Abstract
Carcinogenesis results from the sequential acquisition of oncogenic mutations that convert normal cells into invasive, metastasizing cancer cells. Colorectal cancer exemplifies this process through its well-described adenoma–carcinoma sequence, modeled previously using clustered regularly interspaced short palindromic repeats (CRISPR) to induce four consecutive mutations in wild-type human gut organoids. Here, we demonstrate that long-term culture of mismatch-repair-deficient organoids allows the selection of spontaneous oncogenic mutations through the sequential withdrawal of Wnt agonists, epidermal growth factor (EGF) agonists and the bone morphogenetic protein (BMP) antagonist Noggin, while TP53 mutations were selected through the addition of Nutlin-3. Thus, organoids sequentially acquired mutations in AXIN1 and AXIN2 (Wnt pathway), TP53, ACVR2A and BMPR2 (BMP pathway) and NRAS (EGF pathway), gaining complete independence from stem cell niche factors. Quadruple-pathway (Wnt, EGF receptor, p53 and BMP) mutant organoids formed solid tumors upon xenotransplantation. This demonstrates that carcinogenesis can be recapitulated in a DNA repair-mutant background through in vitro selection that targets four consecutive cancer pathways.
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Main
Colorectal cancer (CRC) remains one of the deadliest cancers1. CRC cases are divided into two major genetic instability categories; 85% of sporadic CRCs display chromosomal instability (CIN), while 15% of CRCs are hypermutated and display microsatellite instability (MSI)2. CIN tumors harbor combinations of common driver mutations in APC, TP53, KRAS, SMAD4 and PIK3CA (ref. 3). Histologically, CRC develops through a well-described adenoma–carcinoma sequence, known as the ‘Vogelgram’ (ref. 4), which coincides with a relatively ordered acquisition of mutations in the previously mentioned genes. Hypermutated MSI CRC occurs in the context of the hereditary Lynch syndrome (LS), which is caused by germline mismatch repair (MMR) gene mutation, or as high-MSI (MSI-H) sporadic CRC caused by somatic MMR silencing typically associated with the BRAFV600E mutation5,6. Of note, the oncogenic driver mutations found in MSI-H CRC (ACVR2A, TGFBR2, AXIN1, AXIN2, BMPR2 and BRAF) tend to be different from those in CIN CRC3,7,8,9,10,11,12.
Recently, we and others introduced CRC driver genes into normal human colon organoids through clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 genome editing and demonstrated that combined APC, SMAD4, TP53, KRAS and/or PIK3CA mutations transform colon epithelial organoids into full-blown cancer cells13,14. Indeed, subsequent in vivo experiments showed that the combination of these mutations enabled the modified organoids to metastasize to distant organs15. These data demonstrated that the sequential introduction of CRC driver mutations into human wild-type (WT) colon organoids does recapitulate the proposed Vogelgram of sequential histological changes toward CRC. While four different pathways (Wnt, epidermal growth factor (EGF), bone morphogenetic protein (BMP) and p53) were specifically targeted by CRISPR constructs, mutant organoids could be selected by removing the corresponding components from the medium (Wnt, R-spondin 1, EGF or the BMP inhibitor Noggin) or by adding the small-molecule p53 inhibitor Nutlin-3 (Nut3)13,14. These findings indicated that the carcinogenic CRC process exploits niche growth factor dependencies to transform normal cells into cancer cells. As said, the genes typically mutated in CIN cancers are less frequently altered in MMR-deficient hereditary CRCs and MSI-H hypermutated CRCs, while these MMR-deficient CRCs are known to harbor specific genetic mutations such as ACVR2A, TGFBR2, RPL22, RNF43, AXIN1, AXIN2 and BRAF mutations3,7,8,9,10,11,12,16.
Previously, we generated MMR-deficient human colon organoids with knockout (KO) of the MLH1 gene and demonstrated that short-term in vitro culture could recapitulate the mutational signature of MMR-deficient CRCs17. MLH1, MSH2, MSH6, PMS1 and PMS2 have been identified as predisposing MMR pathway genes18. Of these, germline mutations in MLH1 and MSH2 are correlated with the highest risk of developing CRC in LS19, while MLH1 promotor hypermethylated silencing is the key mechanism for MMR-deficient hypermutated CRC20,21. We, therefore, chose to exploit the MLH1-mutant background for our study. Here, we build on this experimental approach by selecting spontaneous oncogenic mutations of MLH1-mutant organoids by removing the niche factor components Wnt, R-spondin 1, EGF and Noggin from the organoid culture medium or by adding the small-molecule p53 inhibitor Nut3. Thus, this study asks whether simple growth factor-based selection of MMR-deficient (yet otherwise WT) organoids would allow the derivation of full-blown colon cancer cells in vitro, potentially mimicking features of MSI-related CRCs.
Results
Wnt deprivation selects AXIN1-mutant and AXIN2-mutant organoids
On the basis of our previous study, we knew that MLH1KO human colon organoids recapitulate the MMR-deficient phenotype in vitro and accumulate substantial numbers of substitutions and insertion or deletions (indels) during in vitro culture17. Withdrawal of Wnt3a and R-spondin 1 from the culture medium allowed us previously to select CRISPR-induced APC inactivation mutant organoids13,22. Here, we attempted to similarly select Wnt signal-independent MLH1KO human colon organoids, yet without specific gene targeting (Fig. 1a). As expected, WT and MLH1KO organoids rapidly died after single-cell seeding in a medium lacking Wnt3a and R-spondin 1 (−WR). This allowed us to recover rare MLH1KO subclonal cells (MLH1KO-1) from an MLH1KO organoid line that was continuously cultured for ~350 days after single-cell cloning. No such subclones could be derived from two other MLH1 mutation clones derived from the same donor (MLH1KO-2 and MLH1KO-3) (Fig. 1b,c). No Wnt-independent subclones could be isolated from the MLH1KO-1 line at earlier time points (30, 100 and 200 days). Single surviving organoids were manually picked and expanded for downstream analysis (Fig. 1d). These organoid clones (MLH1KO-1−WR-1 and MLH1KO-1−WR-2) presented as dense and budding structures when cultured in −WR medium, whereas APCKO organoids are reported to display a much more cystic structure23,24. Similar selections were performed using media designed to inhibit or activate key pathways of the stem cell niche (EGF receptor (EGFR), BMP and p53). Despite the limited initial clonal outgrowth, we could not establish long-term expanding cultures under these conditions, suggesting that no mutations affecting these pathways were present at this stage (Extended Data Fig. 1a,b). The emergence of surviving clones in the −WR condition was also observed in the MLH1KO clonal line derived from another donor, which indicated this to be a reproducible phenomenon (Extended Data Fig. 1c,d).
a, Strategy to select the survived organoids with deprivation of Wnt pathway factors. Organoids were dissociated into single cells and then 200,000 single cells were cultured in niche-factor-deprived medium to select mutated stem cells (red) out from WT stem cells (blue) and differentiated cells (white). W, Wnt3a; R, R-spondin 1; N, Noggin; E, EGF. Surviving clone organoids were expanded briefly for WGS and WES analysis. Accumulated mutations were determined by subtracting the mutations in the original clone (blue) from the mutations in the selected subclone (red). b, WT organoids and MLH1KO-1 organoids grew in complete medium (WRNE) but some of organoids grew out only from MLH1KO-1 organoids in −WR medium (representative pictures from n = 3 clonal organoid lines). Scale bars, 500 µm. (c) Quantification of survived organoids in −WR medium. Mean and s.d. (error bars) of n = 4 clonal organoid lines. P values (two-sided Welch’s t-test): MLH1KO-1 versus MLH1KO-2, P = 0.0001; MLH1KO-1 versus MLH1KO-3, P = 0.0002. d, Single survived organoids (MLH1KO-1−WR-1 and MLH1KO-1−WR-2) were expanded and passaged in −WR medium (representative pictures from n = 3 independent replicates). Scale bars, 500 µm. e, The Venn diagrams illustrate the number of base substitutions and indels found in WGS data of survived clones (MLH1KO-1−WR-1 and MLH1KO-1−WR-2). AXIN1 and AXIN2 candidate gene mutations are listed in the overlapping section. The numbers indicate nonsynonymous mutations and the numbers in brackets indicate all mutations found in WGS. f, RT–qPCR analysis of AXIN2 mRNA levels. AXIN2 transcripts were compared between each clone cultured in the medium with or without Wnt3a and R-spondin 1. Mean and s.d. (error bars) of n = 3 biologically independent experiments. P values (two-sided Welch’s t-test): MLH1KO-1 versus MLH1KO-1−WR-1 in −WR medium, P = 0.0048; MLH1KO-1 versus MLH1KO-1−WR-2 in −WR medium, P = 0.0037. g, Representative images of Sanger DNA sequencing of AXIN1 and AXIN2 mutations in Wnt-independent clones (MLH1KO-1−WR-1 and MLH1KO-1−WR-2).
