High-density ZnO nanowires for cellular biointerfaces: a new role as myogenic differentiation switch

The design of artificial platforms for expanding undifferentiated stem cells is of tremendous importance for regenerative medicine [1]. We have recently demonstrated that a ZnO nanowires (NWs) decorated glass support permits to obtain a differentiation switch during proliferation for mesoangioblasts (MABs)– i.e. multipotent progenitor cells which are remarkable candidates for the therapy of muscle diseases [2]. We have optimized the ZnO NWs synthesis on glass surfaces by numerical simulations and experimental systematic investigations, considering zinc speciation and supersaturation [3]. In particular, we demonstrated by numerical simulations that the ligand ethylenediamine, at the isoelectric point of the ZnO NWs tips, can effectively control – at 1:1 stoichiometric ratio with zinc – both speciation and supersaturation of zinc in the nutrient solution. In this regard, we employed ethanolamine (a safer precursor) for in-situ producing ethylenediamine by means of a zinc-catalysed amination reaction of ethanolamine by ammonia. The obtained highquality ZnONWs-cells biointerface allows cells to maintain viability and a spherical viable undifferentiated state during the 8 days observation time. Simulations of the interface by theoretical models [4] and our experimental investigations by SEM and confocal microscopy demonstrate that NWs do not induce any damage on the cellular membrane, whilst blocking their differentiation. More specifically, the myosin heavy chain, typically expressed in differentiated myogenic progenitors, is completely absent. Interestingly, the differentiation capabilities are completely restored upon cell removal from the NW-functionalized substrate and regrowing onto a standard culture glass dish. These results open the way towards unprecedented applications of ZnO NWs for cell-based therapy and tissue engineering [5]. References [1] G. Cossu, P. Bianco, Curr. Opin. Genet. Dev. 2003, 13, 537-542. [2] V. Errico, G. Arrabito, E. Fornetti, C. Fuoco, S. Testa, G. Saggio, S. Rufini, S. M. Cannata, A. Desideri, C. Falconi, C. Gargioli, ACS Appl. Mater. Interfaces, 2018, 10, 14097- 14107. [3] G. Arrabito, V. Errico, Z. Zhang, W. Han, C. Falconi, Nano Energy, 2018, 46, 54-62. [4] N. Buch-Månson, S. Bonde, J. Bolinsson, T. Berthing, J. Nygård, K.L. Martinez, Adv. Funct. Mater. 2015, 25, 3246-3255. [5] Y. Su, I. Cockerill, Y. Wang, Y.-X. Qin, L. Chang, Y. Zheng, and D. Zhu, Trends in Biotechnology, 2019, 37, 428-441

Single-cell mass cytometry reveals the impact of graphene nanomaterials with human primary immune cells

Understanding the interaction of nanomaterials and immune cells at the biomolecular level is of great significance in therapeutic applications. Here, the authors investigated the interaction of graphene oxide nanomaterials and several immune cell subpopulations using single-cell mass cytometry and genome-wide transcriptome analysis

Exploiting Mass Spectrometry to Unlock the Mechanism of Nanoparticle-Induced Inflammasome Activation

Nanoparticles (NPs) elicit sterile inflammation, but the underlying signaling pathways are poorly understood. Here, we report that human monocytes are particularly vulnerable to amorphous silica NPs, as evidenced by single-cell-based analysis of peripheral blood mononuclear cells using cytometry by time-of-flight (CyToF), while silane modification of the NPs mitigated their toxicity. Using human THP-1 cells as a model, we observed cellular internalization of silica NPs by nanoscale secondary ion mass spectrometry (nanoSIMS) and this was confirmed by transmission electron microscopy. Lipid droplet accumulation was also noted in the exposed cells. Furthermore, time-of-flight secondary ion mass spectrometry (ToF-SIMS) revealed specific changes in plasma membrane lipids, including phosphatidylcholine (PC) in silica NP-exposed cells, and subsequent studies suggested that lysophosphatidylcholine (LPC) acts as a cell autonomous signal for inflammasome activation in the absence of priming with a microbial ligand. Moreover, we found that silica NPs elicited NLRP3 inflammasome activation in monocytes, whereas cell death transpired through a non-apoptotic, lipid peroxidation-dependent mechanism. Together, these data further our understanding of the mechanism of sterile inflammation

Ejection of damaged mitochondria and their removal by macrophages ensure efficient thermogenesis in brown adipose tissue.

