Antitumoral effects of attenuated Listeria monocytogenes in a genetically engineered mouse model of melanoma

Attenuated Listeria monocytogenes (Lmat-LLO) represents a valuable anticancer vaccine and drug delivery platform. Here we show that in vitro Lmat-LLO causes ROS production and, in turn, apoptotic killing of a wide variety of melanoma cells, irrespectively of their stage, mutational status, sensitivity to BRAF inhibitors or degree of stemness. We also show that, when administered in the therapeutic setting to Braf/Pten genetically engineered mice, Lmat-LLO causes a strong decrease in the size and volume of primary melanoma tumors, as well as a reduction of the metastatic burden. At the molecular level, we confirm that the anti-melanoma activity exerted in vivo by Lmat-LLO depends also on its ability to potentiate the immune response of the organism against the infected tumor. Our data pave the way to the preclinical testing of listeria-based immunotherapeutic strategies against metastatic melanoma, using a genetically engineered mouse rather than xenograft models

Neurosteroidogenesis-based therapeutic strategies for the control of neuroinflammation: the role of the 18-kDa Translocator Protein (TSPO)

Neuroinflammation is a physiological response occurring in the CNS to restore homeostasis following tissue damage. However, altered and chronic neuroinflammatory conditions are considered to contribute to the onset of several neurodegenerative disorders, as well as preventing the repair of the affected tissue. Although the neuroinflammatory response involves numerous central and peripheral reactive elements, microglia play a crucial role. Microglia, through the release of a large amount and type of inflammatory cytokines, are able to influence the resulting microenvironment, determining the fate of the regenerative processes. Over the years, numerous phenotypes and stages of activated microglia have been observed with the identification of a myriad of transcriptional patterns associated with a specific pathological condition. These types of studies result to be fundamental to the identification of targets for the control of pathological states of microglia and neuroinflammation. Within these, one of the markers expressed exclusively in activated microglia appears to be the 18 kDa translocator protein - TSPO expressed at the contact sites between the outer and inner membrane of the mitochondria. TSPO appears to be involved in the rate-limiting step in the de novo production of neurosteroids, as potent anti-inflammatory molecules produced at the CNS level, via the translocation of cholesterol within the mitochondrion. However, the neurosteroidogenic capacity of microglia has been questioned over the years due to the reduced expression of the CYP11A1 enzyme, which is involved in the conversion of cholesterol into pregnenolone, the precursor metabolite of all neurosteroids. In the present thesis, the role of TSPO-mediated neurosteroidogenesis in promoting the homeostatic/repairing microglial phenotype and preventing the onset of hyperactivated states was investigated. First of all, the neurosteroidogenic capacity of human microglia was demonstrated. Indeed, human microglia expressed all proteins involved in the rate-limiting step of neurosteroid production (StAR, TSPO, and CYP11A1) and enzymes involved in the neurosteroidogenic cascade. We also observed a time-dependent production of pregnenolone confirming the neurosteroidogenic capacity of microglia. Pharmacological stimulation of TSPO by selective ligands led to a significant increase in pregnenolone production. The most promising ligands appeared to be XBD-173 and the newly synthesized ligand PIGA1138, which also demonstrated the ability to promote neurotrophic support of microglia via increased BDNF release. The role of TSPO in microglial neurosteroidogenesis was further investigated by using a TSPO-KD model, in which pregnenolone production was impaired. Secondly, the inflammatory response of human microglia was investigated following immunogenic stimulation with IL-1β. Here, neurosteroidogenesis was inhibited using a