The selected subclones (MLH1KO-1−WR-1 and MLH1KO-1−WR-2) were subjected to whole-genome sequencing (WGS) analyses. The genomic sequence of the parental MLH1KO-1 clone allowed us to subtract somatic mutations that were already present in the parental clone (Fig. 1a). As expected from our previous study, surviving clones accumulated single-base substitutions (SBSs) (8,748 ± 149 mutations) and indels (23,010 ± 110 mutations), which recapitulated the mutator phenotype of MSI-H tumors with high tumor mutational burden (TMB) (Fig. 1e). The number of actual mutations and the previously calculated mutation number per genome division allowed us to calculate an estimated culture duration (354 ± 69 days)17. This matched our actual culture duration (417 days) well. The clonality of selected organoid clones was confirmed by variant allele frequency analyses (Extended Data Fig. 2). We compared the mutations acquired by the two surviving clones to the parental MLH1KO clone. Among 6,372 base substitutions and 18,479 indels shared in two clones, we identified 74 base substitutions and 81 indels as nonsynonymous somatic coding mutations (Supplementary Table 1). Among those listed mutations, we found two compound heterozygous AXIN1 mutations (c.1051C>T, p.Gln351* and c.1523 del, p.Gly508fs*197) and a homozygous AXIN2 mutation (c.1994 del, p.Gly665fs*) carried in both subclones (Fig. 1e). These mutations are reported as hotspot mutations in the cBioPortal and in the Catalog of Somatic Mutations in Cancer (COSMIC) database as cancer-related somatic variants (COSM8405446, COSM1240884 and COSM1385326) (Extended Data Fig. 3). Biallelic loss-of-function mutations of both AXIN1 and AXIN2 lead to Wnt pathway activation and occur in CRC25. Indeed, a recent report confirmed that CRISPR KO of AXIN1 of human intestinal organoids allows growth in the absence of Wnt and R-spondin 1, similar to APCKO organoids24. In contrast, the common co-occurrence of AXIN2 mutations with AXIN1 and other Wnt pathway mutations suggests that weak pathway activation caused by AXIN2 loss of function may synergistically upregulate Wnt signal26,27,28.
We performed a medium selection experiment comparing the AXIN-mutant subclones with organoids that were CRISPR-engineered to carry mutations in other driver genes. This confirmed that organoids with AXIN1 and AXIN2 mutations upon MLH1KO acquire the same Wnt signal independency as APC-mutant organoids (Extended Data Fig. 4). Wnt pathway activation was further confirmed by reverse transcription (RT)–qPCR analysis for the Wnt target gene AXIN2, as AXIN2 was expressed at higher levels in the selected clones (MLH1KO-1−WR-1 and MLH1KO-1−WR-2) than in the WT and MLH1KO clone without Wnt3a and R-spondin 1 (Fig. 1f). The SBS in AXIN1 p.Gln351* (GCA>GTA) matched the MMR-linked mutational signature SBS 20 observed in MLH1KO organoid17,29. Both AXIN1 p.Gly508fs* and AXIN2 p.Gly655fs* result from single-nucleotide deletion in homopolymer cytosine repeats, representative of the mutation pattern known as slippage in MMR-deficient and MSI-H tumors (Fig. 1g)8,30. Importantly, these types of AXIN1 and AXIN2 mutations are reported in MSI-H CRCs and gastric cancers7,8,9,30,31. Thus, MLH1KO organoids allowed the faithful selection of some of the specific mutations seen in MMR-deficient and MSI-H tumors in in vitro culture.
Sequential mutant selections gain niche independence
Because these observations indicated that functional selection of spontaneous oncogenic mutations in MLH1KO organoids allowed us to model MMR-deficient tumorigenesis in vitro, we performed sequential selection of these mutant clones (MLH1KO-1−WR-1 and MLH1KO-1−WR-2) in −Noggin, −EGF +gefitinib (−EGF+Gef) or +Nut3 medium. We, thus, hoped to find BMP pathway mutations, Ras and Raf pathway mutations and TP53 mutations, respectively. Surviving organoids readily grew out from the MLH1KO-1−WR-1 clone in the −Noggin condition. In contrast, only one organoid survived and grew into a large budding organoid from the MLH1KO-1−WR-1 clone in +Nut3 medium (Fig. 2a,b). These surviving organoids were picked and clonally expanded for further genomic analyses (Extended Data Fig. 5). As expected, a missense mutation in TP53 (c.473G>T, p.Arg158Leu (Hom)) was found in the Nut3-selected clone (MLH1KO-1−WR-1+Nu) by whole-exome sequencing (WES), which was confirmed by Sanger sequencing. Aberrant p53 protein expression and increased p21 expression were confirmed by western blot (Fig. 2c). This TP53 mutation is mostly reported in lung tumors but also seen in persons with CRC (Extended Data Fig. 3)32,33.
a, MLH1KO-1−WR-1 organoid grew in −WR medium but some of organoids grew out in −WR−Noggin medium (representative pictures from n = 3 clonal organoid lines) and a single organoid grew out in −WR+Nut3 medium. Scale bars, 500 µm. All cells died out in −WR−EGF+Gef medium. b, Quantification of survived organoids in each selection medium. Mean and s.d. (error bars) of n = 4 clonal organoid lines. P values (two-sided Welch’s t-test): −WR−Noggin versus −WR−EGF+Gef, P < 0.0001; −WR−Noggin versus −WR+Nut3, P < 0.0001; −WR−EGF+Gef versus −WR+Nut3, P = 0.0002. c, Representative western blot analysis to detect p53, p21 and GAPDH in WT and MLH1KO-1 organoid lines with WT p53 (MLH1KO-1 and MLH1KO-1−WR-1) and with mutant p53 (MLH1KO-1−WR-1+Nut3) treated with DMSO or 10 μM Nut3 for 24 h. The analysis was performed with n = 2 biologically independent experiments for one organoid clone. d, MLH1KO-1−WR-1+Nut3 organoids were selected in −WR+Nut3−Noggin or −WR+Nut3−Noggin−EGF+Gef medium (representative pictures from n = 3 clonal organoid lines). Scale bars, 500 µm. e, Quantification of survived organoids in −Noggin and −EGF+Gef medium. Mean and s.d. (error bars) of n = 4 clonal organoid lines. P values (two-sided Welch’s t-test). f, Mutational spectra of all base substitutions observed for each selected clone. g,h, Heat map showing the cosine similarity scores for each indicated clone and COSMIC SBS signature (g) and indel signature (h). Arrows indicate signatures associated with deficiency in DNA MMR in previous analyses29,38.
