## 3 Results  
The thermal trends recorded during the stimulation procedure confirm a low intensity treatment; in fact, cell death is not caused by thermal effects, but only by the mechanical effects of ultrasound. The temperatures were acquired in water, in correspondence with the probe and inside the target well. Tables 1-3 show the average values calculated from the four stimulations conducted for each frequency. Ten sessions of experimental testing took place. For each treatment, three 96-well plates were used, one for each stimulation frequency, including four containing PANC-1 cell cultures to be treated, and four containing control PANC-1 cell cultures. \begin{table}
\begin{tabular}{c c c c} \hline \hline  & \multicolumn{3}{c}{**Spheroids at 1 MHz**} \\ \hline
**Time (min)** & **Twater (°C)** & **Tpiezo (°C)** & **Ttarget Well (°C)** \\ \hline
0 & 18.9 & 19.1 & 19.0 \\
5 & 19.0 & 19.4 & 19.2 \\
10 & 19.1 & 19.5 & 19.2 \\
15 & 19.1 & 19.5 & 19.2 \\
20 & 19.0 & 19.5 & 19.2 \\ \hline \hline \end{tabular}
\end{table} 
Table 1: Average temperature values calculated at 1 MHz. \begin{table}
\begin{tabular}{c c c c} \hline \hline  & \multicolumn{3}{c}{**Spheroids at 2 MHz**} \\ \hline
**Time (min)** & **Twater (°C)** & **Tpiezo (°C)** & **Ttarget Well (°C)** \\ \hline
0 & 19.5 & 19.8 & 19.7 \\
5 & 19.6 & 21.1 & 19.8 \\
10 & 19.6 & 21.1 & 19.8 \\
15 & 9.6 & 21.0 & 19.8 \\
20 & 19.7 & 21.1 & 19.8 \\ \hline \hline \end{tabular}
\end{table} 
Table 2: Average temperature values calculated at 2 MHz. \begin{table}
\begin{tabular}{c c c c} \hline \hline  & \multicolumn{3}{c}{**Spheroids at 3 MHz**} \\ \hline
**Time (min)** & **Twater (°C)** & **Tpiezo (°C)** & **Ttarget Well (°C)** \\ \hline
0 & 20.4 & 20.8 & 20.6 \\
5 & 20.5 & 22.3 & 20.8 \\
10 & 20.4 & 22.2 & 20.8 \\
15 & 20.4 & 22.4 & 20.8 \\
20 & 20.5 & 22.4 & 20.8 \\ \hline \hline \end{tabular}
\end{table} 
Table 3: Average temperature values calculated at 3 MHz. Cells treated at different frequencies were stained with Calcein AM/PI and images were acquired using Confocal microscopy at a magnification of 10\(\times\) (image size for all pictures: 900 x 900 \(\upmu\)m). The analysis provided cell death index as a ratio between red cells (dead cells) and green cells (live cells), as shown in Figure 7. The percentage of dead cells was shown at three different frequencies for the treated spheroids. On the right side of each panel, the mortality rate of untreated spheroids due to the possible side effects induced at various frequencies at a distance of 25 mm from the stimulation beam was evaluated. Figure 7: Images acquired with Confocal fluorescence microscope SP8 confocal microscope (Leica), showing PDAC cell spheroids. (**A**) PANC-1 spheroids, control upper panel and treated lower panel with 1, 2 and 3 MHz. In each well one spheroid was seeded, and images were acquired at 10\(\times\) magnification. Control cells appear stained in green, which represents viable cells (see white arrows); while treated cells show a marked spotted red staining (see white arrow), which represent dead cells. Quantification of viable and dead cells using the BioFilmAnalyzer software is also reported. (**B**) Mia Paca-2 spheroids, control cells upper panel and treated cells lower panel, with 1, 2 and 3 MHz. In each well one spheroid was seeded, and images were acquired at 10\(\times\) magnification. Control cells appear with a predominant green staining (see white arrow), indicating viable cells; while treated spheroids appear smaller and with a prevalence of red staining dots, indicating cell death (see white arrows). Mia Paca-2 spheroids, treated with 3 MHz frequency, also appear to disaggregate. Quantification of viable and dead cells using Spheroid Sizer (Mathlab) is also reported. (**C**) BxPc3 spheroids, control upper panel and treated lower panel with 1, 2 and 3 MHz. In each well one spheroid was seeded, and  
images were acquired at 10\(\times\) magnification.