CYP11A1 inhibitor to assess its role in influencing the microglial inflammatory response. The resulting microglial population following such inhibition, showed an unbalanced pro-inflammatory response, together with reduced phagocytic capacities, reduced BDNF production, and reduced responsiveness to the homeostatic TGF-β stimulus, suggesting a dysfunctional and hyperreactive phenotype. In contrast, pharmacological pre-activation of TSPO by XBD-173 and PIGA1138 led to the acquisition of a functional and reparative microglial phenotype, characterized by an increase in TGF-β expression and BDNF release. In particular, this effect was observed for the PIGA1138 ligand, suggesting this compound as promising for the control of the neuroinflammatory response. Since the results suggested a control of the inflammatory response by the neurosteroidogenic process, an analysis of neurosteroidogenic protein expression was then conducted. Interestingly, inflammatory conditions increased the expression of TSPO and CYP11A1, whereas gene expression analysis of the steroidogenic enzymes suggested an imbalance towards the production of 3α-reduced progesterone metabolites rather than CYP17A1-mediated ones. Analysis of the neurosteroidome by LC-MS corroborated the results: activated microglia showed higher levels of allopregnanolone and pregnanolone, and reduced levels of testosterone and estradiol compared to CTRL. Regarding the neurosteroidome of microglia pre-treated with ligands, it was observed that the phenotypic shift could be associated with increased production of androstenedione, testosterone, and estradiol. The attention was also directed toward the ability of TSPO to promote endogenous estradiol production. To this end, the neuritogenic effects of TSPO ligands XBD-173 and PIGA1138 were studied in primary cultures of hippocampal neurons, also focusing on possible differences in the pharmacological response between male and female neurons. Greater reactivity to ligands was observed in female neurons, which are known to have a higher capacity to endogenously produce estradiol compared with males, whereas only PIGA1138 had a significant neuritogenic effect in both neuronal populations. To confirm, the effect appeared to be mediated by an increased production of estradiol, which in turn regulates BDNF and Ngn3 levels, involved in neuronal development. The effects of microglia in conditioning the repair processes were studied in a model of human neural progenitor cells (hNPCs) treated with a conditioned microglia medium (MCM). MCM could promote hNPCs cell migration without significantly altering cell viability. MCM from homeostatic microglia also promoted neuronal cell differentiation and development, in contrast to MCM from activated microglia, which promoted astrocytic cell proliferation. Interestingly, MCM derived from activated microglia and pre-treated with TSPO ligands recovered the negative effects of MCM IL-1β. Again, the greatest effects were evidenced in microglia pre-treated with the ligand PIGA1138. The role of the neurosteroidome was assessed by treating hNPCs with an MCM derived from microglia in which neurosteroid production was inhibited. In particular, the inhibition of neurosteroid production by microglia led to the suppression of the positive effects of both MCM from CTRL and ligand-pretreated microglia, and also exacerbated the negative effects of activated MCM on hNPCs migration, viability, and differentiation. As the last part of this thesis, neuroinflammatory processes were investigated in vivo in a model of LPS-induced neuroinflammation. In particular, the inflammatory response and the learning and memory abilities of mice were evaluated in the recovery phase after 5 days of chronic treatment with LPS. Preliminary data have shown alterations in the inflammatory response and reduction of markers of synaptic plasticity, especially in the hippocampal region, accompanied by cognitive deficits of the animal following behavioral test after 5 days of recovery. Experiments concerning the neuroprotective action of PIGA1138, as the most promising ligand, are currently in progress