Interestingly, the Noggin-independent clones arising from the MLH1KO-1−WR-1 clone (MLH1KO-1−WR-1−N) carried ACVR2A mutations (c.285 del, p.Thr96* (Hom)) or BMPR2 mutations (c.255G>A, p.Trp85* (Het) and c.1748 del, p.Asn583fs* (Het)). SMAD4 mutation, the most common BMP pathway driver mutation in sporadic CRC, was not encountered (Extended Data Fig. 3). Another Noggin-independent clone selected from the MLH1KO-1−WR-2 clone (MLH1KO-1−WR-2−N) also carried partially the same ACVR2A (c.285 del, p.Thr96* (Het) and c.1310 del, p.Lys437fs(Het)) and BMPR2 (c.1748 del, p.Asn583fs* (Hom)) mutations. These mutations occurred through one nucleotide deletion in the polyadenine tract (ACVR2 A8, BMPR2 A11), as reported in MSI-H CRCs and recently defined as a representative indel mutational signature for MSI (ID2 signature)3,10,12,29,34,35. In particular, ACVR2A mutations were recently found and functionally validated as driver mutations for CRC36. The BMPR2 mutation is reported to be mutually exclusive with SMAD4 in sporadic CRC34.
These double-mutant lines were selected again in additional conditions. Some surviving clones arose in −Noggin or −EGF+Gef medium from the TP53 mutated clone (MLH1KO-1−WR-1+Nu-1). Again, surviving organoids readily grew out in −Noggin medium (Fig. 2d,e). We selected two Noggin-independent clones in −Noggin medium and sent these for WES analyses (Extended Data Fig. 6). Interestingly, we confirmed that one (MLH1KO-1−WR-1+Nu−N-1) acquired duplication of a somatic mutation in ACVR2A (c.285 del, p.Thr96*(Hom)) and the other one (MLH1KO-1−WR-1+Nu−N-2) acquired compound heterozygous mutations in ACVR2A (c.285 del, p.Thr96*(Het) and c.1310 del, p.Lys437fs). As the earlier clone (MLH1KO-1−WR-1+Nu-1) harbored a single ACVR2A (c.285 del, p.Thr96*) mutation, this means that the clone obtained another ACVR2A mutation during the selection step, resulting in loss of heterozygosity (LOH). Of note, this clone did not carry the BMPR2 homozygous mutations observed in the previously selected Noggin-independent clones (MLH1KO-1−WR-1−N, MLH1KO-1−WR-2−N), suggesting that the clone acquired Noggin independency solely through LOH of the ACVR2A gene. This was consistent with the previous findings identifying ACVR2A as a driver gene in the transforming growth factor-β (TGFβ) pathway36 and reporting the BMPR2 as contributing cooperatively to the development of sporadic CRC but not as single driver mutation10,34,37. We again extracted mutational signatures from each selected clone. All clones showed the expected mutational spectra (Fig. 2f)17. We next extracted mutational signatures for comparison to the COSMIC database, using cosine similarity as a measure of closeness29,38. All selected organoids showed mutational signatures that resembled signatures SBS6, SBS15, SBS20 and SBS44, which are associated with defective DNA MMR (d-MMR) and occur in microsatellite unstable tumors38 (Fig. 2g). Again, signature ID2 (representing the slippage of DNA MMR deficiency) was found in all mutant clones (Fig. 2h). These data clearly suggest that the mutational signature of d-MMR induced by MLH1 mutation remained the dominant driver of mutagenesis regardless of the accumulated genetic mutations.
Long-term cultured d-MMR organoids complete tumor progression
As early attempts to select out mutant clones carrying EGF, Ras and Raf pathway mutations failed, we extended culturing times of the triple-pathway mutant clone (MLH1KO-1−WR-1+Nu−N-1) up to 300 days to allow for the accumulation of further critical driver mutations. Recently, differential drug sensitivities of EGFR inhibition were reported among various different RAS-mutant patient-derived CRC organoids (PDOs). The pan-human EGFR inhibitor afatinib (Afa) can eliminate ERK activity oscillations in KRAS-mutant PDOs39,40. To select Ras and Raf pathway mutant clones more reliably, we used Afa as an alternative EGFR inhibitor in addition to Gef. Some organoids grew out in −EGF+Gef or −EGF+Afa conditions (Fig. 3a). These surviving organoids could be continuously expanded under both −EGF+Gef and −EGF+Afa conditions (Fig. 3b).
a, MLH1KO-1−WR+Nu−N organoids were selected in −WRN+Nu−EGF+Afa/Gef medium (MLH1KO-1−WR+Nu−N−E+Afa/Gef). Outgrowing clones are shown in the inset. Representative pictures from n = 3 independent replicates. Scale bars, 500 µm. b, Survived MLH1KO-1−WRN+Nu−EGF+Afa/Gef organoids were picked up and continuously expanded in each selection medium. Representative pictures from n = 3 clonal organoid lines. Scale bars, 500 µm. c, Sanger DNA sequencing result of NRASQ61K (Het) mutation in the selected clone (MLH1KO-1−WR+Nu−N−E+Afa). d, Representative western blot analysis to detect p44/42 MAPK and β-actin in NRASWT and NRASQ61K organoid lines cultured in WRNE medium or −WRNE medium + 10 μM Nut3 and 1 μM Afa. The analysis was performed with n = 2 biologically independent experiments for one organoid clone. e, Representative H&E staining and IHC analysis of E-cadherin, Ki67, β-catenin and EGFR in the parental clone (NRASWT) and the mutant clone (NRASQ61K). The analysis was performed with n = 2 clonal organoid lines. Scale bar, 100 µm. f, Dose–response curves showing the sensitivity of NRASWT and NRASQ61K clones to inhibitors of the EGFR (Afa), MEK (selumetinib) and SHP2 (SHP099). The y axis represents the viability of the samples while the x axis represents the drug concentration (power of 10 µM). Mean and s.d. (error bars) of n = 3 technical replicates for n = 2 independent organoid clones for each genetic background. g, Heat maps reporting the viability of the drug screening performed on NRASWT and NRASQ61K organoids as normalized to vehicle-treated organoids. Color mapping ranges from low viability (red) to high viability (green). The analysis was performed with n = 3 technical replicates for n = 2 independent organoid clones for each genetic background. h, Timescale phylogenic tree of the selected mutant clones that arose from the MLH1KO-1 single organoid clone. Organoids were selected under each condition (colored rectangles). Circles represent survived clones and dashed-line circles represent nonrecovered clones. Horizontal bars indicate the culture duration of cloning and expanding each clone. All mutations in each clone are listed in Supplementary Table 3.
Through WES analyses, we found that surviving clonal organoids under both inhibitor conditions (MLH1KO-1−WR-1+Nu−N−E+Afa/Gef) acquired a heterozygous somatic mutation in NRAS (c.181C>A, p.Gln61Lys(Het) COSM580) (Fig. 3c and Extended Data Fig. 6), a known oncogenic mutation in the Ras and Raf pathway3,41. While KRAS mutations are reportedly more common in low-MSI (MSI-L) relative to MSI-H CRC, the NRAS mutation is equally frequent between MSI-L and MSI-H CRC tumors42. To confirm that the selected NRASQ61K mutant organoids (MLH1KO-1−WR-1+Nu−N−E+Afa/Gef) indeed exert constitutive ERK signaling activity, transcription of ERK target genes (ETV4, ETV5, DUSP5, DUSP6 and CCND1) was compared between NRASWT and NRASQ61K mutant organoids by RT–qPCR (Extended Data Fig. 7a). Furthermore, western blot analysis confirmed that NRASQ61K mutant organoids increased phosphorylation of ERK1 and ERK2 (Fig. 3d). Taken together, we obtained quadruple-pathway mutant organoids, which sequentially acquired MSI-H mutations in AXIN1 and AXIN2 (Wnt pathway), TP53, ACVR2A and BMPR2 (BMP pathway) and NRAS (Ras and Raf pathway) and, thus, became completely independent from the pertinent niche factors. Both triple-pathway and quadruple-pathway mutant organoids were highly proliferative and, as indicative of an active Wnt signaling pathway, accumulated expression of nuclear β-catenin (Fig. 3e). The quadruple-pathway mutant organoids appeared as more compact/less cystic structures as seen in our previous CRISPR-engineered quadruple-pathway mutant organoids13. To investigate the acquisition of drug resistance in the quadruple-pathway mutant organoids, we performed drug screening with various pathway inhibitors. We, thus, observed the expected sensitivity of NRASWT organoids toward Afa and Gef and the resistance of NRAS-mutant organoids. In contrast, the mutant organoids remained sensitive to the MEK inhibitors selumetinib and trametinib, the mTOR inhibitor everolimus, the PI3Kα inhibitor alpelisib and the multiple-CDK (cyclin-dependent kinase) inhibitor dinaciclib (Fig. 3f,g and Extended Data Fig. 7b–d). Recently, Ras status was reported to correlate with SH2 domain-containing phosphatase 2 (SHP2) inhibitor sensitivity in several cancers; in particular, the NRASQ61K mutation in neuroblastoma confers resistance to SHP2 inhibitors43,44. Consistent with this, our NRASQ61K mutant organoids exhibited resistance to SHP2 inhibitor SHP099 treatment (Fig. 3f,g). We constructed a mutant phylogenic tree for each sequential mutant clone (Fig. 3h and Supplementary Table 3). The acquisition of identical mutations was independently observed in different clones. The available data indicated that these recurrent mutations arose independently yet were because of a shared mutational mechanism. Taken together, simple selection in an MLH1KO mutant organoid background allowed the sequential isolation of spontaneous quadruple-pathway mutant organoids, starting from a single MLH1KO stem cell, over a period of no more than 30 months.
