Canonical TGFβ signaling induces collective invasion in colorectal carcinogenesis through a Snail1- and Zeb1-independent partial EMT

Abstract

Local invasion is the initial step towards metastasis, the main cause of cancer mortality. In human colorectal cancer (CRC), malignant cells predominantly invade as cohesive collectives and may undergo partial epithelial-mesenchymal transition (pEMT) at the invasive front. How this particular mode of stromal infiltration is generated is unknown. Here we investigated the impact of oncogenic transformation and the microenvironment on tumor cell invasion using genetically engineered organoids as CRC models. We found that inactivation of the Apc tumor suppressor combined with expression of oncogenic Kras^G12D and dominant-negative Trp53^R172H did not cell-autonomously induce invasion in vitro. However, oncogenic transformation primed organoids for activation of a collective invasion program upon exposure to the prototypical microenvironmental factor TGFβ1. Execution of this program co-depended on a permissive extracellular matrix which was further actively remodeled by invading organoids. Although organoids shed some epithelial properties particularly at the invasive edge, TGFβ1-stimulated organoids largely maintained epithelial gene expression while additionally implementing a mesenchymal transcription pattern, resulting in a pEMT phenotype that did not progress to a fully mesenchymal state. Notably, while TGFβ1 induced pEMT and promoted collective invasion, it abrogated self-renewal capacity of TKA organoids which correlated with the downregulation of intestinal stem cell (ISC) marker genes. Mechanistically, induction of the non-progressive pEMT required canonical TGFβ signaling mediated by Smad transcription factors (TFs), whereas the EMT master regulators Snail1 and Zeb1 were dispensable. Gene expression profiling provided further evidence for pEMT of TGFβ1-treated organoids and showed that their transcriptomes resemble those of human poor prognosis CMS4 cancers which likewise exhibit pEMT features. We propose that collective invasion in colorectal carcinogenesis is triggered by microenvironmental stimuli through activation of a novel, transcription-mediated form of non-progressive pEMT independently of classical EMT regulators.

Fig. 1. Oncogenically transformed intestinal organoids display no cell-intrinsic invasiveness in vitro.

a Strategy for generating oncogenically transformed small intestinal and colonic organoids from genetically engineered mice carrying a Villin-CreER^T2 transgene, two floxed Apc alleles, as well as heterozygous Kras^LSL-G12D/+ and Trp53^LSL-R172H/+ loci. Floxed stop cassettes (LSL) prevent expression of the mutant Kras and Trp53 alleles. For recombination, organoids were treated with 4-hydroxy-tamoxifen (4-OHT), yielding TKA organoids. b Whole mounts and hematoxylin/eosin (H&E) stained sections of floxed and TKA organoids (line 815) cultured in 7 mg/ml Matrigel and visualized by phase contrast (PhC) or bright field microscopy. Scale bars: 200 µm. c Representative images of immunofluorescence staining of pan-laminin, E-cadherin, atypical protein kinase C (aPKC), and β-catenin in sections of floxed and TKA organoids (line 815) cultured in 7 mg/ml Matrigel. Nuclei were stained by DAPI; n > 3. Scale bars: 50 µm. d Top: Microscopy of floxed and TKA organoids (line 815) in 7 mg/ml Matrigel at the beginning (0 h) and the end (8 h) of forskolin and DMSO treatment. Scale bar: 200 µm. Bottom: Quantification of forskolin-induced organoid swelling. TKA organoids were exposed to DMSO or forskolin for the indicated time periods. Normalized changes in organoid diameter were calculated by first computing at each time point and for each organoid under consideration the increase in diameter relative to the corresponding value at t = 0 h, followed by normalization of forskolin-induced relative changes in diameter to those of DMSO-treated control samples. At least five organoids treated with DMSO or forskolin were analyzed per biological replicate and organoid line. e Top: setup for cultivating organoids in type I collagen at an air-liquid-interface and representative H&E stainings of organoid displaying different histological features (line 815). Scale bar: 200 µm. Bottom: Quantification of organoids following histological classification (morib.: moribund; dyspl.: dysplastic). Quantitative experiments in (d, e) were performed with three floxed/TKA organoid lines (815: n = 3; 931: n = 3; 947: n = 3). Dots represent results of independent biological replicates and dot color identifies the organoid lines.

