Construction of a Murine Obstructive Colon Cancer Model
Animals
Eight to10 weeks-old female BalB/c mice were purchased from Charles River Laboratories (Wilmington, MA, USA). The mice were acclimatized for at least one week before performing the experiments.
Cell culture
Murine colon cancer CT26 cells were obtained from the American Type Culture Collection (ATCC, Rockville, MD, USA). Stable luminescent and fluorescent CT26 cell lines were obtained by infection with lentiviral particles carrying luciferase and Green Fluorescent Protein (GFP) constructs (GeneCopoeiaTM LP-HLUC-LV201-0200). Stably transfected luciferase CT26 cells were kindly provided by Prof. Lea Eisenbach (Weizmann Institute of Science Rehovot, Israel). All cell lines were maintained in 10% CO2 at 37°C in high glucose Dulbecco’s modified Eagle medium (DMEM) supplemented with 10 % foetal bovine serum, 1% penicillin/streptomycin solution, and 1% L-glutamine solution.
Construction of a murine obstructive colon cancer model
We used a miniature endoscope (10 cm length, 1.9 mm outer diameter), a xenon light source, a triple chip camera and an air pump to achieve regulated inflation of the mouse rectum (Karl Storz, Tuttlingen, Germany). All procedures were performed under inhalation anaesthesia with 1.5-2 %isoflurane/O2 mixture. Submucosal injections into the distal posterior rectum (about 2 cm from the anal verge) were accomplished using a 30-gauge, 8 inch long and 45° bevel needle with the open bevel heading up in a flat angle. The injected volume was 50 μl of a solution containing CT26 cells at a concentration of 2x106 cells per ml which was found adequate for the development of a tumor (Supplementary Fig. 1a).
Colonic stenting
Cardiac stents, found suitable due to its length and diameter, were used as a substitute for murine colonic stenting. Dimensions of the stents were 1 cm long and 2 mm in diameter and matched with the size of the mouse rectum. Expansion of the stent was obtained by the use of a hydrostatic balloon. After development of an occlusive rectal tumor, a catheter was inserted across the tumor; balloon was inflated in order to expand the stent (leading at the same time to the compression of the tumor). Then the catheter, and the balloon after deflation were removed (Supplementary Fig.1b). In some cases the colonic stent were not be inserted on account of limitations in rectal tumor size as it could lead to the displacement of the stent.
In vivo bioluminescence
Monitoring of the tumor growth and metastatic dissemination was performed in a non- invasive manner by following luciferase-expressing CT26 cells. Two hundredμl of a 10ng/ml luciferin solution was injected intraperitoneally 5 minutes before stent acquisition, and subsequently the mice were inserted in a closed dark chamber of the IVISLumina II (Calliper Life Sciences). Two acquisitions were systematically performed, the first one in supine position and the second one in prone position. A circular region of interest (ROI) was defined in the abdomen of the mice and quantification of light emission was performed in photons/second (p/s) using the Living Image software (PerkinElmer, Waltham, MA, USA).
Serum samples
Blood samples were obtained from the suborbital sinus of mice under anaesthesia. The samples were centrifuged at 4°C, 12000 g for 5 minutes. Serum was stored at -80°C until analysis. Lactate dehydrogenase (LDH) activity, reflecting cancer activity and progression (Warburg effect), was measured using an Architect C8000 Abbott machine and 2P56-21 mice reagent.
Circulating tumour cells (CTC)
Three hundredμl (or more when possible) of blood was obtained from a cardiac puncture under anaesthesia just before sacrificing the mice. Cells were sorted according to their size and visco-elasticity using the CANOPUS technology. After filtering, cells were labelled with antibodies against GFP or Luciferase and revealed by a secondary antibody coupled to an “alexafluor”.