Ivis spectrum equipment

This study was approved by the Institutional Animal Ethics Committee. We implanted CXCR4-overexpressing SP5 human colorectal tumor line to generate subcutaneous colorectal tumors in swiss nude mice (Charles River, France) as previously described [16 (link), 17 (link)]. When tumors reached ca. 500 mm³, mice were randomly allocated to Origami B, MC4100, KPM335, BW30270 or buffer-treated groups (N = 3–5/group). The experimental mice received a single intravenous bolus of the corresponding nanoparticle (500 μg in carbonate buffer, pH 7.4), whereas control mice received only buffer. Five hours after the administration, we measured ex vivo the fluorescence emitted by the nanoparticles accumulated in the whole and slice sectioned tumor and normal tissues (kidney, lung, and heart, liver and brain) using IVIS^® Spectrum equipment (Perkin Elmer, Waltham, MA, USA). The fluorescence signal was digitalized, and after subtracting the autofluorescence, it was displayed as a pseudocolor overlay and expressed as Radiant efficiency. Data were corrected by the specific fluorescence emitted by the different nanoparticles. Signal difference between groups was determined by applying the non-parametric Mann–Whitney test.

Luminescence and volumetric measurements were used to assess the growth dynamics of tumours. After PEF treatment, the volume of tumours (or forming scabs) was determined by measuring centered tumour length and width with a digital caliper every 2–3 days after the treatment. The volumes of tumours (mm³) were calculated by the formula: V = (Length × Width² × π)/6, where π = 3.1416. When the tumour volume reached about 3000 mm³, the mice were sacrificed by cervical dislocation.
The luminescence of tumours was assessed by imaging tumours with IVIS Spectrum equipment and Living Image Software (Caliper/Perkin Elmer, Akron, OH, USA). Prior to the treatment, mice were intraperitoneally injected with 150 µL (30 mg/mL in PBS) of D-luciferin solution (Promega, Madison, WI, USA). After 10–15 min, mice were visualised under anaesthesia with 3% isoflurane and oxygen gas mixture that was later lowered to 1.5% (Vetpharma Animal Health, S.L., Barcelona, Spain). Then, tumours were imaged before the treatment, right after the bleomycin and PEF treatment, as well as 11 days after the treatment. The bioluminescence of tumours was proportional to the number of live LLC1-Luc cells. Luminescence was expressed as the photons/sec/region of interest (ROI) by subtracting the background luminescence of the same size region.

To assess the antitumor activity of the T22-PE24-H6 nanotoxin we used the SW1417 subcutaneous model in Swiss Nude mice. All procedures were conducted in accordance with the guidelines approved by the institutional animal Ethics Committee of Hospital Sant Pau. Swiss nude female mice (4 weeks old) were obtained from Charles River Laboratories and ten million CXCR4⁺ SW1417 cells were injected in two subcutaneous flanks. Once tumors reached approximately 120 mm³ sizes, mice were randomized into the control or treated group. Treated mice received intravenous doses of 10 μg of T22-PE24-H6 (N = 6 mice), at a repeated dose regime of three times a week, per eight doses. The control group received a buffer using the same administration schedule. Overall the experimental period, mouse body weight and tumor volume were registered three times a week. Tumor bioluminescence was also measured once a week using the IVIS Spectrum equipment (Perkin Elmer, Waltham, MA, USA). When tumors reached a volume of 600 mm³, mice were euthanized and tumor and non-tumor organs were collected for further analyses.