In Vitro Organoid-Based Assays Reveal SMAD4 Tumor-Suppressive Mechanisms for Serrated Colorectal Cancer Invasion

Abstract

Colon cancer is the third most prominent cancer and second leading cause of cancer-related deaths in the United States. Up to 20% of colon cancers follow the serrated tumor pathway driven by mutations in the MAPK pathway. Loss of SMAD4 function occurs in the majority of late-stage colon cancers and is associated with aggressive cancer progression. Therefore, it is important to develop technology to accurately model and better understand the genetic mechanisms behind cancer invasion. Organoids derived from tumors found in the Smad4^KO BRAF^V600E/+ mouse model present multiple phenotypes characteristic of invasion both in ex vivo and in vivo systems. Smad4^KO BRAF^V600E/+ tumor organoids can migrate through 3D culture and infiltrate through transwell membranes. 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.

Keywords: SMAD4; colon cancer; invasion; organoids.

Figure 1

Smad4^KO BRAF^V600E tumor organoids have invasive capability in 3D environment. (A) Representative images of organoids cultured in BME R1. Adjacent normal and tumor organoids were derived from Smad4^KO BRAF^V600E/+ Villin^Cre-ERT2 mice 3–6 months post-tamoxifen injection. Tumor organoids have capability to migrate through Matrigel and develop projections which connect neighboring organoids. Scale bars = 0.5 mm. (B) Tumor organoids with RosaGFP expression reveal that projections are cellular. Scale bars = 0.5 mm (C) Capability to develop projections is unique to tumor organoids (* = p-value < 0.05, Student’s t-test). (D) Smad4^KO BRAF^V600E/+ Villin^Cre-ERT2 Rosa-GFP tumor organoids transplanted into NOD.CB17-Prkdc^scid mice colon for 2 months revealed invasive capability (white arrows). Scale bars = 0.5 mm (E) Smad4^KO BRAF^V6000E/+ β-catenin^gof Villin^Cre-ERT2 organoids developed similar invasive behavior as Smad4^KO BRAF^V600E/+ tumor organoids (red arrows). (F) Capability to form projections was highest in Smad4^KO BRAF^V600E Ctnnb1^Exon3 Villin^Cre-ERT2 organoids (* = p-value < 0.05, Student’s t-test).

Figure 2

SMAD4 expression in tumor organoids suppresses invasive capability. (A) Western Blot of doxycycline induced expression of SMAD4 in pINDUCER-SMAD4 Smad4^KO BRAF^V600E/+ tumor organoids. (B) Immunofluorescence images of wildtype organoids (Control) and untreated (−Dox) and treated (+Dox) pINDUCER-SMAD4 Smad4^KO BRAF^V600E/+ tumor organoids. (C) Representative images displaying growing cellular connections (white arrows) between tumor organoids in either untreated or treated conditions. Scale bar = 0.5 mm. (D) Smad4^KO BRAF^V600E/+ tumor organoids form significantly more connections than Smad4^KO BRAF^V600E/+ organoids with induced expression of Smad4 via doxycycline. (* = p-value = 0.0.017, Student’s t-test). 10× transmission and florescence images representative of 3 biological replicates. (E) EdU stain of tumor organoids treated with either vehicle or doxycycline. 10× images representative of 3 biological replicates (Scale bar = 0.5 mm). (F) Quantification of EdU-positive cells in pINDUCER-SMAD4 Smad4^KO BRAF^V600E/+ organoids.

Figure 3

SMAD4 expression alters ECM and secretory pathway genes. (A) GSEA analysis shows Tgfβ signaling genes are significantly upregulated in SMAD4-expressing tumor organoids. GSEA analysis shows MYC targets are significantly downregulated in SMAD4+ organoids grown in BME. (B) Heatmap of significantly regulated (log2FC < −1 and >1, adj p-value < 0.05) pINDUCER-SMAD4 Smad4^KO BRAF^V600E/+ organoids untreated (−Dox) (n = 4) and treated (+Dox) with 4 μg/mL Doxycycline treatment (n = 2). (C) GO Terms show significant upregulation in gene sets related to extracellular region, intermediate filaments, and n-linked asparagine in tumor organoids grown in BME with expression of SMAD4 induced via doxycycline. (D) GO Terms show significant downregulation in gene sets related to positive regulation of MAPK cascade, cell-to-cell signaling, and n-linked asparagine in tumor organoids grown in BME with expression of SMAD4 induced via doxycycline.

Figure 4

Smad4^KO BRAF^V600E/+ tumor organoids have invasive behavior in 2D culture. (A) Viability of organoids plated without BME (*** = adj p-value = 0.002; **** = adj p-value < 0.0001, ANOVA). (B) Smad4^KO BRAF^V600E/+ adjacent normal and tumor organoids are capable of surviving on plastic and formed a 2D monolayer. Scale bar = 1 mm. (C) Diagram of transwell assay for invasive potential. (D) Smad4^KO BRAF^V600E/+ RosaGFP tumor organoids were capable of migrating through transwell after 3 weeks and forming 2D colonies (Arrows). Images representative of 3 biological replicates. Scale bar = 0.5 mm.

Figure 5

SMAD4 suppresses organoid invasion in 2D culture. (A) Timeline of transwell assay and treatment with doxycycline to induce SMAD4. (B) Images of organoid colonies on plastic (arrows) representative of 3 biological replicates. Scale bar = 1 mm. (C) Tumor organoids expressing SMAD4 prior to transwell removal were unable to migrate. (* = p-value < 0.05, ANOVA).

Figure 6

SMAD4 suppresses cell cycle and proliferation in 2D culture. (A) Heatmap of Dox-treated orgnaoids in 2D monolayer. (B,C) DAVID analysis of genes upregulated (B) and downregulated (C) in SMAD4-expressing tumor organoids grown in the absence of BME R1. (D) EdU stain of tumor organoids in 2D cultures, images representative of 3 biological replicates. Scale bar = 1 mm. (E) Quantificaiton of EdU-positive cells in organoids (p-value reported, Student’s t-test). (F) GSEA analysis reveals cell cycle is downregulated upon expression of SMAD4.

Figure 7

Smad4 directly binds to cell cycle genes downregulated upon re-expression of SMAD4. (A) Smad4 binding sites from mouse whole epithelium (n = 2), or CaCo2 (n = 1) and SW480 (n = 1) human CRC cells. ChIP-Seq data are compared with significantly regulated genes from RNA-Seq of tumor organoids +/− SMAD4 (from Figure 6, log2FC > 1 or <−1, adj p-value < 0.05). (B) DAVID analysis of genes directly bound by SMAD4 and upregulated and (C) downregulated. (D) RNA-Seq of cell cycle genes bound by SMAD4 and significantly downregulated (adj p-value < 0.05). (E) IGV of cell cycle genes in SMAD4 ChIP-Seq from mouse epithelium (n = 2, blue), SW480 (red), and CaCo2 (green).