Tele pack vet x led endoscopic video unit

Colonic sub-mucosal injections of organoids were performed as previously described (Roper et al., 2017 (link)), using a Karl Storz TELE PACK VET X LED endoscopic video unit. KPN liver metastases derived (C57BL/6, N=10) organoids, cultured in conditions as described above without R-spondin, were harvested and dissociated by fiercely pipetting. Organoids were washed twice with PBS before being injected. Approximately 500 organoids in 70 μl PBS were injected in a single injection. At clinical end point tumors and metastasis were quantified.

Immunohistochemical staining to detect pSTAT3^tyr705 was performed on a KPN and an AKPT tumor to determine constitutive activation of the pathway in vivo. A KPN male mouse on a C57BL/6J (generation N5-6) background of 10 weeks of age were induced with a single intraperitoneal injection of 2mg tamoxifen and aged until clinical endpoint as evidenced by anemia, hunching and/or weight loss to generate small intestinal tumor for IHC staining. Intracolonic induction in a 16-week-old female mouse villinCre^ER Apc^fl/+ Kras^G12D/+ Trp53^fl/fl Tgfbr1^fl/fl on a C57BL/6 background (generation N6/N7) was performed under general anesthesia. Three 70µl 100uM dose of 4-hydroxy tamoxifen (H7904-5MG from Sigma) were injected from the mid-colon to distal colon into the sub-mucosa via a colonoscope (Karl Storz TELE PACK VET X LED endoscopic video unit). At clinical endpoint (weight loss with the presentation of hunching) colonic tumor tissue (spontaneous SI tumour) was collected from a single tumour for IHC staining.
Staining was performed using pSTAT3^tyr705 antibody (#9131, Cell signaling, Danvers, MA, USA) at 1:100 on a Leica BOND Rx autostainer (Leica Biosystems, Wetzlar, Germany).

The different intestinal cancer models are listed in Table 3. Two models of tumorigenesis were used: aged genetically engineered mice; and intracolonic transplants of murine-derived organoids (Supplementary Table S1). All genetically engineered mouse models (GEMM) were induced with a single 2 mg intraperitoneal injection of tamoxifen (Sigma-Aldrich T5648) and aged to a clinical endpoint. For transplant mice, murine tumor-derived organoids were injected intracolonically into male immune competent C57BL/6J mice (Charles River strain 632) using previously described methods (24 (link)). Tumor organoids were mechanically dissociated into fragments by pipetting and washed twice in PBS. Each mouse was injected with the equivalent of one well of a 6-well plate in 70 µL of PBS. This was injected into the colonic submucosa using a Karl Storz TELE PACK VET X LED endoscopic video unit with associated needle.