Female nu nu nude mice

Female nude mice were obtained from Jackson Laboratory. Experimental protocols were approved by the Institutional Animal Care and Use Committee at Wake Forest health Science. Female NU/NU Nude Mice from Charles River Laboratories were used for animal experiments. Mice were housed in a 12h light/12 h dark cycle with temperature-controlled room. The animal rooms are provided with 100% fresh, HEPA filtered air at 10-15 air changes per hour. Room temperatures are controlled by reheat units within each room, and are maintained within the range of 70°F ± 2° F. The humidity levels are controlled globally, and it is maintained between 30-70%. The mice were fed with a standard chow (Prolab Isopro RMH 3000, 5P00, LabDiet) and water ad libitum. Approximately 1 million cells were subcutaneously injected into the mammary fat pad of 7- to 9-week-old mice. Tumor length (L) and width (W) were measured by caliper, and tumor size was calculated using the formula, 1/2* LW^{2 (link)}. The maximum tumor size is 2cm of the length and maximal tumour size/burden was not exceeded in this study. Tumor growth were also monitored by bioluminescence imaging using the IVIS lumina III in vivo imaging system (Perkin Elmer). Living Image (Caliper Life Science) version 4.7.3 was used to analyze the bioluminescence level. Tumor wet weight were measured at the endpoint.

Female nu/nu nude mice were obtained from Charles River Laboratories (Montreal, QC, Canada). All studies involving mice were approved by the institutional Animal Care Committee (Protocol: # 237-10). Human HCT116 xenografts were established in 4-week-old nude mice. Mice were subcutaneously inoculated with 0.2 mL of 1 × 10⁶ HCT116 cells in McCoy’s 5A on the right flank. After the formation of 100 mm³ tumors, mice were randomly assigned into 4 groups, control (untreated), MAG-EPA-treated (n = 8 per group), Krill-Oil-treated, and EPA-EE (ethyl ester)-treated. MAG-EPA was administered per os (618 mg/kg) daily. Krill oil and EPA-EE were administered per os at a human equivalent of 3.0 g/day of total omega-3. Tumor volumes (V) were calculated using the formula: V = (a × b²)/2, where “a” is the largest superficial diameter and “b” the smallest. Cleaved caspase-3 ELISA assay was performed on tumor homogenate derived from the control and treated mice according to the manufacturer’s instructions (New England BioLabs, Pickering, ON, Canada). Western blot analyses using specific antibodies against phosphorylated forms of VEGFR (Y1059 and Y951), EGFR, and AKT (at serine and threonine residues) as well as total form of VEGFR, EGFR, AKT, β-actin, VEGF and HIF1α were performed on tumor homogenates derived from control and MAG-EPA-treated mice [8 (link),15 (link)].

Female NU/NU nude mice (Charles River Laboratories, Wilmington, MA, USA) were used as an in vivo model. Images were acquired with the Li-Cor Pearl Impulse imaging system using the same settings as in the case of phantoms. DiR-loaded nanodroplets were injected subcutaneously in both hind sides of each mouse. Mice were sedated with 2.5% vol isofluorane (Fluoriso, VetOne, Boise, ID, USA) in O₂ with a flow rate of 1.5 L/min for the duration of the procedure. Images were acquired prior to injection, immediately after injection and after pulsed laser activation of the droplets. One of the injection sites was activated, while the other one was used as an internal control to compare the changes in dye intensity over time. The fluorescence in the spots was quantified before and after activation by summing the intensity of pixels in the ROI around each of the spots. Four mice were used in total in this work. All animal experiments were approved by Dartmouth IACUC, as outlined in the corresponding protocol # 00002170(a).