MSI status and mutational tendency in d-MMR organoids
To assess the MSI profile of our MMR-deficient organoids, we compared the somatic mutations acquired in our quadruple-pathway mutant organoids to those of CRCs from The Cancer Genome Atlas (TCGA) database (TCGA-COAD). Leveraging two bioinformatic MSI predicting tools, MSMuTect and MANTIS45,46,47, we derived MSI scores from WES data. Notably, MANTIS enabled the extraction of TCGA-COAD samples annotated as MSI-H and microsatellite-stable (MSS), facilitating group-specific comparisons. In the MANTIS analysis, the MSI scores of the quadruple-pathway mutant organoids mirrored those of the MSI-H group, significantly diverging from the MSS counterparts (Fig. 4a). This observation clearly indicated that MLH1KO mutant organoids recapitulated the phenotype of MMR-deficient colorectal tumors. Furthermore, to evaluate the fidelity of the MSI-H tumor landscape in the MMR-deficient organoids, we checked whether the frequent microsatellite loci48 of MSI-H cancers were targeted in our organoids. As expected, culturing of the mutant clones over time led to increased numbers of somatic mutations in the microsatellite loci found in MSI cancers (Fig. 4b). We then calculated the expected probability for acquiring specific mutations in in vitro cultured MLH1KO organoids, taking into account the specific mutational signatures and the kinetics of mutation accumulation. First, by analyzing all mutational signatures in collected mutant clones, we visualized the ‘average’ mutational signature as the mutational frequency in 96 channels (Fig. 4c). The mutational signature of the mutant clones was compared to the mutational signatures of in vitro cultured organoids and in vivo tissue derived from small intestine and colon, as previously reported (Fig. 4d)49. Next, candidate mutations known as driver mutations in the Ras, Raf, MAPK, PI3K and ErbB pathways were listed from the previous report in MSI-H cancers50. The relative probability of occurrence in each specific mutation was calculated from the average mutational signature derived from the previous approach51 and compared to the mutational rate in normal intestinal tissue and organoids (Fig. 5). As expected, KRASG12D and KRASG12V, known as the most frequent mutations in the Ras pathway, were unlikely to occur in an MMR-deficient background. BRAF mutations, which represent the most common mutations of the Ras and Raf pathway in sporadic MSI-H, were also calculated as being of low probability, which is consistent with a previous report that BRAFV600E mutation frequency is low in MMR mutation carriers3,5,52. Selection in −EGF+Afa medium resulted in NRASQ61K mutation despite being previously reported as of lower probability albeit detected in MMR-deficient CRCs53. In contrast, the high relative probability of AXIN1Q351fs corresponded to our selection result (described above). The TP53R158L mutation found in our clone showed a relatively low probability, corresponding to the occurrence of a single surviving clone in the Nut3 selection step (Fig. 2a,b). The report of TP53R158L mutations specifically in carcinogenesis related to inflammatory bowel disease suggests that the TP53 mutation is influenced by various backgrounds and mutational signatures32. Despite the low probability of TP53 mutations in our MLH1KO mutant organoids, we were able to select a TP53 missense mutation. Together, these data suggest that driver mutations inferred from the mutational signature are acquired with high probability while other oncogenic mutations with relatively low probability can also be acquired (albeit at lower frequency), shaping the genetic diversity of MSI-H CRCs.
a, MANTIS scores were calculated in the mutant organoid clones (n = 8 samples) and MSI-H (n = 38 samples) and MSS (n = 38 samples) of TCGA-COAD. We used the stepwise difference threshold set by MANTIS, which defines a score above 0.4 as MSI-H (unstable) and below this threshold as MSS (low) using TCGA-COAD. MSMuteTect2 scores were also calculated in the mutant organoid clones (n = 8 samples) and all samples used in TCGA as MSMuTect2 (n = 262 samples). The minimum values are the smallest number per MANTIS and MSMuTect2 scores. Sample numbers are written below each group. The first quartile above the whiskers represents the data point that separates the lowest 25% of the data from the rest. The center line per box plot represents the median value among the data points. The third quartile just on top of the box plot separates the lowest 75% of the data points from the highest 25%. The maximum value represents the largest score of each tool. b, MSI mutations (pink, mutated) harbored in each mutant organoid clone (columns). The top 50 most differentially unstable microsatellite loci (rows) were selected from the previous study48. c, Mutational spectra of all base substitutions observed for each selected clone. d, The average mutational signature of the mutant clones extracted from c are shown on top (samples). The mutational signatures of in vitro cultured organoids and in vivo tissue derived from the small intestine (SI) and colon (Col) were referred from the previous result49.
Relative risk of frequent driver mutations for Ras, Raf, MAPK, PI3K and ErbB pathways listed from the previous report in MSI-H cancers50. Each mean relative risk was calculated from mutational signatures of normal human colonic organoid (C in vitro, n = 1), normal human colonic tissue (C in vivo, n = 21), normal human small intestinal organoid (SI in vitro, n = 6), normal human small intestinal tissue (SI in vivo, n = 14) and the average mutant organoids (samples, n = 12). Left, SBS mutations found in the mutant organoids (AXIN1Q351* and TP53R158L) are shown together. Data are presented as mean values ± s.d.
Xenotransplantation of quadruple-pathway mutant organoids
To characterize the molecular features of the sequential mutant organoids, we compared the gene expression patterns for each mutated driver pathway. Given that the NRASQ61K mutant organoids (being the quadruple-pathway mutant clone) displayed niche factor independency for all four pathways, we compared gene expressions of each mutant clones cultured for 24 h in the condition that inhibited all four pathways (that is, in the absence of WRNE and in the presence of p53 activator Nut3, EGFR inhibitor Afa, Wnt antagonist Dickkopf 1 (DKK1) and BMP2 receptor ligand BMP2). Comparison of the representative gene expression patterns related to each pathway confirmed their activation by the specific gene mutations affecting each pathway in the sequential driver gene mutant clones (Fig. 6a). We then sought to investigate whether the spontaneous quadruple-pathway mutant clone was tumorigenic in vivo. To compare the tumorigenic activity between NRASQ61K and KRASG12D, the most prevalent mutation of sporadic CRCs and hypermutated CRCs in the Ras pathway, we introduced the KRASG12D activating mutation into our NRASWT triple-pathway mutant clone through previously used CRISPR–Cas9-mediated gene editing13. KRASG12D mutants were selected by withdrawing EGF and adding Gef to the culture medium (Extended Data Fig. 8a–c). Genotyping of clonally expanded organoids confirmed that the clone harbored the KRASG12D heterozygous mutation together with the acquired AXIN1, AXIN2, TP53, ACVR2A and MLH1 mutations (Extended Data Fig. 8d). We subcutaneously injected triple-pathway mutants (NRASWT), quadruple-pathway mutants (NRASQ61K), triple-pathway + KRASG12D engineered mutants and the previously engineered AKPS (APCKO;KRASG12D;TP53KO;SMAD4KO) mutants derived from the same donor13 into immunodeficient mice.
