0 or EDTA solution pH 9. 0. An automated de tection using a Leica Bond Autostainer was employed. Normal expression was defined as nuclear staining within tumor cells, using infil trating lymphocytes as positive internal control. Negative protein expression was defined as complete absence of nu clear staining within tumor cells in the face of concurrent positive labeling in internal non neoplastic tissues. Gastric organoid cancer modeling in mice and functional analysis All procedure involving animal were approved the Stanford University Administrative Panel on Laboratory Animal Care and was fully compliant with the USDA Animal Wel fare Act, and our Assurance of Compliance with the PHS Policy on Human Care and Use of Laboratory Animals. Air liquid interface organoid culture was performed as de scribed.

Cdh1flox/flox.Trp53flox/flox mice were generated by crossing Cdh1flox/flox mice, obtained from Jackson La boratory, and Trp53flox/flox mice, kindly provided by Dr. Anton Berns NOD. Cg Prkdcscid Il2rgtm1Sug/JicTac mice were obtained from Taconic Farms, Inc. We dis sected stomachs from neonatal mice and washed them in cold PBS to remove all luminal con tents. We extensively minced either large 25% sections or any entire neonatal stomach per dish and embedded the minced tissues in a 3D collagen gel using a double dish air liquid interface culture system as previously described. To maintain the organoids, we applied fresh medium every week. Tgfbr2 shRNAs were obtained from Origene. Retroviral plasmids were cotransfected with pCL Eco into 293 T cells by Lipofectamine2000.

Retroviral supernatants were collected 48 and 72 h post transfection and concentrated by PEG it virus precipitation solution. Virus titer was determined by infection of NIH3T3 cells and FACS analysis of GFP positive cells 48 h post infection. Cdh1flox/flox.Trp53flox/flox gastric organoids were in fected at day 0 with adenovirus Ad Cre GFP or control adenovirus Ad Fc encoding a mouse immunoglobulin IgG2 Fc fragment by layering viral particles suspended in 500 uL culture media over the top of the collagen matrix con taining primary tissue. For retrovirus infection of sec ondary organoids, primary organoids at 14 to 20 days of growth were recovered from collagen gel by collagenase IV incubation followed by 0. 05% trypsin/ EDTA incubation to dissociate organoids into a single cell suspension.

Following extensive washing with 10% FBS to inactivate collagenase/trypsin, cells were pelleted by centrifugation and incubated with retroviral particles encoding Tgfbr2 shRNA in the presence of growth medium and TransDux at room temperature for 60 min before serial replating into 3D collagen gel air liquid interface culture. Samples were fixed with 4% paraformaldehyde overnight, Batimastat paraffin embedded, sectioned, and sections stained by H E for initial histology analysis. Further immunohistochemistry analysis, used the following antibodies PCNA, CDH1, TGFBR2, p53.