglia from the hippocampus remain rather immune supressed whereas myeloid infiltrates display a strong inflammatory profile. GLIA 2016 GLIA 2016;64:1350-1362.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/glia.23008DOI Listing
August 2016

Tumor Stroma Manipulation By MSC.

Curr Drug Targets 2016 ;17(10):1111-26

Laboratory of Cellular Therapy Department of Medical and Surgical Sciences for Children & Adults University-Hospital of Modena and Reggio Emilia Via del Pozzo 71, 41124 Modena, Italy.

Tumor stroma (TS) plays relevant roles in all steps of cancer development. We here address several fundamental aspects related with the interaction between cancer cells and their stromal counterparts. Dissecting these players is of pivotal importance to understand oncogenesis, immunoescape and drug resistance. In addition, this better comprehension will allow the introduction of novel and more effective therapeutic approaches where manipulated stromal elements may become detrimental for tumor growth. Our group and others rely on the use of multipotent mesenchymal stromal/stem cells (MSC) as anti-cancer tools, since these putative TS cell precursors can deliver potent apoptosis-inducing agents. Multimodal-armed MSC can target a variety of cancers in vitro and, when injected in vivo, they localize into tumors mediating cell death without evident toxicities to normal tissues. While several aspects of these strategies shall require further investigations, these approaches collectively indicate how TS manipulation by MSC represents a tool to influence the fate of cancer cells, creating a new generation of anti-cancer strategies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2174/1389450117666160307143226DOI Listing
August 2017

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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.18632/oncotarget.4670DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4694800PMC
September 2015

Mesenchymal stem/stromal cells as a delivery platform in cell and gene therapies.

BMC Med 2015 Aug 12;13:186. Epub 2015 Aug 12.

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

Regenerative medicine relying on cell and gene therapies is one of the most promising approaches to repair tissues. Multipotent mesenchymal stem/stromal cells (MSC), a population of progenitors committing into mesoderm lineages, are progressively demonstrating therapeutic capabilities far beyond their differentiation capacities. The mechanisms by which MSC exert these actions include the release of biomolecules with anti-inflammatory, immunomodulating, anti-fibrogenic, and trophic functions. While we expect the spectra of these molecules with a therapeutic profile to progressively expand, several human pathological conditions have begun to benefit from these biomolecule-delivering properties. In addition, MSC have also been proposed to vehicle genes capable of further empowering these functions. This review deals with the therapeutic properties of MSC, focusing on their ability to secrete naturally produced or gene-induced factors that can be used in the treatment of kidney, lung, heart, liver, pancreas, nervous system, and skeletal diseases. We specifically focus on the different modalities by which MSC can exert these functions. We aim to provide an updated understanding of these paracrine mechanisms as a prerequisite to broadening the therapeutic potential and clinical impact of MSC.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1186/s12916-015-0426-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4534031PMC
August 2015

Mesenchymal progenitors aging highlights a miR-196 switch targeting HOXB7 as master regulator of proliferation and osteogenesis.

Stem Cells 2015 Mar;33(3):939-50

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

Human aging is associated with a decrease in tissue functions combined with a decline in stem cells frequency and activity followed by a loss of regenerative capacity. The molecular mechanisms behind this senescence remain largely obscure, precluding targeted approaches to counteract aging. Focusing on mesenchymal stromal/stem cells (MSC) as known adult progenitors, we identified a specific switch in miRNA expression during aging, revealing a miR-196a upregulation which was inversely correlated with MSC proliferation through HOXB7 targeting. A forced HOXB7 expression was associated with an improved cell growth, a reduction of senescence, and an improved osteogenesis linked to a dramatic increase of autocrine basic fibroblast growth factor secretion. These findings, along with the progressive decrease of HOXB7 levels observed during skeletal aging in mice, indicate HOXB7 as a master factor driving progenitors behavior lifetime, providing a better understanding of bone senescence and leading to an optimization of MSC performance.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/stem.1897DOI Listing
March 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.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1002/stem.1903DOI Listing
March 2015

Detection of microparticles from human red blood cells by multiparametric flow cytometry.

Blood Transfus 2015 Apr 23;13(2):274-80. Epub 2014 Oct 23.

