Sh30910

Patient tumors were processed as described previously (66 (link)). Adjustments are described: tumors were obtained on the day of Mohs micrographic surgery and washed in cold DMEM [Gibco 11995-065] supplemented with 10% FBS [Thermo Scientific SH30910.03]. Any fat was removed from tumor samples using a scalpel. Then, tumors were then cut into small pieces using a razor and resuspended in 10 mL of DMEM/10% FBS, 10 mg/mL collagenase II [Sigma C2674] and 10 U DNase I [Sigma 00453869] for 20 min at 37°C. After incubation, the suspension was vortexed 1× for 30 s followed by pipetting sequentially through 25, 10 and 5 ml pipettes for 1 min each. Next, the cell suspension was filtered through a 70-μm filter [Fisher 22363548] and spun at 300 g for 5 min. After spinning, the supernatant was removed, and ACK red blood cell lysis was performed according to the manufacturer's protocol as needed [Gibco a10492-01].

An orthotopic mouse model of human rectal carcinoma was prepared as follows. A BALB/c nude mouse (5-week-old, female) was anesthetized with pentobarbital sodium (40 mg/kg body weight, intraperitoneal), and the anorectal wall was cut to prevent colonic obstruction resulting from the rectal tumor progression. HT-29 cells (5×10⁶ total), cultured with McCoy’s medium (12330–031, Gibco) containing 10% FBS (SH30910.03, Thermo Fisher Scientific) and 1% penicillin streptomycin (15140–122, Gibco) in a 5% CO₂ incubator at 37°C, were injected submucosally into the rectum of the mouse. After the surgery, tumor size was monitored throughout the tumor progression daily. If the tumor size reached to 17 mm in diameter, animals were sacrificed with an overdose of pentobarbital sodium (200 mg/kg body weight, intraperitoneal) as humane endpoint. After 6 weeks, the rectal tumor and normal rectal tissue were excised from the mouse after euthanasia with an overdose of pentobarbital sodium (200 mg/kg body weight, intraperitoneal). The tumor and the normal tissue were frozen and embedded in O.C.T. The frozen sections were sliced to a thickness of 10-μm by a cryostat (CM1900, Leica) and attached to a cover glass (No. 1 grade, Matsunami Glass Ind., LTD.). The samples were embedded into Gelvatol and covered by a cover glass with 10-μm-thick double-sided tape.