"Systematic evaluation of oncogene modules of increasing complexity in primary colon organoids (a,b) In vitro organoid Apc deletion. Primary small intestine (a) or colon (b) organoids from neonatal Apc flox/flox ; villin-CreER mice were treated with tamoxifen (2 μM, 7 days) followed by analysis at day 20. Tamoxifen-mediated Apc deletion induced overall growth, proliferative index (middle, PCNA) and APC deletion (bottom, APC IHC). Scale bars, top, 5 mm; middle, 50 μm; bottom, 50 μm. (c,d) In situ polyposis within long-term intestinal organoid cultures. Small intestine organoids from neonatal Apc flox/flox ; villin-CreER mice induced without (c) or with (d) tamoxifen followed by culture for 105 days and analysis by PCNA. An in situ tubular adenomatous polyp within the organoid wall in (d) exhibits a polypoid proliferation of PCNA-positive closely spaced tubules lined by enlarged, crowded nuclei. H E staining of the yellow-boxed region is depicted. Scale bars, 50 μm. (e) one to four oncogene CRC modules were created by infection of tamoxifen-treated Apc flox/flox ; villin-CreER adult colon organoids with appropriate combinations of control LMP retrovirus (with GFP cassette) or retroviruses encoding Kras G12D , LMP p53 shRNA/GFP or LMP Smad4 shRNA/GFP followed by H E staining at day 50 post-infection. The 1-gene Apc (""A"") and 2-gene colon modules ( Apc −/− /Kras G12D (AK), Apc −/− /p53 shRNA (AP) or Apc −/− /Smad4 shRNA (AS)) only exhibited minimal dysplasia. High-grade focal dysplasia was exhibited by the 3-gene module Apc −/− /Kras G12D /p53 shRNA (AKP). The four-gene module Apc −/− /Kras G12D /p53 shRNA/ Smad4 shRNA (AKPS) induced adenocarcinoma characterized by atypia, confluent sheets of cells, cribriform growth patterns, luminal necrosis, jagged infiltrating growth patterns and frank invasion. Scale bars, 50 μm."