Recent findings have demonstrated that mitochondria can be transferred between cells to control metabolic homeostasis. Although the mitochondria of brown adipocytes comprise a large component of the cell volume and undergo reorganization to sustain thermogenesis, it remains unclear whether an intercellular mitochondrial transfer occurs in brown adipose tissue (BAT) and regulates adaptive thermogenesis. Herein, we demonstrated that thermogenically stressed brown adipocytes release extracellular vesicles (EVs) that contain oxidatively damaged mitochondrial parts to avoid failure of the thermogenic program. When re-uptaken by parental brown adipocytes, mitochondria-derived EVs reduced peroxisome proliferator-activated receptor-γ signaling and the levels of mitochondrial proteins, including UCP1. Their removal via the phagocytic activity of BAT-resident macrophages is instrumental in preserving BAT physiology. Depletion of macrophages in vivo causes the abnormal accumulation of extracellular mitochondrial vesicles in BAT, impairing the thermogenic response to cold exposure. These findings reveal a homeostatic role of tissue-resident macrophages in the mitochondrial quality control of BAT.This work was partially supported by the European Foundation for the Study of Diabetes (EFSD/Lilly, 2017 and EFSD/Boehringer Ingelheim European Research Programme on ‘‘Multi-System Challenges in Diabetes’’) and the Italian Ministry of Health (GR-2018-12367588) to D.L.-B.; Associazione Italiana per la Ricerca sul Cancro (AIRC) under IG 2019 - ID. 23562 project to K.A.; MIUR ‘‘Progetto Eccellenza’’ to Dipartimento di Scienze Farmacologiche e Biomolecolari, Universita` degli Studi di Milano, and NUTRAGE (CNR FOE 2019, DSB.AD004.271) to A.S; Italian Foundation of Multiple Sclerosis (grant 2017/R/8), the Italian Ministry of Health (grant GR-2016-02362380) and the MAI Award grant to V. Chiurchiu; National Institutes of Health (NIH) common fund (DP5 OD028125) and the Burroughs Wellcome Fund (1019648) to J.R.B.; NIH K01DK125608 to F.S.; R01DK102898 and R01DK122808 to Y.- H.T.; and NIH RO1 DK121805 and AHA 19TPA34910079 to B.Z. A.H. was supported by RTI2018-095497-B-I00 from MICINN, HR17_00527 from La Caixa Foundation, and TNE-18CVD04 from the Leducq Foundation. M.S-D was supported by a fellowship PRE2019-08746 from the Ministerio de Ciencia e Innovacio´ n. M.R. was partially supported by a fellowship from AIRC (IG 2019 - ID. 23562) and by the Italian Ministry of Health (SG-2019-12368589). V.C. is part of the PhD Program in Evolutionary Biology and Ecology, Department of Biology, University of Rome Tor Vergata.S

31st Annual Meeting and Associated Programs of the Society for Immunotherapy of Cancer (SITC 2016) : part two

Background The immunological escape of tumors represents one of the main ob- stacles to the treatment of malignancies. The blockade of PD-1 or CTLA-4 receptors represented a milestone in the history of immunotherapy. However, immune checkpoint inhibitors seem to be effective in specific cohorts of patients. It has been proposed that their efficacy relies on the presence of an immunological response. Thus, we hypothesized that disruption of the PD-L1/PD-1 axis would synergize with our oncolytic vaccine platform PeptiCRAd. Methods We used murine B16OVA in vivo tumor models and flow cytometry analysis to investigate the immunological background. Results First, we found that high-burden B16OVA tumors were refractory to combination immunotherapy. However, with a more aggressive schedule, tumors with a lower burden were more susceptible to the combination of PeptiCRAd and PD-L1 blockade. The therapy signifi- cantly increased the median survival of mice (Fig. 7). Interestingly, the reduced growth of contralaterally injected B16F10 cells sug- gested the presence of a long lasting immunological memory also against non-targeted antigens. Concerning the functional state of tumor infiltrating lymphocytes (TILs), we found that all the immune therapies would enhance the percentage of activated (PD-1pos TIM- 3neg) T lymphocytes and reduce the amount of exhausted (PD-1pos TIM-3pos) cells compared to placebo. As expected, we found that PeptiCRAd monotherapy could increase the number of antigen spe- cific CD8+ T cells compared to other treatments. However, only the combination with PD-L1 blockade could significantly increase the ra- tio between activated and exhausted pentamer positive cells (p= 0.0058), suggesting that by disrupting the PD-1/PD-L1 axis we could decrease the amount of dysfunctional antigen specific T cells. We ob- served that the anatomical location deeply influenced the state of CD4+ and CD8+ T lymphocytes. In fact, TIM-3 expression was in- creased by 2 fold on TILs compared to splenic and lymphoid T cells. In the CD8+ compartment, the expression of PD-1 on the surface seemed to be restricted to the tumor micro-environment, while CD4 + T cells had a high expression of PD-1 also in lymphoid organs. Interestingly, we found that the levels of PD-1 were significantly higher on CD8+ T cells than on CD4+ T cells into the tumor micro- environment (p < 0.0001). Conclusions In conclusion, we demonstrated that the efficacy of immune check- point inhibitors might be strongly enhanced by their combination with cancer vaccines. PeptiCRAd was able to increase the number of antigen-specific T cells and PD-L1 blockade prevented their exhaus- tion, resulting in long-lasting immunological memory and increased median survival

