In vitro organoid-based assays reveal SMAD4 tumor-suppressive mechanisms for serrated colorectal cancer invasion [ChIP-seq]

We establish a model system where organoids derived from tumors found in the Smad4KO BRAFV600E/+ mouse model present multiple phenotypes characteristic of invasion both in ex vivo and in vivo systems. Additionally, Smad4KO BRAFV600E/+ tumor organoids have the ability to infiltrate a transwell and initiate colonies on the culture plate below. This invasive behavior can be suppressed when SMAD4 is re-expressed in the tumor organoids. RNA-Seq analysis reveals that SMAD4 expression in organoids rapidly regulates transcripts associated with extracellular matrix and secreted proteins, suggesting that the mechanisms employed by SMAD4 to inhibit invasion are associated with regulation of extracellular matrix and secretory pathways. These findings indicate new models to study SMAD4 regulation of tumor invasion and an additional layer of complexity in the tumor-suppressive function of the SMAD4/Tgfβ pathway. SW480 cells were then transduced serially with lentiviral vectors carrying avi-tagged SMAD4 expression constructs (pReceiver-Lv108, GeneCopoeia) with puromycin resistance cassette and BirA expression plasmid with hygromycin B (Addgene plasmid 29649). SW480 cells were cultured in RPMI, containing 10% FBS and 1% penicillin and streptomycin. Cells stably expressing both avi-tagged SMAD4 and BirA were selected with medium containing 2 μg/ml puromycin and 0.4 mg/ml Hygromycin B, respectively. Cells were fed 24 hours prior to harvest at approximately 95% confluency for ChIP-Seq.