Fig. 2. TGFβ1 triggers collective invasion of TKA organoids.

a Morphological appearance of TKA organoids (line 815) cultured in 3 mg/ml Matrigel and treated with solvent or TGFβ1 for the indicated time periods. Boxed areas are shown at higher magnification on the right. Similar TGFβ1-induced morphological changes were observed with TKA organoid lines from five different founder mice. Scale bars: 100 µm. b Boyden chamber invasion assays with TKA organoids (line 931) seeded in 3 mg/ml Matrigel. Top: bright field (BF) images taken at 0 h of TGFβ1 and solvent treatment. Inserts show magnified views of the upper chambers. Bottom: crystal violet (CV) staining of invaded cells after 96 h of treatment. Inserts show magnified views of the bottom faces of the Boyden chambers. c Quantification of invasion experiments as shown in (b) performed with TKA organoid lines 815 (n = 3), 931 (n = 3), and 947 (n = 3). Each dot represents the result of a single invasion assay while dot color identifies the organoid lines. ***p = 0.0004; Mann–Whitney U test. d Phase contrast microscopy of TKA organoids cultured in type I collagen in presence of solvent or TGFβ1. Arrows point at sites of collective invasion. Independent experiments were performed in three different organoid lines (815: n = 3; 931: n = 3; 947: n = 3). Scale bar: 100 µm.

Fig. 3. TGFβ1-stimulated TKA organoids interact with and remodel the ECM.

a TKA organoids (line 815) expressing nuclear H2B-GFP and membrane-bound mTomato were seeded in 3 mg/ml Matrigel, treated with solvent or TGFβ1 for 48 h, and subjected to live imaging using confocal microscopy. Displayed are 3D reconstructions and orthogonal views of organoid cross-sections (yellow lines: positions of the cross-sections along the x-, y- and z-axes). Scale bars: 100 µm (n = 3). Bottom: schematic representations of TKA organoid morphologies. Arrows: proposed streaming of cells. b Whole-mount immunofluorescence staining and confocal microscopy of TKA organoids (line 815) seeded in 3 mg/ml Matrigel and treated with solvent or TGFβ1 for 72 h. Organoids were stained with antibodies against the indicated antigens. Actin was visualized by phalloidin staining. Nuclei were labeled using DAPI. Boxed areas are shown at higher magnification on the right. Pictures are representative for results obtained with three TKA organoid lines (815: n = 1; 931: n = 1; 947: n = 1). Scale bars: 100 µm. c Top: strategy for quantification of picrosirius red staining: (I) organoids were visualized by bright field illumination and the outline of the organoids (A) and a surrounding area (B) with an approximate width of 65 µm were marked. (II) Collagen was stained by picrosirius red and larger or parallel collagen bundles were visualized by polarizing light and signal intensities across the entire image were recorded. (III) For the final quantifications, only signals within area B and exceeding a defined threshold were considered. To allow comparison of organoids with different sizes, signal intensities were normalized to the size of area B. Middle: quantifications of picrosirius red-stained TKA organoids cultured in type I collagen and treated with solvent (solv) or TGFβ1 (TGF) for 96 h. Colored dots represent individual measurements from at least two independent experiments. Mann–Whitney U test. TKA organoid line 815: ***p = 0.0007 (solvent: n = 10, TGFβ1: n = 13); TKA organoid line 931: ***p = 0.0005 (solvent: n = 6, TGFβ1: n = 16); TKA organoid line 947: ***p = 0.0002 (solvent: n = 6, TGFβ1: n = 27). Scale bar: 100 µm. Bottom: exemplary pictures of TKA organoids (line 815) treated with solvent or TGFβ1.