Study of chemical functionalization of an Attenuated Listeria Monocytogenes Strain able to infect Metastatic Melanoma and assessment of its potential as tracer and platform for antitumoral drug release

The following study is based on the research of new possible methods for the chemical functionalization of attenuated Listeria Monocytogenes Lmat as tracer of primary tumor and metastatic sites, and as drug carrier in particular against Metastatic Melanoma. Cutaneous Melanoma is a relatively common, potential lethal skin tumor of increasing incidence even in young patients. The main concern regarding Melanoma is its high propensity for metastasis especially in visceral organs which occurs also in its early growth phase. Once reached its metastatic stage, Melanoma results a particular aggressive type of cancer and resistant even to the most recent pharmacological treatment, leading to decrease of 5-year survival rate to 15%-20%. Recently, Immunotherapy has attracted scientific world since when it was clear that cancerous cells can escape from Immune System and even inhibit it, a process that has been called Immunoediting. So rearming the inhibited Immune System specifically against cancerous cells has become one of the most promising line of thought in cancer therapy. Metastatic melanoma is well known for its resistance to traditional cancer treatments, including chemotherapy and radiotherapy, as well as its relative susceptibility to immunotherapy compared with other cancer types. A promising tool in Cancer Immunotherapy, not only for Melanoma, is represented by Bacteria. Nowadays many clinical trials are based on the vaccination with attenuated bacteria able to selectively colonize primary and metastatic tumor for its Immune-privileged microenvironment, reactivating host immune system specifically against cancer cells. In particular, the subject of this work is Listeria Monocytogenes, a foodborne pathogen, Gram positive, facultative anaerobic bacterium which, used in its attenuated form (Lmat) , can be a valuable tool for Cancer Vaccines. Beyond its intrinsic toxicity given by the production of ROS in cancer cells, the main advantage of using Lmat consists in the possibility of its genetic engineerization as an antigenic vector able to stimulate immune system against cancer cells that bring on their surface one or more specific antigens. Given Lmat selective antitumoral effect, we started exploring the possibility of using Lmat as drug carrier and tumor tracer. Different approaches were tested to label Lmat with Fluorescent Probes and labelling protocols were developed. The attention was focus both on compounds that can specifically label Listeria on cell wall or inside bacteria. Fluorescent Aptamers were tested to reach a surface labelling while Fluorescent β-Cyclodextrin or Fluorescent Gentamicin for the “inner” uptake approach. A comparison between the three different selected Probes was done, paying particular attention to the extent of labelling and the viability of labelled Lmat. Once obtained a satisfactory labelling of Lmat, other features were investigated. First of all, the relation between growth and loss of fluorescence in various culture media was evaluated in order to demonstrate the stability of Lmat fluorescence over time. Afterwards, labelled Lmat was tested in vitro in human Metastatic Melanoma “501-Mel” cell line using various protocols. These essays were performed to evaluate if labelled Lmat maintains unaffected the capacity to infect, proliferate and kill tumor cells of unlabelled Lmat , which is used as positive control for cell infection. Moreover, thanks to the use of Immunofluorescence protocols, it was possible to evaluate the tumor cell labelling after infection by Lmat and eventually its potential both as tracer and drug carrier. As a first practical application, the last part of this work was focused on the possibility of Lmat functionalization as Photoacoustic tracer for primary and metastatic tumor. The early detection of tumor and metastatic sites is still one of the cardinal point in the fight against cancer, in particular for Melanoma because of its high propensity to metastasise and resistance to pharmacological treatments in its advanced stage. Photoacoustic Imaging represent a novel non-invasive diagnostic procedure complementary to the current Imaging technique of tumor sites , useful for their characterization and therapeutic monitoring. So the selected Probe has been functionalized with dye suitable for Photoacoustic Imaging Technique and Photoacoustic signal of labelled Lmat has been evaluated using appropriate Photoacoustic Laser system. Of course this thesis work represents just a first step for the realization of a promising theranostic tool against Melanoma, combining the selective antitumoral effect of Lmat and the possibility of its functionalization both as tumor tracer and as antitumoral drug carrier. Interesting future perspective could be brought by this thesis work. In fact, the study can be extended with the development of synthesis techniques for the conjugation of the selected Probe with drugs and the in vitro evaluation of the effective increase of the antitumor action of Lmat. Regarding the use of Lmat as a tumor tracer, once the marking protocol has been optimized, the study can be carried out on animal models for the evaluation of the photoacoustic signal and therefore of the actual diagnostic potential of Lmat

Translocator Protein 18-kDa: a promising target to treat neuroinflammation-related degenerative diseases

In the nervous system, inflammatory responses physiologically occur as defense mechanisms triggered by damaging events. If improperly regulated, neuroinflammation can contribute to the development of chronically activated states of glial cells, with the perpetuation of inflammation and neuronal damage, thus leading to neurological and neurodegenerative disorders. Interestingly, neuroinflammation is associated with the overexpression of the mitochondrial translocator protein (TSPO) in activated glia. Despite the precise role of TSPO in the immunomodulatory mechanisms during active disease states is still unclear, it has emerged as a promising target to promote neuroprotection. Indeed, TSPO ligands have been shown to exert beneficial effects in counteracting neuroinflammation and neuronal damage in several in vitro and in vivo models of neurodegenerative diseases. In particular, the regulation of neurosteroids' production, cytokine release, metabolism of radical oxidative species, and cellular bioenergetics appear to be the main cellular events that underlie the observed effects. The present review aims to illustrate and summarize recent findings on the potential effect of TSPO ligands against neuroinflammation and related neurodegenerative mechanisms, taking into consideration some pathologies of the nervous system in which inflammatory events are crucial for the onset and progression of the disease, and attempting to shed light on the immunomodulatory effects of TSPO

Translocator Protein Ligand PIGA1138 Reduces Disease Symptoms and Severity in Experimental Autoimmune Encephalomyelitis Model of Primary Progressive Multiple Sclerosis

