Coloview system

To induce colitis, mice were given 2.5% (w/v) DSS (MP Biomedicals) in the drinking water for 6 days, and then switched to regular drinking water until the end of the experiment. Mice were weighed every day to determine percentage body weight changes. Colonoscopy was used to monitor and score the colitis. Colonoscopy was performed in a blinded fashion using the Coloview system (Karl Storz, Germany).^{65 (link)} Briefly, colitis score was addressed considering the consistence of stools, granularity of the mucosal surface, translucency of the colon, fibrin deposit and vascularization of the mucosa (0–3 points for each parameter). After sacrifice of the mice, colon length was measured and colon tissue samples were collected for histology. For IL-18BP level analysis in Fig. 3q, harvested colon tissues were homogenated and the IL-18BP levels in colon homogenates were measured using the Mouse interleukin-18 binding protein (IL-18BP) ELISA Kit (CUSABIO, Cat# CSB-E17797m)

Mice were anaesthetized using intraperitoneal injection of ketamine, xylazine, and acepromazine. The “Coloview system” (Karl Storz) was used for colonoscopy as described in Becker et al (2005). The endoscopic procedure was viewed on a color monitor, and pictures were digitally recorded.

Colorectal tumor development was monitored in vivo using the endoscopic ‘Coloview system’ (Karl Storz), as described before [37 (link),38 (link)]. Mice were anesthetized during endoscopy using Isoflurane (Abbott Laboratories, Lake Bluff, IL, USA).

For anesthesia, medetomidine (0.5 mg/kg, Zoetis, Berlin, Germany), midazolam (5 mg/kg, Ratiopharm, Ulm, Germany), and fentanyl (0.05 mg/kg, Albrecht, Aulendorf, Germany) were administered via intraperitoneal injection. The depth of anesthesia was assessed using toe pinch observing no withdraw reflex (stage of surgical tolerance). Peri-interventionally, mice were placed on a heated pad (Witte & Sutor, Murrhardt, Germany) for the maintenance of homeostasis. For endoscopy and endoscopy-guided tumor cell implantation, the Coloview^® system (Storz, Tuttlingen, Germany) with a zero-degree optic (diameter 1.9 mm) was utilized (Figure 6A). The colon was then washed gently with 2 mL of DPBS at 37 °C using a soft pipette (Nerbe plus, Winsen/Luhe, Germany). For endoscopy without cell implantation, a 7 Fr. examination sheath was used. The valve of the Luer lock adapter (Figure 6A, black asterisk) was adjusted to create a slow constant air flow which made the mucosa just become flattened after gentle trans-anal insertion of the endoscope. This enabled a clear 360-degree view without inflating the gastrointestinal tract too severely. The extent of insertion was controlled endoscopically by using gradations on the endoscope (Figure 6A, red asterisk).

The colitis protocol was based on preliminary toxicity data in the Lgr5-lacZ strain. The standard protocol to induce acute colitis uses 2%–3% DSS for 7 consecutive days dependent on mouse strain sensitivity (84 (link)). As we found approximately 30%–40% of Lgr5-lacZ mice died at day 6 or 7 after DSS treatment because of severe colon damage at ≥2% DSS, we elected to use 1% DSS in drinking water ad libitum (MP Biomedicals or TdB, molecular mass 36–50 kDa) for colitis induction in this strain. Adenoma induction used a standard AOM-DSS protocol (21 (link), 84 (link)) modified as follows: electing a schedule of decreasing DSS dosage to mitigate toxicity, 6- to 8-week-old male Lgr5-lacZ mice were injected intraperitoneally with AOM (MilliporeSigma) at a dose of 12.5 mg/kg body weight and after 5 days were fed 2% DSS in the drinking water for 5 days, followed by 14 days of regular water. This cycle was repeated twice with 1.5% DSS and 1.0% DSS, respectively. Colonoscopy was performed at day 90 on anesthetized mice to check for tumor formation using the Coloview System (Karl Storz). Our data show a single AOM injection does not induce colon tumorigenesis in C57BL/6J mice (data not shown) (40 (link), 85 (link), 86 (link)).