Fig. 4. TGFβ1 induces a pEMT in TKA organoids.

a Time-resolved gene expression analyses of TKA organoids seeded in 3 mg/ml Matrigel and treated with solvent (solv) or TGFβ1 for the indicated time periods. RNA levels of EMT-TFs and EMT-associated genes were quantified by qRT-PCR and normalized to transcript levels of Eef1a1. Each dot represents the result of a single measurement while dot color identifies the organoid lines. Three independent biological replicates were performed for each organoid line (815: n = 3; 931: n = 3; 947: n = 3). ***p < 0.001; statistical significance was analyzed using a linear regression model combined with Bonferroni correction for multiple comparisons. Exact p-values are provided in Supplementary Table 7. b Western blot analyses of phospho-Smad2/3 (pSmad2/3), total Smad2/3, EMT-TFs, and EMT-associated genes in TKA organoids (line 815) treated as in (a). Gsk3β and α-tubulin were included as loading control. Molecular weights of size standards are given in kDa. Results are representative for experiments performed with three TKA organoid lines (815: n = 2; 931: n = 2; 947: n = 2); E-cad: E-cadherin. c Whole-mount immunofluorescence staining and confocal microscopy of TKA organoid lines 815 and 931 seeded in 3 mg/ml Matrigel and treated with solvent or TGFβ1 for 72 h. Organoids were stained for E-cadherin, claudin-7, β-catenin, and atypical protein kinase C (aPKC). Arrowheads indicate membranous E-cadherin, claudin-7, and β-catenin in the central part of the TGFβ1-treated organoid. Arrows highlight reduced staining of aPKC in the peripheral region of TGFβ1-stimulated organoids. Boxed areas are shown at higher magnification below. Reduced staining intensities in some central regions of organoids might be caused by technical issues related to whole-mount staining and confocal microscopy of organoids. Similar results were obtained with three different organoid lines (815: n = 1, 931: n = 1, 947: n = 1). Scale bars: 100 µm.

Fig. 5. The TGFβ1 response in TKA organoids is mediated by canonical TGFβ-receptor/Smad signaling.

a Whole-mount phase contrast microscopy of TKA organoids transduced with an empty vector (EV) or an expression vector for dominant negative TGFBR2 (TGFBR2DN), seeded in 3 mg/ml Matrigel, and treated with solvent or TGFβ1 for 72 h. Scale bar: 200 µm. b Western blot expression analysis of the indicated proteins in control (EV) and TGFBR2DN-expressing TKA organoids (line 947) treated as in (a). Gsk3β detection served as loading control. Molecular weights of size standards are given in kDa. Panels (a, b) show representative results of two independent biological replicates for TKA organoid lines 931 (n = 2) and 947 (n = 2). c Exemplary Boyden chamber invasion assays with control (EV) and TGFBR2DN-expressing TKA organoids (line 931) seeded in 3 mg/ml Matrigel. Bright-field (BF) images were taken at 0 h of solvent and TGFβ1 treatment. Inserts: magnified views of the upper chambers. Invaded cells were visualized by crystal violet (CV) staining after 96 h of treatment. Inserts: magnified views of the bottom face of the Boyden chambers. d Quantification of invasion experiments as shown in (c). Dots represent results of independent biological replicates (line 931: n = 3; line 947: n = 4). Dot color identifies the organoid lines. ***p < 0.001; Mann–Whitney U test. e Scheme of the Smad4 locus showing sgRNA target positions (red arrows) flanking exon 9 (black box) and the location of PCR primers used for genotyping. The distance between the sgRNA targets and the length of the PCR amplicon in Smad4 wt organoids are given in base pairs (bp). f Results of genotyping PCRs with genomic DNA from Smad4 wt TKA and Smad4 mutant TKAS organoid lines as indicated. Sizes of DNA standards are given in kilobase pairs (kbp). g Western blot expression analyses for Smad4 in TKA and TKAS organoids. Gsk3β detection served as loading control (n = 3). Molecular weights of size standards are given in kDa. h Whole-mount phase contrast microscopy of TKA and TKAS organoid lines seeded in 3 mg/ml Matrigel and treated with solvent or TGFβ1 for 72 h. Inserts display a larger field of view at lower magnification. Scale bars: 200 µm. i Boyden chamber invasion assays performed with TKA and TKAS organoids (line 931) as in (c). j Quantification of invasion experiments as shown in (i) performed with TKA and TKAS organoid lines 931 (n = 4) and 947 (n = 3). Dots represent results of individual experiments. Dot color identifies the organoid lines. ***p = 0.0006, **p = 0.0012, n.s. not significant (p = 0.39); Mann–Whitney U test. For (d and j), exact p-values are provided in Supplementary Table 7.

