M3 vision

For bioluminescence, mice received an intraperitoneal injection (150 mg/kg) of VivoGlo™ Luciferin (Promega) 5 min before imaging. For fluorescence, mice received an intravenous injection (2 nmol per mouse) of MMPSense™ 750 FAST (PerkinElmer) 18 h before imaging. Signals plus grey-scale photographic images were acquired using a Photon Imager™ (Biospace) and M3 Vision (Biospace). We maintained animals under general anaesthesia with 1–2% isoflurane, plus warming and monitoring, during image acquisition. We carried out signal quantification (photons/s/cm²/sr) using M3 Vision (Biospace).

Fluorescence acquisition were performed using the planar optical camera PhotonImager RT (Biospace Lab, France). Mice were narcotised with isoflurane (2–3% isoflurane, 0.5 l per minute air) and injected intravenously with 10 nmol of AngioStamp^®700 (50 μmol L⁻¹ in PBS; Fluoptics, France) or with 2 nmol of HypoxiSense 680 (20 µmol L⁻¹ in PBS; Perkin Elmer, USA) for imaging angiogenesis (expression of αvβ integrin, overexpressed in neovessel endothelial cells during angiogenesis) or hypoxia (expression of carbonic anhydrase 9 protein, which increases in hypoxic regions within tumours), respectively. Images were acquired before and 5 min, 1, 2, 4, 6, 12, 24, 48 post tracer injection. Filters were set as following: excitation filter: 650 nm, emission filter: 575 nm, background filter: 575 nm. Images analyses was performed using the software M3Vision (Biospace Lab, France). Grayscale photographic images and fluorescence colour images were superimposed. Regions of interest were drawn over each tumour to determine the signal intensity.

Mice at days 17, 27 and 37 post-implantation were anesthetized with 2% isoflurane, injected intraperitoneally with 150 mg/kg D-luciferin (Promega) and placed in a photonIMAGER™ chamber (Biospace Lab). White light and luciferase activity images were monitored at 30 sec intervals for 5 min and images were analyzed using the Living Image software M3 Vision (Biospace Lab). Signal intensity was quantified as the sum of all detected photon counts from tumors. Signal average for each group of mice (+Dox) was compared with those from control animals (−Dox). Apoptosis was monitored one day before tumor growth imaging. Mice were injected i.p. with VivoGlo™ Caspase-3/7 substrate (50 mg/kg; Promega) and subjected to a 5 min imaging session. Acquisition of the Luciferase apoptotic signal and ROI measurement were then performed. Z-DEVD-aminoluciferin signal was normalized with the D-luciferin signal. Signal average for each group was calculated and results were analyzed for statistical significance using a Mann-Whitney t-test. Values are the mean ± SD.