a, Heat map diagram of expression levels for each representative genes in CRC driver pathways. Columns and rows in the heat map represent mutant clones and genes, respectively. Color scale indicates fold changes in gene expression (n = 3 clonal organoid lines). b, Triple-pathway mutant organoids (NRASWT) and quadruple-pathway mutant organoids (NRASQ61K) were injected subcutaneously in NSG immunodeficient mice. Both triple-pathway mutant (NRASWT) and quadruple-pathway mutant (NRASQ61K) organoids developed visible nodules. NRASQ61K organoids grew as tumors with vasculature in the subcutaneous lesion. Representative pictures from n = 6–12 tumors. Scale bars, 5 mm. c, Representative images of extracted tumors of NRASWT organoids (top) and NRASQ61K organoids (bottom). Representative pictures from n = 6–12 tumors. d, Tumor size measurement of xenografts at 8 weeks after xenotransplantation. Mean and s.e.m. (error bars) of n = 6–12 tumors. P values (two-sided Welch t-test): N-1 NRASWT versus N-1 KRASG12D, P = 0.0023; N-2 NRASWT versus N-2 NRASQ61K 1, P = 0.0473; N-2 NRASWT versus N-2 NRASQ61K 2, P = 0.0334; N-2 NRASQ61K 2 versus N-1 KRASG12D, P = 0.2812. e, Representative H&E stain, hNA, E-cadherin and Ki67 immunostaining on nodules isolated from triple-pathway mutant (NRASWT) and quadruple-pathway mutant (NRASQ61K) injected mice. Triple-pathway mutant organoids did engraft but remained thin with a cystic structure of organoids, similar to adenoma features. Quadruple-pathway mutant organoid-derived tumors showed a thick and irregular multilayered epithelium. The analysis was performed on n = 3 tumors. Scale bars, 100 mm.
After 8 weeks, most of the mice injected with quadruple-pathway mutants, triple-pathway + KRASG12D engineered mutants and AKPS-mutant organoids developed visible nodules. By contrast, some of the mice injected with triple-pathway mutants showed very small nodules. Compared to tumors of triple-pathway mutants, quadruple-pathway mutant tumors were larger and well vascularized (Fig. 6b,c). Tumor size analysis confirmed that some of the triple-pathway organoids engrafted but remained small, while the quadruple-pathway mutant clones had a higher rate of tumor formation and larger tumor size. No significant differences were found between KRASG12D engineered mutant clones and the NRASQ61K mutant clones (Fig. 6d). Histological analysis revealed that triple-pathway tumors grew as thin, cystic organoids encapsulated in the injected subcutaneous region, while quadruple-pathway tumors were larger and all exhibited an irregular multilayered epithelium. The tumor origin was confirmed using a human-specific nuclear antigen (hNA) (Fig. 6e). These data implied that MLH1KO organoids carrying quadruple-pathway mutations (Wnt, EGFR, p53 and BMP) form malignant tumors upon xenotransplantation. Notably, a comparison of growth rates during in vitro culture confirmed no apparent difference between AKPS-mutant organoids and the quadruple-pathway mutant organoids (Extended Data Fig. 9a,b). The AKPS organoid line was engineered to carry the strongest mutations in each of the four pathways, while the quadruple-pathway mutant MLH1KO carries much weaker versions. We do not know how each of these mutations contributes to the observed in vivo growth difference, yet they apparently add up to a notably more malignant phenotype of the AKPS organoids upon transplantation.
Discussion
Here, we apply long-term culture of MLH1KO organoids with sequential clonal selection for CRC driver mutations using niche factor deprivation in an attempt to obtain spontaneous tumor organoids in vitro. Previously, we demonstrated that CRISPR-mediated KO of the MLH1 gene in human colon organoids recapitulates the mutational signature of MMR-deficient tumors even in short-term culture17. We now extend this experimental approach by using MMR-deficient organoids in long-term culture to recapitulate the acquisition of CRC driver mutations by clonal selection using niche factor selection. Organoid culture is now a well-established method for the robust culture of normal human epithelial stem cells in vitro, which typically remain genetically stable during prolonged culturing54. On the other hand, tumor organoids retain the genetic mutations and phenotypic characteristics of the original tumor55,56. Having said this, SBSs and indels stochastically occur in human intestinal organoids, just as these occur in human adult stem cells over time17,49.
MLH1KO intestinal organoids acquire mutations at an accelerated rate compared to normal stem cells and the resulting mutation spectrum mimics the characteristic mutational signature seen in MSI-H CRCs17. As mutations accumulate at a considerably faster rate than in normal tissues, this suggests that MLH1KO organoids can be used as an in vitro model for MSI-H tumorigenesis. After nearly 1 year of in vitro culture, we were able to isolate clones with oncogenic mutations in AXIN1 and 2, negative regulators of the Wnt pathway. Such mutations are generally considered to establish the first step in colon tumorigenesis. Further selection identified oncogenic mutations in ACVR2A and BMPR2 upon BMP inhibition, TP53 upon Nut3 selection and NRAS upon EGF and Ras pathway inhibition. All observed mutations fit the MMR-deficient mutational signature and the single-base slippage patterns, as these occur in MSI-H tumors. These results, thus, imply that MLH1KO organoids faithfully recapitulate the specific mutational patterns of MMR-deficient and MSI-H tumors. Organoid culture is based on the in vitro recapitulation of stem cell niche factor conditions and it has been shown previously that tumor organoid culture condition reflects independency from the niche factors13,14,55,57. Consistent with these reports, quadruple-pathway mutant clones isolated in this study were tumorigenic in vivo upon subcutaneous transplantation.
Our approach has some limitations. As only pure epithelial cell components can be evaluated in tumor epithelial organoids, the organoid culture system used in this study is limited in its ability to replicate the tumor microenvironment or high neoantigen immune response observed in MSI-H tumors. T cell infiltration in the rectal mucosa of persons with LS was recently reported to shape carcinogenesis regardless of mutation burden58. Indeed, the abnormal immune environment in LS was proposed to represent the initial step of MMR-deficient cancer59. Because of the time and scale constraints in our experimental scheme, we were not able to assess different combinations of driver mutations or various patterns of mutational acquisition order. However, the conservation of the d-MMR mutation signature in all progeny clones of MLH1-mutant organoids suggests that the d-MMR mutation signature derived from MLH1 mutation is strongly retained as the mutation signature shaping the tendency to acquire subsequent mutations, regardless of accumulated genetic mutations, suggesting that the mutation signature, rather than the acquired gene mutation, is the strongest factor influencing the propensity to acquire mutations. Indeed, a recent report showed that long-term culturing of TP53-deficient gastric organoids leads to a CIN phenotype60, but not to the sequence changes observed in the current study. Our approach is not easily generalizable to other tumor types. CRC appears relatively unique in that the four most commonly mutated pathways can be easily and specifically selected for: Three of these pertain to growth factor dependencies (Wnt, EGF and BMP), while Nut3 allows for selection of mutations in the fourth pathway (TP53).
To our knowledge, this study provides the example of the derivation of full-blown cancers from WT cells, simply by providing selective pressure for four well-defined oncogenic pathways. We chose to perform our study in an MLH1-mutant background, which has an increased rate of accumulation of genetic mutations and is well known to confer CRC predisposition. Similar approaches with WT cells or with organoids carrying other cancer predisposing mutations will likely yield similar results.
Methods
Ethics statement
The study was approved by the UMC Utrecht and Diakonessenhuis ethical committee (TC-Bio 12-093) and was in accordance with the Declaration of Helsinki and according to Dutch law. This study is compliant with all relevant ethical regulations regarding research involving human participants.
Animals
All mouse experiments were conducted under a project license (AVD 8010020209924) granted by the Central Committee Animal Experimentation of the Dutch government and approved by the KNAW–Hubrecht Institute Animal Welfare Body (HI 21.39.04). NOD scid gamma (NSG; NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ; 8 weeks old) mice were used in the study. The mice were kept in a constant-temperature environment of 21 °C (40–60% humidity) with a natural day–night light cycle in a conventional animal colony with free access to food and water. All mice were housed in a pathogen-free vivarium in sterile, disposable microisolator cages and fed a sterile, irradiated diet with free access to sterile, irradiated water.
