• metastatic melanoma
• immunotherapy
• Listeria Monocytogenes
• metastasis tracer
• theranostic agent
• drug carrier

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.