# Effects of Low Intensity Continuous Ultrasound (LICU) on Mouse Pancreatic Tumor Explants
## 3 Results and Discussion
### LICU Effects on Cytokine Secretion, Tumor Vasculature, and Collagen I Production  
The results of the cytokine array (Figure 2) showed that after LICU stimulation, IFN-\(\gamma\), was significantly upregulated by 3-fold compared to control, non-LICU-treated samples. In addition, IL1-\(\beta\) was significantly upregulated by 16-fold, while TNF-\(\alpha\) was upregulated 17-fold compared to control, non LICU-treated samples (Figure 2). In contrast, IL-10, IL-12, IL-2, IL-4, IL-5, and IL-6 did not significantly increase following LICU treatment. Cytokines are particularly important in the neoplastic initiation; they are aberrantly produced by tumor cells, macrophages, and other phagocytic cells. In pancreatic cancer, various signaling pathways are perturbed, and this not only affects the tumor cells directly but also influences the stromal cells within and around the tumor [32]. In particular, NF-kB signaling is commonly deregulated in PDAC [33]. A major activator of NF-kB is the cytokine tumor necrosis factor (TNF), which is mainly produced by activated immune cells, especially macrophages and T cells, but can also be expressed by tumor cells [34]. However, the role of TNF in pancreatic tumor progression still remains controversial. While some studies demonstrated anti-tumorigenic properties of TNF [35,36], others have shown the opposite results [37,38]. The 17-fold increase, in TNF-\(\alpha\) following LICU thus remains to be further explored. Figure 2: Enzyme linked immunosorbent assay (ELISA) results of the V-PLEX Proinflammatory panel 1. IFN-\(\gamma\) was significantly upregulated 3-fold; IL-\(\beta\) was significantly upregulated by 16-fold, while TNF-\(\alpha\) was upregulated 17-fold compared to control, non-low intensity continuous ultrasound (LICU)-treated samples. IL-10, IL-12, IL-2, IL-4, IL-5, and IL-6 did not significantly increase following LICU treatment. **: \(p<0.005\); *: \(p<0.05\). One of the key features of pancreatic cancer is extended fibrosis, which has been linked to the activation of pancreatic stellate cells (PSCs) [39]. The desmoplastic reaction not only accompanies the disease but plays an active role in its progression and reduces the efficiency of cytostatic drugs [40; 41]. Interferon-\(\gamma\) (IFN\(\gamma\)) has been shown to inhibit fibrogenesis by targeting PSCs [42], and thus it has been proposed as an active component for the treatment of pancreatic cancer as part of a chemoradiation protocol [43; 44]. Here, we report on a 3-fold increase in IFN\(\gamma\), suggesting that LICU localized application could be employed as a method to enhance the anti-fibrotic and anti-proliferative effect of IFN\(\gamma\). We also demonstrate a significant increase in IL-1\(\beta\) secretion. High plasma IL-1\(\beta\) levels are associated with a significantly increased risk of cancer, and tumor patients with high IL-1 expression have worse prognosis [45]. IL-1 is a key modulator for induction of innate immunity and inflammation and is a major pathogenic mediator of autoimmune, inflammatory, and infectious diseases [46]. IL-1\(\beta\) promotes invasiveness, including tumor angiogenesis, and also induces immune suppression in the host [38; 47]. In pancreatic cancer cells, IL-1\(\beta\) mediates adhesion and invasion, and modulates chemoresistance by activating the NF-\(\kappa\)B and ERK signaling pathways [41; 48]. Finally, in order to investigate the effects of LICU on the vasculature, as well as the collagen production of PANC-1 tumor explants, we exposed the tumor explants to 2 h of LICU. Tumor explants were subsequently fixed and stained for CD31 and Collagen I.