Multiple sclerosis (MS) is an autoimmune and demyelinating disease of the central nervous system (CNS) caused by CNS infiltration of peripheral immune cells, immune-mediated attack of the myelin sheath, neuroinflammation, and/or axonal/neuronal dysfunctions. Some drugs are available to cope with relapsing-remitting MS (RRMS) but there is no therapy for the primary progressive MS (PPMS). Because growing evidence supports a regulatory role of the translocator protein (TSPO) in neuroinflammatory, demyelinating, and neurodegenerative processes, we investigated the therapeutic potential of phenylindolyilglyoxylamydes (PIGAs) TSPO ligands in myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) mice mimicking the human PPMS. MOG-EAE C57Bl/6-mice were treated by TSPO ligands PIGA839, PIGA1138, or the vehicle. Several methods were combined to evaluate PIGAs-TSPO ligand effects on MOG-EAE symptoms, CNS infiltration by immune cells, demyelination, and axonal damages. PIGA1138 (15 mg/kg) drastically reduced MOG-EAE mice clinical scores, ameliorated motor dysfunctions assessed with the Catwalk device, and counteracted MOG-EAE-induced demyelination by preserving Myelin basic protein (MBP) expression in the CNS. Furthermore, PIGA1138-treatment prevented EAE-evoked decreased neurofilament-200 expression in spinal and cerebellar axons. Moreover, PIGA1138 inhibited peripheral immune-CD45 + cell infiltration in the CNS, suggesting that it may control inflammatory mechanisms involved in PPMS. Concordantly, PIGA1138 enhanced anti-inflammatory interleukin-10 serum level in MOG-EAE mice. PIGA1138-treatment, which increased neurosteroid allopregnanolone production, ameliorated all pathological biomarkers, while PIGA839, unable to activate neurosteroidogenesis in vivo, exerted only moderate/partial effects in MOG-EAE mice. Altogether, our results suggest that PIGA1138-based treatment may represent an interesting possibility to be explored for the innovation of effective therapies against PPMS

De novo neurosteroidogenesis in human microglia: Involvement of the 18 kda translocator protein

Neuroactive steroids are potent modulators of microglial functions and are capable of counteracting their excessive reactivity. This action has mainly been ascribed to neuroactive steroids released from other sources, as microglia have been defined unable to produce neurosteroids de novo. Unexpectedly, immortalized murine microglia recently exhibited this de novo biosynthesis; herein, de novo neurosteroidogenesis was characterized in immortalized human microglia. The results demonstrated that C20 and HMC3 microglial cells constitutively express members of the neurosteroidogenesis multiprotein machinery—in particular, the transduceosome members StAR and TSPO, and the enzyme CYP11A1. Moreover, both cell lines produce pregnenolone and transcriptionally express the enzymes involved in neurosteroidogenesis. The high TSPO expression levels observed in microglia prompted us to assess its role in de novo neurosteroidogenesis. TSPO siRNA and TSPO synthetic ligand treatments were used to reduce and prompt TSPO function, respectively. The TSPO expression downregulation compromised the de novo neurosteroidogenesis and led to an increase in StAR expression, probably as a compensatory mechanism. The pharmacological TSPO stimulation the de novo neurosteroidogenesis improved in turn the neurosteroid‐mediated release of Brain‐Derived Neurotrophic Factor. In conclusion, these results demonstrated that de novo neurosteroidogenesis occurs in human microglia, unravelling a new mechanism potentially useful for future therapeutic purposes

Carbonic anhydrase activation profile of indole-based derivatives

Carbonic Anhydrase Activators (CAAs) could represent a novel approach for the treatment of Alzheimer's disease, ageing, and other conditions that require remedial achievement of spatial learning and memory therapy. Within a research project aimed at developing novel CAAs selective for certain isoforms, three series of indole-based derivatives were investigated. Enzyme activation assay on human CA I, II, VA, and VII isoforms revealed several effective micromolar activators, with promising selectivity profiles towards the brain-associated cytosolic isoform hCA VII. Molecular modelling studies suggested a theoretical model of the complex between hCA VII and the new activators and provide a possible explanation for their modulating as well as selectivity properties. Preliminary biological evaluations demonstrated that one of the most potent CAA 7 is not cytotoxic and is able to increase the release of the brain-derived neurotrophic factor (BDNF) from human microglial cells, highlighting its possible application in the treatment of CNS-related disorders

Antitumoral effects of attenuated Listeria monocytogenes in a genetically engineered mouse model of melanoma

Attenuated Listeria monocytogenes (Lmat-LLO) represents a valuable anticancer vaccine and drug delivery platform. Here we show that in vitro Lmat-LLO causes ROS production and, in turn, apoptotic killing of a wide variety of melanoma cells, irrespectively of their stage, mutational status, sensitivity to BRAF inhibitors or degree of stemness. We also show that, when administered in the therapeutic setting to Braf/Pten genetically engineered mice, Lmat-LLO causes a strong decrease in the size and volume of primary melanoma tumors, as well as a reduction of the metastatic burden. At the molecular level, we confirm that the anti-melanoma activity exerted in vivo by Lmat-LLO depends also on its ability to potentiate the immune response of the organism against the infected tumor. Our data pave the way to the preclinical testing of listeria-based immunotherapeutic strategies against metastatic melanoma, using a genetically engineered mouse rather than xenograft models