Ambra1 is a novel autophagy tumour suppressor gene

Macroautophagy is the major regulated catabolic mechanism used by eukaryotic cells to degrade long-lived proteins and organelles. It involves the formation of cytosolic doublemembrane vesicles, called autophagosomes, that sequester portions of cytoplasm and then they fuse with lysosomes to form autolysosomes. Autophagy has a well-documented role in the maintenance of tissue homeostasis and in the response to stressful environments; moreover, this process is often dysregulated in various human diseases, such as neurodegeneration and cancer. In this context, autophagy has been identified as a crucial process in oncogenesis and in tumour progression. Allelic loss of the essential autophagy gene Beclin 1 occurs in human cancers and renders mice tumour-prone. The regulation of the Beclin 1/Vps34 complex lipid kinase activity is a critical step in autophagy signaling pathway. Ambra1 (Activating molecule in Beclin 1-regulated autophagy) has been shown to be an important member of this complex and to be involved in autophagosome formation. This evidence prompted us to investigate a possible role for Ambra1 as a haplo-insufficient tumour suppressor gene. We show that monoallelic deletion of Ambra1 promotes tumorigenesis. We found that Ambra1+/gt mice have a significantly higher probability than Ambra1+/+ mice to develop a malignancy, showing approximately three folds increase of spontaneous tumorigenesis in a number of organs, such as liver, spleen, lymphonodes, and lung. In lung, Ambra1 hemizygous tumours show traits of lung papillary adenocarcinoma. We have previously shown that Ambra1 deficiency during embryogenesis in vivo and in vitro induces an increase in cell proliferation. Therefore, we have also investigated whether the observed tumours could be related to a direct impairment of cell growth control by autophagy. The first evidence that we found was a general increase of the organ size, especially of liver, kidney, heart and spleen of the heterozygous animals in comparison with the wt mice, thus suggesting a role for Ambra1 in cell growth control. Moreover, we elucidate this aspect also in vitro by analysing the proliferation rate and the markers of cell cycle in Ambra1 defective systems. In principle, the demonstration of a haplo-insufficient tumour suppressor phenotype for Ambra1’s reduced function in mice may have direct implications for analysing the molecular pathogenesis of human cancer. Therefore, we isolated mouse embryonic fibroblast (MEFs) from embryos wt, heterozygous and knockout for the Ambra1 gene trap mutation and we evaluated the cell growth rate by BrdU incorporation assay and by cell counting. Both analyses revealed a marked increase in the proliferation rate of Ambra1+/gt and Ambra1gt/gt MEFs when compared with wild-type cells, indicating that the loss of the only one allele of Ambra1’s alleles is sufficient to increase cellular proliferation. Since the molecular mechanism responsible for these results could be due to a deregulation of the cell cycle, we decided to examine the main cell cycle regulators. First of all we focussed our attention on analysing the levels of the four main cyclins: D, E, A, B. During our analysis we have found that in Ambra1 defective systems, unlike the cyclin D and E, high levels of the cyclin A and B were present. These proteins, called mitosis cyclins, are more expressed in the S- and M-phase of cell cycle. Therefore, our finding correlates with the hyperproliferative phenotype specific for Ambra1 deficient cells. Moreover the transcription of the cyclin A gene is under the control of p107 hyperphosphorylation (Zerfass et al., 1996), a protein belongs to the Retinoblastoma protein family. The increased levels of cyclin A could explain the hyperphoshorylate state of p107 that we found in our experimental systems. Moreover, since activities of cyclin/CDK complexes is also mediated by their binding to other proteins, we decided to examine whether the Ambra1 dosage was correlated to the expression of these proteins. In particular, we analyzed the levels of two main members of the Cip/Kip family, p21 and p27. In our studies we found a deregulation of a number of cell cycle regulatory proteins: A and B cyclins, p107, p21 and p27. This may explain the hyperproliferative phenotype observed in Ambra1-defective systems in vitro. In summary, we identified a novel haplo-insufficient tumour suppressor gene. This strongly support for the idea that Ambra1 could play an important role in the regulation of tumour development and that its activity is tightly regulated in coordination with cell growth. The detailed mechanism by which Ambra1 contributes to tumour suppression is still unknown