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

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-1ß and TNFα. Immunofluorescence showed that astrocytes but not microglia expressed IL-1ß. 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.

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

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.

Mesenchymal progenitors expressing TRAIL induce apoptosis in sarcomas.

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.

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

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.

Autologous anti-GD2 CAR T cells efficiently target primary human glioblastoma.

Glioblastoma (GBM) remains a deadly tumor. Treatment with chemo-radiotherapy and corticosteroids is known to impair the functionality of lymphocytes, potentially compromising the development of autologous CAR T cell therapies. We here generated pre-clinical investigations of autologous anti-GD2 CAR T cells tested against 2D and 3D models of GBM primary cells. We detected a robust antitumor effect, highlighting the feasibility of developing an autologous anti-GD2 CAR T cell-based therapy for GBM patients.

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

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.

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

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.

A 3D Platform to Investigate Dynamic Cell-to-Cell Interactions Between Tumor Cells and Mesenchymal Progenitors.

We here investigated the dynamic cell-to-cell interactions between tumor and mesenchymal stromal/stem cells (MSCs) by the novel VITVOⓇ 3D bioreactor that was customized to develop in vivo-like metastatic nodules of Ewing's sarcoma (ES). MSCs are known to contribute to tumor microenvironment as cancer associated fibroblast (CAF) precursors and, for this reason, they have also been used as anti-cancer tools. Using dynamic conditions, the process of tissue colonization and formation of metastatic niches was recreated through tumor cell migration aiming to mimic ES development in patients. ES is an aggressive tumor representing the second most common malignant bone cancer in children and young adults. An urgent and unmet need exists for the development of novel treatment strategies to improve the outcomes of metastatic ES. The tumor-tropic ability of MSCs offers an alternative approach, in which these cells can be used as vehicles for the delivery of antitumor molecules, such as the proapoptotic TNF-related apoptosis inducing ligand (TRAIL). However, the therapeutic targeting of metastases remains challenging and the interaction occurring between tumor cells and MSCs has not yet been deeply investigated. Setting up in vitro and in vivo models to study this interaction is a prerequisite for novel approaches where MSCs affinity for tumor is optimized to ultimately increase their therapeutic efficacy. Here, VITVOⓇ integrating a customized scaffold with an increased inter-fiber distance (VITVO50) was used to develop a dynamic model where MSCs and tumor nodules were evaluated under flow conditions. Colonization and interaction between cell populations were explored by droplet digital PCR (ddPCR). VITVO50 findings were then applied in vivo. An ES metastatic model was established in NSG mice and biodistribution of TRAIL-expressing MSCs in mice organs affected by metastases was investigated using a 4-plex ddPCR assay. VITVOⓇ proved to be an easy handling and versatile bioreactor to develop in vivo-like tumor nodules and investigate dynamic cell-to-cell interactions with MSCs. The proposed fluidic system promises to facilitate the understanding of tumor-stroma interaction for the development of novel tumor targeting strategies, simplifying the analysis of in vivo data, and ultimately accelerating the progress towards the early clinical phase.

GD2 CAR T cells against human glioblastoma.

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 ß1, 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.

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

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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

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.

Inducible Caspase9-mediated suicide gene for MSC-based cancer gene therapy.

Cellular therapies based on mesenchymal stromal/stem cells (MSC) are promising strategies in regenerative medicine and oncology. Despite encouraging results, there is still some level of concerns on inoculating MSC in cancer patients. To face this issue, one possibility resides in engineering MSC by incorporating a suicide gene in order to control their fate once infused. Strategies based on Herpes Simplex Virus Thymidine Kinase (HSV-TK) and the Cytosine Deaminase genes have been developed and more recently a novel suicide gene, namely, iCasp9, has been proposed. This approach is based on a variant of human Caspase9 that binds with high affinity to a synthetic, bioinert small molecule (AP20187) leading to cell death. Based on this technology so far marginally applied to MSC, we tested the suitability of iCasp9 suicide strategy in MSC to further increase their safety. MSC have been transfected by a lentiviral vector carrying iCasp9 gene and then tested for viability after AP20187 treatment in comparison with mock-transfected cells. Moreover, accounting our anti-tumor approaches based on MSC expressing potent anti-cancer ligand TNF-Related Apoptosis-Inducing Ligand (TRAIL), we generated adipose MSC co-expressing iCasp9 and TRAIL successfully targeting an aggressive sarcoma type. These data show that anti-cancer and suicide mechanisms can coexist without affecting cells performance and hampering the tumoricidal activity mediated by TRAIL. In conclusion, this study originally indicates the suitability of combining a MSC-based anti-cancer gene approach with iCasp9 demonstrating efficiency and specificity.

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

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.

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

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. Methods: Starting from the BXPC3 cell line, we generated and profiled a TRAIL-resistant model of pancreatic cancer, testing the impact of the combined treatment in vitro 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. Results: 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 in vitro and in vivo and the combined treatment resulted in an improved cytotoxicity on tumour cells. Conclusion: 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.

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

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.

GD2 expression in breast cancer.

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.

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

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.

Tumor Stroma Manipulation By MSC.

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.

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

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.

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

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.