Fig. 6. TGFβ1-induced global transcriptional deregulation features a pEMT in TKA organoids.

a Principal component analysis (PCA) of RNA-seq data from TKA organoids seeded in 3 mg/ml Matrigel and stimulated with TGFβ1 for up to 72 h as indicated, or harvested at the onset of the experiment (C-0) and after 72 h of cultivation with solvent (C-72) to account for culture-dependent effects. Results are based on two independent biological replicates for organoid lines 931 (n = 2) and 947 (n = 2). b Numbers of differentially expressed genes (DEGs) were determined by performing pairwise comparisons of transcriptomes from TKA organoids treated as in (a). Black and gray segments of the bars: up- and downregulated genes, respectively. c Functional enrichment analysis of genes upregulated upon TGFβ1 treatment (adjusted p value <0.01, log₂(FC) > 1). The top ten GO terms from the indicated categories are listed. Dot size: ratio of upregulated genes compared to all genes within a set. Dot color: -log₁₀(adjusted [adj.] p value) of the enrichments. d Exclusive enrichment of mesenchymal components of published EMT signatures among genes significantly upregulated in TGFβ1-treated TKA organoids. The analyses were conducted for DEGs from TKA organoids treated with TGFβ1 for 72 h compared to cultivation for 72 h in solvent. Published EMT signatures were split into subsets comprising epithelial (Epi) and mesenchymal genes (Mes) and processed separately. The color encodes the gene ratio. The length of the bars depicts the -log₁₀ of the adj. p-values. Dotted line: adj. p value = 0.05. e The independent biological replicates (R1, R2) of the transcriptomes as described in (a, b) were assessed for resemblance to the four consensus molecular subtypes (CMS) of CRC. Non-classification of C-0 and T-6 samples may result from mechanical disruption and reseeding of organoids which likely erased any typifying gene expression. Colored dots and their positions depict the CMS type and the adj. p-value, respectively. NA: no significant classification possible. f Functional enrichment analyses of EMT sub-signatures as described in (d) among genes differentially expressed in colon cancers classified as CMS4 and CMS2. The color encodes the gene ratio. The length of the bars depicts the -log₁₀ of the adj. p-values. Dotted line: adj. p value = 0.05.

Fig. 7. TGFβ1-induced collective invasion occurs independently from the EMT transcription factors Snail1 and Zeb1.

a Schematics of the Snai1 and Zeb1 loci showing sgRNA target positions (red arrows) and relevant exons (black boxes). The size of the expected deletions is given in base pairs (bp). b Western blot expression analyses of the proteins indicated in TKA-Snai1^wt, TKA-Snai1^KO, TKA-Zeb1^wt, and TKA-Zeb1^KO organoids derived from line 815 seeded in 3 mg/ml Matrigel and treated with solvent (solv) or TGFβ1 (TGF) for 72 h. Gsk3β detection served as loading control (n = 3). Molecular weights of size standards are given in kDa. c Morphological appearance of TKA-Snai1^wt (#5, #7, #48), TKA-Snai1^KO (#18, #65, #75), TKA-Zeb1^wt (#44, #145, #171), and TKA-Zeb1^KO (#9, #34, #180) organoids derived from line 815 cultured in 3 mg/ml Matrigel and treated with solvent or TGFβ1 for 72 h. Scale bars: 200 µm.