Theranostic Nanoengineered Lymphocyte-Based System for Melanoma Treatment

Melanoma is a type of cancer that develops from melanocytes and typically occurs in the skin but may occur also in the mouth, intestine, or eye. Melanoma is recognized to be the most dangerous type of skin cancer. Globally, in 2012, it newly occurred in 232,000 people while in 2015 within 3.1 million people with active disease, 59,800 died. Apart from the surgical removal of the melanoma tissue, several pharmaceutical treatments are available: some of these include Vemurafenib, Dabrafenib, Trametinib, Ipilimumab, Pembrolizumab, Nivolumab, alone or in combination. New experimental approaches include the use of nanoparticles capable of carrying drugs or causing hyperthermia in the cancer area, however the side effects caused by toxicity of nanoparticles and their accumulation in non-specific tissues and organs has limited further development of this treatment. Another interesting approach is represented by the autologous transplant of anti-melanoma specific immune cells, bringing to a reduction of the melanoma with good tolerability property. Within the INSIDE (Development of diagNostic and theranoStic targeting systems, based on nano-systems and/or nanoengineered lymphocytes for the early Diagnosis and treatment of mElanoma and multiple sclerosis) consortium the idea to combine the use of nanoparticles and immune approaches was thought to be a good chance to develop a new approach to melanoma treatment. Poly-lactic-co-glycolic acid polyethylene glycol (PLGA-PEG) -magnetite and PLAGA-PEG-gold nanorod nanoparticles were included inside specific T lymphocytes from patients, proliferated and activated in vitro against the MAGE antigens, expressed by melanoma (A375 melanoma cell line). The nanoparticles containing lymphocytes could reach specifically the tumor site and eventual metastasis, where the lymphocytes can play their cytotoxic role, while the nanoparticles can be used as a tracing tool, or be radio-induced to produce a hyper-thermic effect on the specific area or even to deliver conjugated drugs. The first step of the investigation of this new approach was represented by checking the biodistribution of nanoparticles containing lymphocytes. The biodistribution was evaluated in a newly set-up in vivo mouse model of human melanoma: nude mice were inoculated with A375 melanoma cell line and, after 14 days, 8.0x106 specifically nanoengineered T-lymphocytes were administered to the animals. The dose of lymphocyte was calculated after in vitro experiments as the number of engineered lymphocytes needed to increase the temperature up to 43-44°C (in 20 minutes when subjected to radio- hyperthermia). After 24 and 48 hours from lymphocyte inoculum, the animals were sacrificed and the tumor and other organs were removed to check the lymphocytes infiltration using flow cytometry. The preliminary results confirmed that nanoparticles loaded lymphocytes specifically accumulated into the tumor site with a higher percentage respect to other organs. Additional experiments to confirm the possibility to use the approach also as an imaging platform (by Nuclear Magnetic Resonance Imaging (MRI) and Photo Acoustic imaging (PA) were performed with the support of National Research Council (CNR) of Pisa including 5.0x107 lymphocytes as improvement: these preliminary results supported the possibility to use the new platform as MRI and PA imaging system. Moreover, experiments to demonstrate the efficacy of the system in reducing the tumor size after radio-hyperthermia were executed on the generated mouse model. In two of the experimental groups (N=8), magnetite nanoparticles were conjugate to curcumin, which has been demonstrated to be effective in inducing melanoma cells apoptosis in vitro. Preliminary findings on groups of 8 animals demonstrated the efficacy of the curcumin-conjugated magnetite nanoparticles, either subjected to radio-hyperthermia or not, to reduce the tumor growth respect to the non-loaded lymphocyte group, in a 21-day experimental window. This new approach could represent a useful instrument to diagnose and treat melanoma patients in short times, as well as discover recurrencies, saving lives and avoiding side effects of current therapies.