# Morphological Analysis of US Treated PANC-1 Spheroids
## 7 Discussion  
The results indicate a strong correlation between LICU treatment at specific frequencies and the observed changes in spheroid behavior, as demonstrated in Figure 6.  
At a frequency of 2 MHz, a substantial reduction in spheroid volume was recorded. This effect was accompanied by a smaller peak in mortality rates, as depicted in Figure 5, alongside a noticeable decrease in the disgregation index (Figure 7). Such findings suggest that LICU at 2 MHz induces measurable alterations in three-dimensional spheroid cultures. These observations hint at the potential for off-target effects at 2 MHz, which may influence spheroid architecture without completely compromising cell viability. Figure 6: The volume mean ratio obtained by making the evaluation of treated Volume on Control volume. The standard deviation is also displayed for each histogram column. Figure 7: The disgregation index is obtained by making the evaluation of the mean treated perimeter on area on the mean control perimeter on area. The standard deviation is also displayed for each histogram column. In contrast, the effects observed at 3.5 MHz were particularly striking. This frequency was associated with a marked increase in mortality rates, as illustrated in Figure 5, coupled with a marked reduction in the volume of the spheroid (Figure 6). Furthermore, there is a significant decrease in the disgregation index (Figure 7) at 3.5 MHz underscores. Unlike the effects at 2 MHz, treatment at 3.5 MHz appeared to cause higher mortality rates, still preventing disaggregation. This distinction is crucial, as excessive disgregation could facilitate metastasis, a concern supported by the literature that highlights the mechanical and structural consequences of spheroid disaggregation [48; 49; 50; 51]. Thus, LICU at 3.5 MHz demonstrates a dual capability: it induces significant cellular mortality while promoting spheroid destabilization, with potential implications for therapeutic strategies targeting tumor cohesion and survival. These findings highlight the nuanced interplay between frequency-specific LICU effects on spheroids. Although 2 MHz elicits subtle structural changes with limited mortality, 3.5 MHz emerges as a more potent frequency to cause cell death, potentially offering a targeted approach to therapeutic interventions. Future investigations should focus on further elucidating the mechanistic underpinnings of these frequency-dependent effects, with an emphasis on optimizing therapeutic windows to balance efficacy and safety. The following section provides a concise yet comprehensive statistical analysis of the data presented in the figures. This analysis aims to assess the significance of the observed differences and to identify the correlations. Regarding the mortality data it is noted that the sample size exceeds 250 and the \(p\) value is significantly less than 0.05. Regarding the statistical analysis of the indices, the standard deviation of the volume index is consistently below 0.2, indicating relatively low variability. The statistical correlation values derived from the volume index suggest a significant relationship with mortality. Specifically, the volume index shows a correlation coefficient of \(-0.496\), indicating a correlation. The corresponding \(t\)-score is \(-3.023\) and the \(p\)-value for this result is 0.0053, which is well below the commonly used significance threshold of 0.05. This confirms that the correlation is statistically significant and suggests that changes in volume index may be related to mortality. For the disgregation index, the standard deviation is smaller, remaining below 0.09, suggesting even less variability in these measurements. This index shows a stronger negative correlation with mortality, with a correlation coefficient of \(-0.800\). The \(t\)-score is \(-7.052\). The corresponding \(p\)-value is \(1.14\times 10^{-7}\), which is extremely small, indicating an exceptionally strong and statistically significant relationship.