Purpose: Due to the many similarities with its human counterpart, canine malignant melanoma (cMM) is a valuable model in which to assess the efficacy of novel therapeutic strategies. The model is herein used to evaluate the immunogenicity, safety, and therapeutic efficacy of a human chondroitin sulfate proteoglycan-4 (hCSPG4) DNA-based vaccine. The fact that homology between hCSPG4 and cCSPG4 amino-acidic sequences stands at more than 80% provides the rationale for using an hCSPG4 DNA vaccine in the cMM model.Experimental Design: Dogs with stage II-III surgically resected CSPG4-positive oral MM were subjected to monthly intramuscular plasmid administration, which was followed immediately by electroporation (electrovaccination) for at least 6, and up to 20, months. The immunogenicity, safety, and therapeutic efficacy of the vaccine have been evaluated.Results: hCSPG4 electrovaccination caused no clinically relevant local or systemic side effects and resulted in significantly longer overall and disease-free survival times in 14 vaccinated dogs as compared with 13 nonvaccinated controls. All vaccinated dogs developed antibodies against both hCSPG4 and cCSPG4. Seven vaccinated dogs were also tested for a cCSPG4-specific T-cell response and only two gave a detectable interferon (IFN)g response.Conclusion: Xenogeneic electrovaccination against CSPG4 is able to overcome host unresponsiveness to the "self" antigen and seems to be effective in treating cMM, laying the foundation for its translation to a human clinical setting.

Thanks to striking progress in both the understanding of anti-tumor immune response and the characterization of several tumor associated antigens (TAA), a more rational design and more sophisticated strategies for anti-tumor vaccination have been possible. However, the effectiveness of cancer vaccines in clinical trial is still partial, indicating that additional studies are needed to optimize their design and their pre-clinical testing. Indeed, anti-tumor vaccination success relies on the choice of the best TAA to be targeted and on the translational power of the pre-clinical model used to assess its efficacy. The chondroitin sulfate proteoglycan-4 (CSPG4) is a cell surface proteoglycan overexpressed in a huge range of human and canine neoplastic lesions by tumor cells, tumor microenvironment and cancer initiating cells. CSPG4 plays a central role in the oncogenic pathways required for malignant progression and metastatization. Thanks to these features and to its poor expression in adult healthy tissues, CSPG4 represents an ideal oncoantigen and thus an attractive target for anti-tumor immunotherapy. In this review we explore the potential of CSPG4 immune-targeting. Moreover, since it has been clearly demonstrated that spontaneous canine tumors mimic the progression of human malignancies better than any other pre-clinical model available so far, we reported also our results indicating that CSPG4 DNA vaccination is safe and effective in significantly increasing the survival of canine melanoma patients. Therefore, anti-CSPG4 vaccination strategy could have a substantial impact for the treatment of the wider population of spontaneous CSPG4-positive tumor affected dogs with a priceless translational value and a revolutionary implication for human oncological patients.

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The role of non-coding RNAs in different biological processes and diseases is continuously expanding. Next-generation sequencing together with the parallel improvement of bioinformatics analyses allows the accurate detection and quantification of an increasing number of RNA species. With the aim of exploring new potential biomarkers for disease classification, a clear overview of the expression levels of common/unique small RNA species among different biospecimens is necessary. However, except for miRNAs in plasma, there are no substantial indications about the pattern of expression of various small RNAs in multiple specimens among healthy humans.By analysing small RNA-sequencing data from 243 samples, we have identified and compared the most abundantly and uniformly expressed miRNAs and non-miRNA species of comparable size with the library preparation in four different specimens (plasma exosomes, stool, urine, and cervical scrapes).Eleven miRNAs were commonly detected among all different specimens while 231 miRNAs were globally unique across them. Classification analysis using these miRNAs provided an accuracy of 99.6% to recognize the sample types. piRNAs and tRNAs were the most represented non-miRNA small RNAs detected in all specimen types that were analysed, particularly in urine samples. With the present data, the most uniformly expressed small RNAs in each sample type were also identified. A signature of small RNAs for each specimen could represent a reference gene set in validation studies by RT-qPCR.Overall, the data reported hereby provide an insight of the constitution of the human miRNome and of other small non-coding RNAs in various specimens of healthy individuals.

In an attempt to reveal deregulated miRNAs associated with the progression of carcinomas developed in BALB-neuT transgenic mice, we found increased expression of miR-135b during malignancy. Relevantly, we observed that miR-135b is up-regulated in basal or normal-like human breast cancers, and it correlates with patient survival and early metastatization. Therefore, we investigated its biological functions by modulating its expression (up- or down-regulation) in mammary tumor cells. Although no effect was observed on proliferation in cell culture and in orthotopically injected mice, miR-135b was able to control cancer cell stemness in a mammosphere assay, anchorage-independent growth in vitro, and lung cancer cell dissemination in mice after tail vein injections. Focusing on the miR-135b molecular mechanism, we observed that miR-135b controls malignancy via its direct targets, midline 1 (MID1) and mitochondrial carrier homolog 2 (MTCH2), as proved by biochemical and functional rescuing/phenocopying experiments. Consistently, an anti-correlation between miR-135b and MID1 or MTCH2 was found in human primary tumor samples. In conclusion, our research led us to the identification of miR-135b and its targets, MID1 and MTCH2, as relevant coordinators of mammary gland tumor progression.