All the mice were monitored and weighed once a week. Mice with symptoms of severe discomfort (for example, >20% weight loss compared to beginning or >15% weight loss in 2 days) with tumor burden or with measured tumors larger than 1,000 mm3 were humanely killed. In this study, three mice in the subcutaneous transplantation experiment were killed with the estimation of exceeded maximal tumor burden at an earlier time point. As tumor size was weekly checked by manual palpation, it was only possible to estimate the size of the tumors ahead of killing. Indeed, we noted that n = 1 tumor of the killed animals exceeded the size limit.
Organoid culture establishment
MLH1KO colon organoids were established from two WT human colon organoid cultures in a previous study17. These were derived from endoscopy material of a healthy donor (female, age 31 years)15 and from the normal tissue of a resected colon segment derived from a person (female, age 60 years) diagnosed with CRC (sigmoid)55. As the differences by sex in mutational progression were not expected, we did not consider sex or gender in our study design. Organoid culture establishment and medium conditions were described previously13. Briefly, basal culture medium (AdDMF+++) contained advanced DMEM/F12 (Invitrogen) supplemented with 10 mM HEPES, 2 mM GlutaMAX, 100 U per ml penicillin and 100 μg ml−1 streptomycin. The culture medium (WRNE) contained AdDMF+++ including B27 (Invitrogen), nicotinamide (Sigma-Aldrich), N-acetylcysteine (Sigma-Aldrich), Noggin (Peprotech), R-Spondin 1-conditioned medium (20%, produced using R-spondin 1 cells (Trevigen)), EGF (Peprotech), Wnt3a-conditioned medium (50%, produced using stably transfected L cells), TGFβ type I receptor inhibitor A83-01 (Tocris) and p38 inhibitor SB202190 (Sigma-Aldrich). Organoids were embedded in domes of Cultrex β-mercaptoethanol (BME; Trevigen) and covered by culture medium (WRNE). Established MLH1KO colon organoids were cultured in two wells of a six-well plate (480 μl of BME, approximately 160,000–200,000 cells before passaging). Organoids were passaged weekly at a 1:2 split ratio, keeping multiclonal cell diversity without selective pressure.
Organoid selection
Organoids were dissociated into single cells using TrypLE Express (Thermo Fisher Scientific) in the presence of 10 µM Y-27632 (Tocris Bioscience) for 20 min and mechanically dissociated into single cells. A total of 120,000 single cells were counted, centrifuged at 600g for 1 min, resuspended in 240 μl of Cultrex BME and seeded at low density on a prewarmed six-well culture plate (Corning). Survived organoids derived from single cells were manually picked up, resuspended in 20 μl of Cultrex BME and seeded on a 48-well culture plate (Corning). Clonally selected organoids were cultured in selection medium to confirm as they grow in that culture condition. For selection of Wnt pathway or BMP pathway mutants, organoids were grown in culture medium lacking Wnt3a-conditioned medium and R-spondin 1-conditioned medium or Noggin, respectively. For mutant p53 selection, organoids were cultured in the presence of 10 mM Nut3 (Cayman Chemical) and maintained in the presence of 1 mM Nut3. For selection of Ras and Raf pathway mutants, organoids were grown in culture medium lacking EGF and containing 1 µM Gef (Selleck Chemicals) or 1 µM Afa (Selleck Chemicals).
WGS and WES
Individual outgrowing organoids surviving the medium-based selection were manually picked and propagated as clonal lines for a couple of passages to avoid unexpected mutation accumulation before being harvested for WGS and WES. In brief, organoids were collected in ice-cold AdDMF+++, centrifuged at 400g for 3 min and washed twice in ice-cold PBS to ensure the removal of all remaining BME. The DNA was extracted using the Quick-DNA miniprep kit (Zymo research, D3024) following the manufacturer’s instructions. Then, 1 µg of DNA was used for WES performed with the Agilent SureSelect V7 kit, using 8 Gb per sample (50× average mapped) data throughput, with 150-bp paired-end sequencing on NovaSeq. Sequence reads were mapped against human reference genome GRCh37 using the Burrows–Wheeler Aligner version 0.7.17 mapping tool61 with settings ‘bwa mem -c 100 -M’. Sequence reads were marked for duplicates using Sambamba version 0.6.8 and realigned per donor using the Genome Analysis Toolkit (GATK) version 4.13.0. A description of the complete data analysis pipeline is available on GitHub (https://github.com/ToolsVanBox/NF-IAP). Raw variants were called in multisample mode using the GATK HaplotypeCaller and GATK-Queue with default settings and additional option ‘EMIT_ALL_CONFIDENT_SITES’. The quality of variant and reference positions was evaluated using GATK VariantFiltration with options ‘--filter-expression QD < 2.0 --filter-expression MQ < 40.0 --filter-expression FS > 60.0 --filter-expression HaplotypeScore > 13.0 --filter-expression MQRankSum < 12.5 --filter-expression ReadPosRankSum < 8.0 --filter-expression MQ0 R 4 && ((MQ0/(1.0 * DP)) > 0.1) --filter-expression DP < 5 --filter-expression QUAL 4.0 --filter-name SNP_LowQualityDepth --filter-name SNP_MappingQuality --filter-name SNP_StrandBias --filter-name SNP_HaplotypeScoreHigh --filter-name SNP_MQRankSumLow --filter-name SNP_ReadPosRankSumLow --filter-name SNP_HardToValidate --filter-name SNP_ LowCoverage --filter-name SNP_VeryLowQual --filter-name SNP_LowQual --filter-name SNP_SOR -cluster 3 -window 10’. To obtain high-quality somatic mutation catalogs, the somatic variant filtering tool SMuRF (https://github.com/ToolsVanBox/SMuRF) was applied. We applied postprocessing filters as described49,62,63. Somatic variants that passed all these filters were annotated with ‘PASS’ or with the respective parameter that did not meet any of the previous thresholds. All samples are listed in Supplementary Table 2.
Mutation calling and filtering
Raw variants were called in multisample mode using the GATK HaplotypeCaller version 3.4-46 (ref. 64) and GATK-Queue version 3.4-46 with default settings and additional option ‘EMIT_ALL_CONFIDENT_SITES’. The quality of variant and reference positions was evaluated using GATK VariantFiltration version 3.4-46 with options ‘--snpFilterName LowQualityDepth --snpFilterExpression QD < 2.0 --snpFilterName MappingQuality --snpFilterExpression MQ < 40.0 --snpFilterName StrandBias --snpFilterExpression FS > 60.0 --snpFilterName HaplotypeScoreHigh --snpFilterExpression HaplotypeScore > 13.0 --snpFilterName MQRankSumLow --snpFilterExpression MQRankSum < −12.5 --snpFilterName ReadPosRankSumLow --snpFilterExpression ReadPosRankSum < −8.0 -cluster 3 -window 35’. To obtain high-quality somatic mutation catalogs, we applied postprocessing filters as described49. Briefly, we considered variants at autosomal chromosomes without any evidence from a paired control sample (mesenchymal stem cells isolated from the same bone marrow), passed by VariantFiltration with a GATK phred-scaled quality score that was ≥100 for base substitutions and ≥250 for indels, a base coverage of at least 20× in the clonal and paired control sample, no overlap with single-nucleotide polymorphisms (SNPs) in the SNP Database version 137.b3730 and absence of the variant in a panel of unmatched normal human genomes. We additionally filtered to exclude in vitro accumulated mutations as described49,62,63. All mutations are listed in Supplementary Table 3.
Driver mutations
SBSs or indels were considered as driver mutations if they had an MQ of 30, had a minimum of 10× base coverage or were found in the driver genes COSMIC cancer gene consensus (version from 9 May 2019). Additionally, these driver mutations were annotated as missense, frameshift, stop-gain, insertion or deletion, had either a high or moderate effect as annotated by SnpEff and were not in the SNP Database version 146 or a panel of unmatched normal human mesenchymal stem cells and fetal genomes (BED file available upon request).