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

Background: During storage, red blood cells (RBC) undergo chemical and biochemical changes referred to as "storage lesions". These events determine the loss of RBC integrity, resulting in lysis and release of microparticles. There is growing evidence of the clinical importance of microparticles and their role in blood transfusion-related side effects and pathogen transmission. Flow cytometry is currently one of the most common techniques used to quantify and characterise microparticles. Here we propose multiparametric staining to monitor and quantify the dynamic release of microparticles by stored human RBC.

Material And Methods: RBC units (n=10) were stored under blood bank conditions for up to 42 days. Samples were tested at different time points to detect microparticles and determine the haemolysis rate (HR%). Microparticles were identified by flow cytometry combining carboxyfluorescein diacetate succinimidyl ester (CFSE) dye, annexin V and anti-glycophorin A antibody.

Results: We demonstrated that CFSE can be successfully used to label closed vesicles with an intact membrane. The combination of CFSE and glycophorin A antibody was effective for monitoring and quantifying the dynamic release of microparticles from RBC during storage. Double staining with CFSE/glycophorin A was a more precise approach, increasing vesicle detection up to 4.7-fold vs the use of glycophorin A/annexin V alone. Moreover, at all the time points tested, we found a robust correlation (R=0.625; p=0.0001) between HR% and number of microparticles detected.

Discussion: Multiparametric staining, based on a combination of CFSE, glycophorin A antibody and annexin V, was able to detect, characterise and monitor the release of microparticles from RBC units during storage, providing a sensitive approach to labelling and identifying microparticles for transfusion medicine and, more broadly, for cell-based therapies.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.2450/2014.0136-14DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4385076PMC
April 2015

Surrounding pancreatic adenocarcinoma by killer mesenchymal stromal/stem cells.

Hum Gene Ther 2014 May;25(5):406-7

Department of Medical and Surgical Sciences for Children and Adults, University Hospital of Modena and Reggio Emilia , Modena 41100, Italy .

View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1089/hum.2014.039DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4027979PMC
May 2014

Adipose-derived mesenchymal stem cells as stable source of tumor necrosis factor-related apoptosis-inducing ligand delivery for cancer therapy.

Cancer Res 2010 May 13;70(9):3718-29. Epub 2010 Apr 13.

Department of Oncology, Hematology and Respiratory Diseases, Plastic Surgery Unit, University-Hospital of Modena and Reggio Emilia, Modena, Italy.

Adipose-derived mesenchymal stromal/stem cells (AD-MSC) may offer efficient tools for cell-based gene therapy approaches. In this study, we evaluated whether AD-MSC could deliver proapoptotic molecules for cancer treatment. Human AD-MSCs were isolated and transduced with a retroviral vector encoding full-length human tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), a proapoptotic ligand that induces apoptosis in a variety of human cancers but not normal tissues. Although several studies have documented the antitumor activity of recombinant human TRAIL, its use in vivo is limited by a short half-life in plasma due to a rapid clearance by the kidney. We found that these limitations can be overcome using stably transduced AD-MSC, which could serve as a constant source of TRAIL production. AD-MSC armed with TRAIL targeted a variety of tumor cell lines in vitro, including human cervical carcinoma, pancreatic cancer, colon cancer, and, in combination with bortezomib, TRAIL-resistant breast cancer cells. Killing activity was associated with activation of caspase-8 as expected. When injected i.v. or s.c. into mice, AD-MSC armed with TRAIL localized into tumors and mediated apoptosis without significant apparent toxicities to normal tissues. Collectively, our results provide preclinical support for a model of TRAIL-based cancer therapy relying on the use of adipose-derived mesenchymal progenitors as cellular vectors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1158/0008-5472.CAN-09-1865DOI Listing
May 2010

Restoration and reversible expansion of the osteoblastic hematopoietic stem cell niche after marrow radioablation.

Blood 2009 Sep 11;114(11):2333-43. Epub 2009 May 11.

Department of Oncology and Hematology, University Hospital of Modena and Reggio Emilia, Modena, Italy.

