Utilizzo degli ultrasuoni su modelli tumorali di epatocarcinoma e glioblastoma: approcci innovativi per l’identificazione di biomarcatori diagnostici

Background. Hepatocellular carcinoma (HCC) and glioblastoma (GBM) are highly aggressive tumors frequently diagnosed at late stages and associated with poor prognosis. Novel diagnostic biomarkers and strategies to enhance therapeutic delivery are urgently needed. Ultrasounds (US) and focused ultrasounds (FUS) emerged as promising tools to modulate cell membrane permeability, enabling biomarker release and improving drug uptake. Methods and Materials. HCC cell lines (HepG2 and SNU-387) were treated with US to assess the release of microRNAs and proteins into the culture supernatant. A normal liver cell line, THLE-2, was used as negative control. After fine-tuning US parameters, microRNA profiling was performed by qRT-PCR using TaqMan-based microfluidic cards. Public datasets were used for validation and ROC and AUC were considered for analyses. Protein analysis was evaluated in cell supernatants by antibody array. To evaluate the ability of US to enhance small-molecule uptake, FAM-labeled AS1411 aptamer and sorafenib, both in combination with US, were tested on HCC cell lines. Uptake enhancement was assessed by measuring FAM fluorescence, while treatment efficacy was evaluated through cell viability analysis. Cell viability was assessed after 24 hours of incubation. Pharmacokinetic analyses were performed in an orthotopic human GBM xenograft mouse model. A pilot study was conducted to determine optimal FUS parameters. FUS treatment was applied to transiently open the blood–brain barrier and facilitate delivery of the radiolabeled antibody xiRA63 targeting Endothelin A receptor. Biodistribution was quantified by PET imaging at different timepoints post-injection. Results. US stimulation induced significant release of four microRNAs compared to untreated samples: miR-145-5p and miR-29a-3p, miR-532-5p, miR-106b-3p. These microRNAs were also significantly upregulated in patient sera across public datasets, with miR-532-5p showing the highest diagnostic performance. The control cell line THLE-2 showed no significant microRNA release after US treatment. Moreover, US promoted the release of nine protein factors increased after treatment. Among these proteins, FasL and Livin were significantly increased. US enhanced sorafenib uptake and drug efficacy in HepG2 cells, significantly reducing cell viability. In the GBM mouse model, a mild non-significant increase in xiRA63 distribution volume within glioma tissue after FUS was observed, while irreversible uptake parameters and plasma AUC remained unchanged. Conclusion. These findings demonstrate that US and FUS are promising tools for identifying novel biomarkers and enhancing therapeutic efficacy. US enabled selective release of microRNAs and proteins, identifying candidate biomarkers miR-532-5p, miR-29a-3p, miR-145-5p, miR-106b-3p, FasL and Livin. Moreover, US improved the in vitro response to sorafenib, suggesting its potential to enhance drug uptake. In the GBM model, FUS induced a transient trend toward increased distribution volume without statistical significance, with no differences in irreversible uptake. Overall, US promotes biomarker release and drug uptake in vitro, while further research is needed to optimize FUS for blood–brain barrier opening and improve xiRA63 distribution.