Mutational signature analysis
Mutational patterns was used for the SNV signature analysis with the developed R package (MutationalPatterns)65, First, we extract the 96-trinucleotide mutational count matrix from the somatic mutation catalog with the mut_mat() function and the relative contribution plot was extracted with plot_96_profile(). The COSMIC version 3 signatures were used and identified on the basis of a cosine similarity > 0.85.
Variant allele frequency calculations
The variant allele frequency (VAF) is a metric (range 0–1) that describes the fraction of reads covering a particular variant position and how much the coverage supports it. VAFs are expected to be around ~0.5 because a heterozygous mutation means that only one of the two alleles is altered, whereas a VAF of 0 shows no support in a variant and a VAF close to 1 shows that all reads support that variant. We plotted the VAF distribution for each clone including all variants (Extended Data Figs. 2, 5 and 6). A VAF filtering threshold of 0.3 was applied to discard mutations that were introduced after the single-cell sort according to a previous method63.
Evaluation of MSI
We performed the comparison of two bioinformatics tools to calculate MSI: MANTIS45 and MSMuTect version 2 (ref. 46). Both tools use normal-tumor-paired, aligned and processed BAM files (hg19). The WGS clone CJMLH1C1 was used as normal. For MANTIS, we used the recommended settings for whole-exome usage (--mrq = 20.0 --mlq = 25.0 --mlc = 20 --mrr = 1) with a WES locus hg19 panel in BED format (included in the MANTIS repository). To run MSMutect2, we created a reference hg19 locus table with Phobos (version 3.3.12; http://www.rub.de/ecoevo/cm/cm_phobos.htm) with the same WES locus panel from MANTIS. We compared our clone or organoids to TCGA-COAD (MSI-H, MSS and MSMutect2). In the case of MANTIS, we recommend the use of the stepwise difference metric where any value greater 0.4 is considered MS-unstable (Fig. 4a).
Relative risk analysis
The relative mutation probability of driver gene mutations was calculated as described in the previous report51. Frequent mutations of MSI-H cancers in the Ras, Raf, MAPK, PI3K and ErbB pathways were selected from a previous study50. Mutational signatures in in vivo colonic and small intestinal tissue mutational signatures and in vitro colonic and small intestinal organoids were taken from our previous work17,49.
Simultaneous inhibition of all four major pathways in vitro
Gene expression and protein analyses were prepared as follows. Organoids were passaged to single cells following an enzymatic dissociation with TrypLE for 10 min at 37 °C. Cells were then resuspended in Matrigel and cultured in full human colon organoid expansion medium for 5 days to allow organoid formation. Fully formed organoids were then washed three times in PBS and cultured for 24 h in either full expansion medium or selection medium (−WRNE + 1 mM Nut3, 1 µM Afa, 50 ng ml−1 BMP2 and 200 ng ml−1 DKK1). After 24 h, the organoids were collected and subjected to either RNA or protein extraction and used for RT–qPCR and western blot analyses.
RNA isolation, complementary DNA preparation and RT–qPCR
Organoids were collected in RLT lysis buffer supplemented with BME and mRNA was isolated using the QIAGEN RNeasy micro kit (QIAGEN) following the manufacturer’s instructions. Following isolation, 1 µg of RNA was used for cDNA synthesis using GoScript reverse transcriptase (Promega) following the manufacturer’s instructions. RT–qPCR was performed for a selection of genes using IQ SYBR green mix (Bio-Rad). Results were quantified using the ΔΔCt method. All primer sequences are listed in Supplementary Table 4.
Western blotting
Cells were lysed in radioimmunoprecipitation assay buffer containing cOmplete EDTA-free protease inhibitor cocktail tablets (4693132001, Sigma-Aldrich). Protein quantification was performed using a standard Bradford assay (Bio-Rad) and equal amounts of protein were run on SDS–PAGE gels and transferred to PVDF membranes (Millipore). After transfer, membranes were blocked, washed and incubated with antibodies against p53 (1:1,000; DO-1, Santa Cruz Biotechnology), p21 (1:1,000; F-5, Santa Cruz Biotechnology), glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:5,000; ab9485, Abcam), phospho-p44/42 MAPK (ERK1 and ERK2) (Thr202, Tyr204) (1:1,000; CST9101, Cell Signaling Technology) and β-actin (1:5,000; 13E5, Cell Signaling Technology). The signals were detected using enhanced chemiluminescence (SuperSignal West Femto maximum sensitivity substrate, Thermo Fisher Scientific).
Drug screening
Drug screenings were performed as described elsewhere66. In brief, outgrowing organoids were recovered from BME and enzymatically dissociated in TrypLE for 5 min at 37 °C. Following TrypLE inactivation, dissociated organoids were resuspended in BME and allowed to recover for 2 days in a medium containing a low concentration of EGF (0.5 ng ml−1 before being cultured in a medium lacking EGF for two additional days. On the day of dispense, prewarmed dispase was added and the organoids were incubated for 1 h at 37 °C, after which the organoids were collected and washed with ice-cold AdDMF+++. The organoids were counted and dispensed at a density of 1,000 organoids per well in 40 µl of culture medium lacking EGF and containing 5% (v/v) BME using a multidrop dispenser (Thermo Fisher Scientific) in a 384-well plate. The inhibitors and the vehicle for the normalization were added with an HP D300e digital dispenser (Hewlett-Packard). After 5 days of treatment, 30 µl of Cell Titer Glo (Promega) was added to each well using a multidrop dispenser and the plate was shaken for 5 min and incubated for an additional 15 min at room temperature before the luminescence was read using a SPARK luminescence detector (Tecan) with Tecan SparkControl software (version 2.1). The experiments were performed on two different clones, each with three technical replicates; organoids before obtaining NRAS mutations were used as a control.
Generation of KRAS G12D mutant organoid
Single guide RNAs (sgRNAs) targeting the human KRAS gene were cloned into an SpCas9–eGFP vector (Addgene, plasmid 48138) as previously described67. sgRNA and a homology-directed repair (HDR) donor DNA template for introducing the KRASG12D mutation were selected from the previous study13. To generate KRASG12D mutants, the organoids were dissociated into single cells with TrypLE express and transfected using a NEPA21 super electroporator (NEPA GENE) with SpCas9–eGFP containing the locus-specific sgRNA and the KRASG12D HDR template as described before68. Then, 3 days after transfection, culture medium supplemented with Y-27632 was exchanged with selection medium lacking EGF and containing 1 µM Gef (Selleck Chemicals) (−EGF+Gef). Clonal lines were generated by isolating individual organoids which survived the selection and dissociating them to single cells with TrypLE express. Outgrowing clones were separated and expanded as clonal lines that were used for subsequent analyses. Clonal lines were genotyped using the Zymogen Quick-DNA microprep kit following the manufacturer’s instructions. Correct gene editing was verified by genomic DNA extraction followed by PCR amplification of targeted loci using Q5 high-fidelity polymerase (New England Biolabs). Finally, Sanger sequencing of the generated amplicons by Macrogen confirmed correct gene editing. Primers used for amplification and Sanger sequencing can be found in Supplementary Table 4.
In vivo subcutaneous transplantation
Clonal organoids were passaged with TrypLE Express (Thermo Fisher Scientific) and allowed to recover in the appropriate culture medium for 2 days before being collected for the transplantation. The cultured organoids were incubated with TrypLE Express in the presence of 10 μM Y-27632 (Tocris Bioscience) and mechanically dissociated into single cells and counted. A total of 200,000 cells were resuspended in 100 μl of culture medium, mixed with Cultrex BME (Trevigen) at a 1:1 ratio and subcutaneously injected into both sides of the posterior flank of NSG mice (n = 3–6 mice, 6–12 injection sites per organoid line) as previously described13. Then, 8 weeks after transplantation, the mice were killed and analyzed for the presence of a tumor mass. Three mice in the CRISPR-AKPS organoid transplantation condition were killed with the estimation of exceeded maximal tumor burden 7 weeks after transplantation. Tumor volume was calculated using the modified ellipsoidal formula ((length × width2)/2)69. After measuring, the tumor specimens were fixed overnight at 4 °C in 4% PFA, paraffin-embedded and processed for histological analyses.