Adequate recovery of hematopoietic stem cell (HSC) niches after cytotoxic conditioning regimens is essential to successful bone marrow transplantation. Yet, very little is known about the mechanisms that drive the restoration of these niches after bone marrow injury. Here we describe a profound disruption of the marrow microenvironment after lethal total body irradiation of mice that leads to the generation of osteoblasts restoring the HSC niche, followed by a transient, reversible expansion of this niche. Within 48 hours after irradiation, surviving host megakaryocytes were observed close to the endosteal surface of trabecular bone rather than in their normal parasinusoidal site concomitant with an increased stromal-derived factor-1 level. A subsequent increase in 2 megakaryocyte-derived growth factors, platelet-derived growth factor-beta and basic fibroblast growth factor, induces a 2-fold expansion of the population of N-cadherin-/osteopontin-positive osteoblasts, relative to the homeostatic osteoblast population, and hence, increases the number of potential niches for HSC engraftment. After donor cell engraftment, this expanded microenvironment reverts to its homeostatic state. Our results demonstrate the rapid recovery of osteoblastic stem cell niches after marrow radioablation, provide critical insights into the associated mechanisms, and suggest novel means to manipulate the bone marrow microenvironment to promote HSC engraftment.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2008-10-183459DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2745851PMC
September 2009

Donor cell-derived osteopoiesis originates from a self-renewing stem cell with a limited regenerative contribution after transplantation.

Blood 2008 Apr 8;111(8):4386-91. Epub 2008 Jan 8.

Department of Oncology and Hematology, University of Modena and Reggio Emilia, Modena, Italy.

In principle, bone marrow transplantation should offer effective treatment for disorders originating from defects in mesenchymal stem cells. Results with the bone disease osteogenesis imperfecta support this hypothesis, although the rate of clinical improvement seen early after transplantation does not persist long term, raising questions as to the regenerative capacity of the donor-derived mesenchymal progenitors. We therefore studied the kinetics and histologic/anatomic pattern of osteopoietic engraftment after transplantation of GFP-expressing nonadherent marrow cells in mice. Serial tracking of donor-derived GFP(+) cells over 52 weeks showed abundant clusters of donor-derived osteoblasts/osteocytes in the epiphysis and metaphysis but not the diaphysis, a distribution that paralleled the sites of initial hematopoietic engraftment. Osteopoietic chimerism decreased from approximately 30% to 10% by 24 weeks after transplantation, declining to negligible levels thereafter. Secondary transplantation studies provided evidence for a self-renewing osteopoietic stem cell in the marrow graft. We conclude that a transplantable, primitive, self-renewing osteopoietic cell within the nonadherent marrow cell population engrafts in an endosteal niche, like hematopoietic stem cells, and regenerates a significant fraction of all bone cells. The lack of durable donor-derived osteopoiesis may reflect an intrinsic genetic program or exogenous environmental signaling that suppresses the differentiation capacity of the donor stem cells.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1182/blood-2007-10-115725DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2288731PMC
April 2008

Proteasome inhibitors sensitize colon carcinoma cells to TRAIL-induced apoptosis via enhanced release of Smac/DIABLO from the mitochondria.

Pathol Oncol Res 2006 23;12(3):133-42. Epub 2006 Sep 23.

Ist Department of Pathology and Experimental Cancer Research, Semmelweis University, Budapest, Hungary.

The synergistic interaction between proteasome inhibitors and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) is a promising approach to induce cell death in tumor cells. However, the molecular and biochemical mechanisms of this synergism have been proven to be cell type specific. We therefore focused our investigation on TRAIL-resistant colon carcinoma cells in this study. DNA fragmentation, mitochondrial membrane depolarization and increased caspase-3-like enzyme activity was exclusively induced only by combined treatment with proteasome inhibitors (epoxomicin, MG132, bortezomib/PS-341) and TRAIL. The expression level of anti-apoptotic proteins (XIAP, survivin, Bcl-2, Bcl-XL), regulated by NF-kappaB transcription factor, was not effected by any of these treatments. TRAIL alone induced only partial activation of caspase-3 (p20), while the combination of TRAIL and proteasome inhibition led to the full proteolytic activation of caspase-3 (p17). Only the combination treatment induced marked membrane depolarization and the release of cytochrome c, HtrA2/Omi and Smac/DIABLO. Apoptosis-inducing factor (AIF) was not released in any of these conditions. These results are consistent with a model where the full activation of caspase-3 by caspase-8 is dependent on the release of Smac/DIABLO in response to the combined treatment. This molecular mechanism, independent of the inhibition NF-kappaB activity, may provide rationale for the combination treatment of colon carcinomas with proteasome inhibitors and recombinant TRAIL or agonistic antibody of TRAIL receptors.
View Article and Find Full Text PDF

Download full-text PDF

Source
http://dx.doi.org/10.1007/BF02893359DOI Listing
February 2007
-->