BRAFV600E drives dedifferentiation in small intestinal and colonic organoids and cooperates with mutant p53 and Apc loss in transformation

BRAFV600E confers poor prognosis and is associated with a distinct subtype of colorectal cancer (CRC). Little is known, however, about the genetic events driving the initiation and progression of BRAFV600E mutant CRCs. Recent genetic analyses of CRCs indicate that BRAFV600E often coexists with alterations in the WNT- and p53 pathways, but their cooperation remains ill-defined. Therefore, we systematically compared small and large intestinal organoids from mice harboring conditional BraffloxV600E, Trp53LSL-R172H, and/or Apcflox/flox alleles. Using these isogenic models, we observe tissue-specific differences toward sudden BRAFV600E expression, which can be attributed to different ERK-pathway ground states in small and large intestinal crypts. BRAFV600E alone causes transient proliferation and suppresses epithelial organization, followed by organoid disintegration. Moreover, BRAFV600E induces a fetal-like dedifferentiation transcriptional program in colonic organoids, which resembles human BRAFV600E-driven CRC. Co-expression of p53R172H delays organoid disintegration, confers anchorage-independent growth, and induces invasive properties. Interestingly, p53R172H cooperates with BRAFV600E to modulate the abundance of transcripts linked to carcinogenesis, in particular within colonic organoids. Remarkably, WNT-pathway activation by Apc deletion fully protects organoids against BRAFV600E-induced disintegration and confers growth/niche factor independence. Still, Apc-deficient BRAFV600E-mutant organoids remain sensitive toward the MEK inhibitor trametinib, albeit p53R172H confers partial resistance against this clinically relevant compound. In summary, our systematic comparison of the response of small and large intestinal organoids to oncogenic alterations suggests colonic organoids to be better suited to model the human situation. In addition, our work on BRAF-, p53-, and WNT-pathway mutations provides new insights into their cooperation and for the design of targeted therapies.


Establishment of organoids
The proximal small intestine or colon were isolated, flushed with ice-cold PBS and opened longitudinally. In the small intestine, the villi were scraped off using a glass slide. Afterwards, the intestine was put into a falcon tube containing 20 ml ice-cold PBS and vortexed for 5-10 sec. This washing was repeated 5-10 times until the supernatant was almost clear. The intestine was subsequently incubated in 30 ml ice-cold crypt isolation buffer (small intestine: 2 mM EDTA in PBS for 30 min; colon: 15 mM EDTA in PBS for 1 h) at 4 °C on a rotating mixer. The intestine was placed into 20 ml ice-cold PBS, inverted 10 times and then transferred into a fresh falcon tube with 10 ml ice-cold PBS and shaken about 10 times (= supernatant 1).
Subsequently, the intestine was transferred into fresh 10 ml ice-cold PBS and shaken again. The crypt releasing procedure was repeated 10 times and each supernatant was inspected under the microscope. The crypt containing fractions were pooled, centrifuged at 100 x g for 10 min and the supernatant was discarded. Afterwards, the crypt pellet was resuspended with 10 ml Advanced DMEM/F12 containing 2 mM Lglutamine, 10 mM HEPES, 100 U/ml penicillin and 100 µg/ml streptomycin (all from PAN-Biotech GmbH), passed through a 70-µm cell strainer and collected in a 1% BSA-coated falcon tube. The crypts were spun down at 300 x g for 5 min, resuspended with Matrigel (Corning) and seeded in 50 µl onto a pre-warmed 24-well culture dish. After the Matrigel had solidified, the crypts were overlaid with 500 µl complete crypt culture medium (Advanced DMEM/F12 with 2 mM L-glutamine, 10 mM HEPES, 100 U/ml penicillin, 100 µg/ml streptomycin, 1xN2, 1xB27 (both from Thermo Fisher), 81.5 mg/ml N-Acetylcystein (Sigma-Aldrich)), supplemented with 100 ng/ml murine recombinant Noggin, 500 ng/ml human recombinant R-Spondin1, 50 ng/ml murine recombinant EGF (all from Peprotech) and 10 µM Rho kinase inhibitor Y27632 (TOCRIS). For colonic crypts 50 ng/ml recombinant murine Wnt3a (Peprotech) and 2.5 µM GSK3i CHIR99021 (Cayman Chemical) were added.
Medium was changed every 3-4 days and crypts were passaged once a week to remove dead cell debris.
Cre mediated recombination of Braf floxV600E and Apc flox was assessed by genomic PCR using primers that were described previously [1,2] and Cre mediated recombination of Trp53 LSL-R172H/+ was assessed using the following primers: 5'- MTT staining of organoids Organoids were incubated with 500 µg/ml MTT solution diluted in DMEM/F12 for 2 h at 37 °C.

