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Liquid biopsies, as blood and urine, could offer an invaluable, easily accessible source of biomarkers, and evidences for elucidating the pathological processes. Only few studies integrated the proteomes driven by more than one biofluid. Furthermore, it is not clear which biofluid better mirrors the alterations triggered by disease. Venous infiltrating RCC(Renal Cell Carcinoma) could represent an advantageous model for exploring this aspect. Herein, we investigate how blood and urine “proteomically” reflect the changes occurring during RCC infiltration into renal vein(RV) by label-free nLC-ESI-MS/MS. We found 574 and 58 differentially expressed proteins(DEPs) in response to vascular involvement. To the augment of vascular involvement, the abundance of only three proteins in urine(UROM,RALA,CNDP1) and two in plasma(APOA1,K2C1) diminished while increased for twenty-six urinary proteins. 80 proteins were found both in urine and plasma, among which twenty-eight were DEPs. A huge overlap between the two biofluids was highlighted, as expected, being urine the filtrate of blood. However, this consistency decreases when RV-occlusion occurs suggesting alternative protein releases, and a loss of kidney architecture. Moreover, several proteomic and functional signatures were biofluid-specific. In conclusion, the complementarity between the specimens allowed to achieve a deeper level of molecular complexity of the RCC venous infiltration. Significance: Although plasma and urine are strongly interconnected, only few proteomic studies investigated the complementarity of these fluids as bio-sources of information. Moreover, none of them was focused to their analysis and comparison in the context of vascular infiltration of renal cancer. Herein, new insights were gained regarding the impact into urinary and plasma proteome of the changes triggered by the ccRCC invasion into vascular system and renal vein. Furthermore, the integration of the information driven by the two liquid biopsies permits to unravel biological processes otherwise lost.

Renal Cell Carcinoma (RCC) is the most frequent form of kidney cancer and approximately 80% of cases are defined as clear cell RCC (ccRCC). Among the histopathological factors, tumour grade represents one of the most important parameters to evaluate ccRCC progression. Nonetheless, the molecular processes associated with the grading classification haven't been deeply investigated thus far. Therefore, the aim of this study was to uncover protein alterations associated with different ccRCC grade lesions. Formalin-fixed paraffin-embedded samples from ccRCC patients were analysed by histology-guided MALDI-MSI and shotgun proteomics in order to study the biological processes implicated in ccRCC. MALDI-MSI data highlighted signals able to discriminate among different grades (AUC > 0.8). The ion at m/z 1428.92 was identified as Vimentin and was overexpressed in grade 4 lesions, whereas ions at m/z 944.71, m/z 1032.78 and m/z 1325,99 were identified as histones H2A, H3, and H4, respectively. nLC-ESI-MS/MS analysis provided a further list of proteins and their abundances, showing a difference in protein content among the four grades. Moreover, the obtained molecular profiles showed a correspondence with the different Cancer-Specific Survival rate at 10 years post-surgery, as reported in literature. Significance Despite the generally accepted role of tumour grade in ccRCC diagnosis, the proteomic processes associated with the different tumour grades has not been extensively studied and doing so may provide insights into the development of the disease. In the current study, data obtained using MALDI-MSI was integrated with that obtained using nLC-ESI-MS/MS to highlight the proteomic alterations underlying the different ccRCC grades. The combined approach identified vimentin and three histones (H2A, H3 and H4) that were able to discriminate among the four grades whilst the nLC-ESI-MS/MS analysis alone provided a further list of proteins with an altered abundance. Furthermore, there was a good correlation between the molecular profiles generated for each grade and the different Cancer-Specific Survival rate at 10 years post-surgery. Such findings could be a valuable starting point for further studies aimed at clarifying the molecular events that occur during the development of ccRCC.