## 4 Discussion  
Literature describes the use of LIPUS in four major areas of cancer therapy: sonodynamic therapy, ultrasound-mediated chemotherapy, ultrasound-mediated gene delivery and anti-vascular ultrasound therapy. It is known that each technique has resulted in the death of cancer cells, with bio-effects attributed primarily to thermal actions and inertial cavitation. Nevertheless, protocols using ultrasounds and avoiding thermal draw backs are needed. Moreover, the application of these agents is important as it establishes a  
Figure 9: Results of Annexin Pi staining performed on PANC-1, Mia Paca 2 and BxPc3 cells. Viable cells are negative for both Annexin V and PI (Q3 quadrant gate in the dot plots); cells that are in the early phases of apoptosis are Annexin V positive and PI negative (Q4 quadrant gate in the dot plots); cells that are in the late phases of apoptosis are both Annexin V and PI positive (Q2 quadrant gate in the dot plots); and dead (nerotic) cells are Annexin V negative and PI positive (Q1 quadrant gate in the dot plots). therapeutic-diagnostic platform for anti-cancer therapy [15]. In this scenario, we have set up a protocol to test LICUS effects on a PDAC, or pancreatic cancer tumor model. The latter was chosen with the ultimate goal to address the need of therapeutic approaches to treat this oncological malignancy. It is known that pancreatic cancer has a very high mortality rate, with an incidence that is increasing in economically developed countries, such as the USA, due to lifestyle habits [16]. We have found that the executed treatments are capable of avoiding thermal damage to the cells and that, according to literature, cell mortality can be ascribed only to LICUS effect. Cell mortality was assessed using the BioFilmAnalyzer software, analyzing the spheroid images acquired with the confocal fluorescence microscope. We have found that the frequency capable of inducing cell death through LICUS treatment is 3 MHz. The analysis of cell mortality allows for the definition that the 3 MHz stimulation frequency is the most effective in inducing cancer cell death. With this set up, further frequencies and intensities will be investigated to optimize treatment times. Moreover, data obtained with the Annexin PI staining suggest that the 3 MHz treatment frequency is capable of inducing cell death, which can be conceivably interpreted as a necrotosis process. This is evident from the plots in all three pancreatic cancer cell lines, where a strong increase in cells in the Q3 quadrant is evident. Such findings were also confirmed from the analysis of the brightfield images of spheroids, showing an enlarged shape of 3 MHz treated cells, which is a sign suggesting the cell transition towards necrotosis. The high percentage of dead cells emerging from the Annexin PI analysis is justified because the staining allows for the quantification of dead cells in the external surface of the spheroids, while for apoptosis analysis, spheroids are disrupted, being able to also quantify dead cells in the central core of the spheroids. Such consideration holds true both for treated and control cells. Moreover, in the brightfield image cells in spheroids treated with 3 MHz manifested a migratory behavior, which is consistent with the effect on proliferation and migration reported in literature and we truly believe it is directly due to the US treatment [9]. We believe we have obtained preliminary but significant results in LICUS treatment of pancreatic tumor spheroids with a single US treatment. The setup and therapeutic protocol represent an innovative strategy for cancer treatment per se and we are confident that, after a proper validation, this can be useful to achieve at least 40% reduction in grafted tumor mass, comparable to that obtained with immunotherapeutic approaches tested in vivo with the administration of nanoparticles [17].