RNA-sequencing
For the RNA-Seq of small intestine (SI) and colon (COL), RNA was extracted from freshly isolated SI and COL crypts of four (two female and male, respectively) mice. Enter token avgfcaecltkpbmf into the box.
To review GEO accession GSE132546: Go to https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE132546 Enter token mlyzwysyltuhfoj into the box TCGA data RNA-Seq raw counts were downloaded from TCGA using TCGAbiolinks R package [9]. BRAF V600E mutant colorectal adenocarcinoma were selected (n=54), as well as healthy colon samples (n=41). Counts were converted into log2 count per million (CPM) for further analysis. GSEA analysis was performed on the average log2 foldchange between COAD and healthy colon using fgsea R package.   (a) Induction of Trp53 R172H/+ neither leads to disintegration of SI nor COL organoids,
Scale bar: 50 µm. (e) Choleratoxin (CTX; 0.1 µg/ml) was used to assess the integrity of tight junctions in spheroid culture, as this toxin triggers ion mediated water efflux into the spheroid lumen [11]. Only if tight junctions are sealed, the lumen will expand in response to CTX. In line with our previous data showing that BRAF V600E impairs the seal of tight junctions in human CRC cell line spheroids [10,12], we demonstrate that CTX triggers luminal expansion in control and p53 R172H expressing SI and COL organoids but not in those expressing BRAF V600E . This indicates that BRAF V600E impairs epithelial sealing.
(f) IF staining for Lysozyme C in SI organoids. The niche of SI stem cells is organized by Paneth cells, which supply the crypt base with growth factors and can be visualized by lysozyme expression [13]. In agreement with the loss of morphologically defined crypts and the disappearance of Paneth cells in bright-field micrographs (Fig. S1d), lysozyme positive Paneth cells were almost completely absent in organoids expressing BRAF V600E . This suggests that Paneth cells are lost upon BRAF V600E expression, which agrees with previous findings that ERK promotes Goblet cell generation at the expense of Paneth cells [14].       Heatmap of the log2 fold-changes (4-HT induced vs. non-induced, color coded) of the genes listed in the EMT hallmark gene set.

Fig. S7. Braf V600E/+ ,Trp53 R172H/+ -mutant COL organoids show enhanced invasive morphology.
Bright-field images at a higher magnification of the invaded organoids shown in Fig. 4 G/H. Double-mutant COL organoids present with faster and increased attachment to the plastic surface and with pseudopodia formation (marked with white arrows).
Time lapse videos (every 4h) of SI (Video S1) and COL (Video S2) organoids at days 1-6 after oncogene induction. Note the fast proliferation of mutant organoids, especially of COL organoids in video supplement 2. Scale bars: 250 µm.

Video S3.
Time lapse videos (every 1 h) of COL organoids with the indicated genotypes at days 2-5 after oncogene induction. Scale bars: 250 µm.
Potential marker for tumor differentiation/neoplastic change and significant association with prognostic factors such as tumor grade, tumor stage and tumor location [30][31][32]34].

CAV1 upregulated
Increased expression in high grade tumors with greater depth of invasiveness [35].

EPHA2 upregulated
High mRNA and protein expression are associated with poor overall survival in stage II/III CRC (shown using a CRC microarray dataset and IHC) [39]. High expression correlates with metastatic spread [40].
Poor prognostic marker in stage II/III CRC and EPHA2targeted agents as potential treatment strategy [39].

ITGB1 upregulated
High expression in advanced tumors and liver metastases as shown by IHC. High expression correlates with shortened OS and DFS [41].
Mucinous CRCs often present at an advanced stage and correlate with poor survival [43,44].

MYOF upregulated
Overexpressed in human CRC as shown by IHC staining and TCGA gene expression data sets [45].
High expression is associated with lower survival in CRC patients [45].

NR2E3 downregulated
No data for CRC so far. Low expression in ERαpositive breast cancer patients associated with worse recurrence-free survival [46]. Low expression in liver tumors as assessed by IHC [47].
OLFM4 downregulated IHC and proteomic approaches show upregulation of OLFM4 in adenoma and early CRC stages compared to normal epithelium. There is no difference between OLFM4 expression in stage III/IV CRC vs. normal tissue [48,49].
Reduced OLFM4 (also known as hGC-1) expression as potential marker for poor differentiation and malignant progression of CRC [49].