IDENTIFICATION OF TUMOR-ASSOCIATED MOLECULES AS SUITABLE BIOMARKERS OF CANCER IN DOGS USING OMIC APPROACHES

Omics techniques have been widely applied to veterinary science, although mostly on farm animal productions and infectious diseases. In canine oncology, on the contrary, the use of omics methodologies is still far behind. This thesis aims to fill in the gap in the molecular background of canine cancer tumors and to apply this new knowledge for the identification of suitable biomarkers mainly based on the identification and quantification of microRNAs. The thesis applied next generation sequencing (NGS) to investigate onco-miRNome and skin microbiota of one of the most relevant, highly metastatic canine malignant tumor, the mast cell tumor. The results of miRNomic have been validated by RT-qPCR to develop molecular tools to enhance the robustness of clinical decision-making. To elucidate the host-tumor interaction, the skin and dermis microbiota of MCT-affected dogs has been investigated as well. This thesis aims also to characterized the proteomic profiles of exosomes purified from plasma of MCT-affected dogs. Due to the pandemic situation, the protocol for exosome purification has been developed, while the proteomic analysis is still ongoing. Finally, a very preliminary evaluation of miRNA dysregulation in canine oral and cutaneous melanoma was carried out. To characterize the epigenetic modulation of MCT microenvironment and to identify a panel of miRNAs dysregulated during the neoplastic process and suitable as biomarkers, the miRNome of canine MCT was profiled on FFPE samples using an NGS approach; the result was then investigated also in saliva samples of MCT-affected dogs. The NGS findings revealed that 63 miRNAs were differentially expressed between MCT and healthy adjacent margins (45 down-regulated and 18 up-regulated). A panel of nine miRNAs was validated by qPCR, of which five were dysregulated in the tumor. Specifically, miR-21 and miR-379 were up-regulated while miR-885, miR-338 and miR-92a were down-regulated in the MCT compare to the healthy margins. A further analysis including the variable of the lymph node involvement in the tumor, revealing that a panel of three miRNAs, miR-21, miR-379, and miR-885, was able to discriminate metastatic from non-metastatic MCTs. The diagnostic potential of the panel was also explored in the saliva of MCT-affected dogs, showing that miR-21 and miR-885 discriminated tumor-affected from healthy dogs; miR-885 could also discriminate the metastatic from the non-metastatic MCTs with good diagnostic potential, make this miRNA attractive for a future diagnostic purpose starting form a less-invasive matrix. The microbiota of the MCT associated with the skin surface and dermis was characterized for the first time investigating alpha diversity which describes the differences of taxa by qualitative and quantitative approaches and beta diversity which describes the differences between groups generating distance matrices between samples. The data showed that a reduction of taxa was present in the tumor skin surface compared with the healthy contralateral part with an increase of Firmicutes phylum and Corynebacteriaceae family. A similar reduction of taxa found out in the tumor dermis compared with the overlying tumor skin surface. These differences exerted an impact also in beta diversity, which highlighted the differences between the samples. The characterization of the core microbiota of the tumor and healthy skin surface and the tumor dermis showed that the healthy skin was characterized by a higher number of ASVs (27 ASVs) compared to the tumor skin surface (12 ASVs) and dermis (16 ASVs) and that 10 ASVs were present only in the tumor site. The data proved that the presence of the tumor interfered in some way with the microbial population, paving the way for further investigations on host-microbiota interaction. Moreover, the reduction of microbiota diversity and taxa was seen to be associated with an unhealthy status. A protocol for the purification of plasmatic exosome from dog affected by MCT was developed to obtain samples with a good degree of purity and suitable for proteomic analysis, that may elucidate the cell-to-cell communication strategy used by MCT to prepare the metastatic niches, and may promote the identification of suitable biomarkers for the MCTs diagnosis and prognosis. The Size Exclusion Chromatography purification approach was identified as the best approach to obtain samples depleted from the most abundant plasmatic protein, including albumin, and exosomes in the right size range and with concertation feasible for further proteomic analysis. Finally, the potential of a target miRNAs panel was also evaluated in canine cutaneous and oral malignant melanoma. The data showed that, among six miRNAs selected from the bibliography, miR-145, miR-365, miR-146a, and miR-425 were differentially expressed in canine melanoma. In detail, miR-145 was down-regulated in cutaneous and oral melanoma and miR-365 was down-regulated in cutaneous melanoma. On the other hand, miR- 146a and miR-425 were up-regulated in cutaneous melanoma. The gene ontology analysis suggested that these miRNAs may have a role in tumor progression and cell proliferation.