# Effects of Low Intensity Continuous Ultrasound (LICU) on Mouse Pancreatic Tumor Explants
## 1 Introduction  
Low-Intensity Pulsed Ultrasound (LIPUS) is widely used as an imaging tool in medicine at low intensities (<3 W/cm\({}^{2}\)). It is a non-invasive and safe technique used extensively as a diagnostic and therapeutic tool [1; 2; 3; 4; 5]. In regular clinical applications, the intensity of the ultrasound applied ranges from about 0.03-1.0 W/cm\({}^{2}\). LIPUS based on contrast-agents has been also applied for imaging [6; 7], with high efficiency results at frequencies ranging from 500 kHz and 2 MHz. Different intensities of exposure have been used in the literature for therapeutic purposes such as healing of bone-fracture or soft-tissue lesions, with dosages up to 2 W/cm\({}^{2}\) without tissue damage, and frequencies between 0.7 and 3.0 MHz. Various studies have reported LIPUS-induced cell growth with proliferation and promotion of multi-lineage differentiation with cell expansion and differentiation in tissue culture, including gingival cells [8; 9], periodontal cells [10; 11; 12], cementoblastic cells [13; 14], chondrocytes [15; 16] or mesenchymal stem cells [17; 18]. Also, LIPUS effects on osteoblasts and enhancement of angiogenesis [19; 20] were reported. Some studies showed that LIPUS enhanced cell expansion and differentiation in tissue culture [10; 21; 22]. However, the underlying molecular mechanisms governing these LIPUS-induced effects on tissues and cells [4; 5] still need to be investigated. In addition, LIPUS effects on tumor samples have not been extensively investigated. All these studies describe the use of conventional pulsed wave generators, typically used for diagnosis methods, to know possible therapeutic effects in cells and tissues. They assume the advantages of non-heating effects on the tissues due to these ultrasonic actuators and the time-gaps between consecutive wave trains. However, cellular responses to low-intensity US are parameter-dependent, especially in the case of low-intensity pulsed US. Very few studies have reported the influence of LIPUS treatment on the effects induced in bio-samples, including the intensity of the acoustic wave and the pulse repetition frequency [23; 24; 25] at frequencies close to 1 MHz. In particular, [PERSON] et al. [25] recently investigated (2016) the systematic effects of low-intensity pulsed US on the proliferation of HepG2 and 3T3 cells in vitro by changing the intensity in the range of 0-1.2 W/cm\({}^{2}\), PRF (1 and 100 Hz), and the duty cycle (10%, 20%, and 50%) with a 1.06 MHz generator. They found increased cell proliferation at intensities between 0.4 W/cm\({}^{2}\) and 0.8 W/cm\({}^{2}\) and PRF \(\sim\) 100 Hz, but higher intensities generated cell death, while lower pulse repetition frequencies did not induce any detectable effect on the cells. This recent study evidenced the need to know how the duty cycle affects the sample response. It is a challenge to know the ultrasound effects by elongating the pulse repetition frequency until reaching a limit condition of duty cycle of 100%, at which the distance between consecutive wave trains disappears and the pulsed wave becomes a continuous wave. This challenge led us to perform the current study. In this paper, we have replaced pulsed waves with continuous low intensity waves at similar frequency and intensity amplitudes. Hereafter, they will be referred to as Low-Intensity Continuous Ultrasound (LICU). LICU maintains the cell vibration throughout the whole acoustic treatment, instead of the intermittent oscillations induced by LIPUS with temporal gaps relaxing the cell oscillatory motion. Thus, the limit condition of an infinite wave-train length was assumed for the continuous wave used in our experiments. Inflammation is critical for tumor progression. Many cancers arise in sites of infection, chronic irritation, and inflammation. In addition, tumor cells have co-opted some of the signaling molecules of the innate immune system, such as selectins, chemokines, and their receptors for invasion, migration, and metastasis [26].