Histology
Tissues and organoids were fixed in 4% PFA, dehydrated in ethanol and embedded in paraffin. Sections were subjected to hematoxylin and eosin (H&E) and immunohistochemical (IHC) staining. Slides were cut at 5-μm thickness and used for histology. Organoids were stained with H&E or primary antibodies for IHC. In brief, the organoids were dehydrated in an ethanol gradient and then rinsed in PBS. Heat-induced antigen retrieval was performed and the slides were sequentially incubated with the primary and secondary antibodies. Finally, DAB was applied on the slides, which were then counterstained with hematoxylin, dehydrated and mounted with permanent mounting medium. The imaging was performed on a LEICA microscope (DM6000) and images were processed using Leica LAS-X (version 1.1) software. The following primary antibodies were used for IHC staining: mouse anti-E-cadherin clone 36 (1:1,000; 610182, BD Biosciences), mouse anti-Ki67 clone MM1 (1:1,000; 550609, BD Pharmingen), rabbit anti-β-catenin H-102 (1:500; sc-7199, Santa Cruz Biotechnology) and rabbit anti-EGFR clone EP38Y (1:1,000; ab52894, Abcam).
Statistics and reproducibility
No statistical method was used to predetermine sample size but our sample sizes are similar to those reported in a previous publication13. No data were excluded from the analyses. The experiments were not randomized. The investigators were not blinded to allocation during experiments and outcome assessment. Data collection and analysis were not performed blind to the conditions of the experiments. Data distribution was assumed to be normal but this was not formally tested. Statistical analysis was performed with Prism 9.0 (GraphPad, Dotmatics). For comparisons between two independent groups, a two-tailed unpaired Welch’s t-test was used. P values < 0.05 were defined as statistically significant.
Reporting summary
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Data availability
The sequencing data were deposited to the European Genome–Phenome Archive (https://ega-archive.org/) under accession numbers EGAS00001001682, EGAS00001000881 and EGAS50000000114. All other data supporting the findings of this study are available from the corresponding authors upon request. Source data are provided with this paper.
Code availability
Mutation calling and filtering pipeline NF-IAP is available from GitHub (https://github.com/ToolsVanBox/NF-IAP). Somatic variants were extracted with SMuRF version 3.0.0 (available from GitHub: https://github.com/ToolsVanBox/SMuRF) and MutationalPatterns (available from GitHub: https://github.com/ToolsVanBox/MutationalPatterns). In-house R scripts (version 4.2.3) were used to generate VAF plots and the comparison of MANTIS and MSMutect2 with library ggplo2.
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Acknowledgements
We thank A. Brousali, J. Salij and O. Kranenburg of the Utrecht Platform for Organoid Technology (UMC Utrecht) for patient inclusion and tissue acquisition, S. van den Brink for Wnt3a-conditioned medium and R-spondin 1-conditioned medium production and S. van der Elst for help with fluorescence-activated cell sorting. We also thank the members of the contributing labs for helpful suggestions and discussions. This work was supported by an award from the Cancer Research UK Grand Challenge (C6307/A29058), the Mark Foundation for Cancer Research to the SPECIFICANCER team, the ‘Sta op tegen Kanker (Stand Up To Cancer)’ International Translational Cancer Research grant to T.M. and H.C. and Oncode Accelerator, a Dutch National Growth Fund project under grant number NGFOP22201 (Organoid Group). M.B. is a postdoctoral fellow supported by a long-term EMBO fellowship (ALTF 765-2019). R.v.B. is a New York Stem Cell Foundation Robertson Investigator and is supported by The New York Stem Cell Foundation.
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T.M., J.D. and H.C. conceptualized and designed the project. T.M. and M.B. performed major experiments and analyzed the data. S.L. performed the organoid culture and western blotting experiments. M.H.G. assisted with CRISPR–Cas9 genome editing. H.B. and J.K. performed the IHC staining. J.K. and J.H.v.E. performed the transplantation experiments. D.M.G., R.O. and R.v.B. performed the WGS data analyses. T.M., M.B. and H.C. wrote the paper. H.C. supervised the study. All authors discussed the results and commented on the manuscript.
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H.C. is the inventor of several patents related to organoid technology; his full disclosure is given at https://www.uu.nl/staff/JCClevers/. The other authors declare no competing interests.
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Extended data
Extended Data Fig. 1 Various niche factor selection of MLH1KO human colon organoids.
(a) MLH1KO-1 organoids were selected by inhibition of other niche factor pathways. Triple independent MLH1KO-1 organoid lines grew in complete medium (WRNE), but all of small organoids formed in all conditions died within a week (representative pictures from n = 3 clonal organoid lines). W, wnt3a; R, R-spondin1; N, Noggin; E, EGF; Gef, gefitinib. Scale bars, 500 µm. (b) Quantification of formed organoids in each condition. Mean and SD (error bars) of n = 4 clonal organoid lines. -EGF+Gef (P = 0.0004, P < 0.0001, P < 0.0001), -Noggin (P < 0.0001, P = 0.0011, P < 0.0001), +Nut3 (P < 0.0001, P < 0.0001, P = 0.0941), two-sided Welch’s t-test. (c) MLH1KO-1 organoid clones established from another individual were selected with the deprivation of Wnt factor. Some of organoids grew out only from MLH1KO-1 clone in -WR medium. Outgrowing clones are shown in inset. Scale bars, 500 µm. (d) Quantification of survived organoids in each condition. Mean and SD (error bars) of n = 4 clonal organoid lines. P < 0.0001, P < 0.0001, two-sided Welch’s t-test.
Extended Data Fig. 2 Variant Allele Frequency distribution of mutant clones.
Graphs show Variant Allele Frequency (VAF) distribution of somatic mutations per organoid clone (MLH1KO-1-WR-1 and MLH1KO-1-WR-2) in WGS. A VAF-filtering threshold of 0.3 (dotted line) is applied to discard mutations that are introduced after the single-cell sort.
Extended Data Fig. 3 Mutational spectrum of AXIN1, AXIN2, BMPR2, ACVR2A, and TP53 in CRC.
Lollipop diagram corresponding colorectal cancer studies selected in cBioPortal (https://www.cbioportal.org). A black dot indicated a truncating mutation; a green dot indicated a missense mutation; and a purple dot indicated other types of mutation. The length of each lollipop represents the number of patients with the specific mutation. Specific mutations found in MLH1KO-1 organoid are represented on top of each lollipop.
Extended Data Fig. 4 MLH1KO AXIN1/2-mutant organoids only gained Wnt-pathway independency.
The medium selection experiments with the MLH1KO AXIN1/2-mutant organoids (MLH1KO-1) and each pathway driver CRISPR-mutant organoids (CRISPR-APCKO, CRISPR-KRASG12D, CRISPR-P53KO) in WRNE (complete medium), -WR, -EGF +Afatinib, +Nutulin3, and -Noggin+BMP2. Representative pictures from n = 3 clonal organoid lines. Scale bars, 200 µm.
Extended Data Fig. 5 Variant Allele Frequency distribution of mutant clones.
Graphs show Variant Allele Frequency (VAF) distribution of somatic mutations per organoid clone (MLH1KO-1-WR-1-N, MLH1KO-1-WR-1+Nu, and MLH1KO-1-WR-2-N) in WES. A VAF-filtering threshold of 0.3 (dotted line) is applied to discard mutations that are introduced after the single-cell sort.
Extended Data Fig. 6 Variant Allele Frequency distribution of mutant clones.
Graphs show Variant Allele Frequency (VAF) distribution of somatic mutations per organoid clone (MLH1KO-1-WR-1+Nu-N-1, MLH1KO-1-WR-1+Nu-N-2, and MLH1KO-1-WR-1+Nu-N-E+Afa) in WES. A VAF-filtering threshold of 0.3 (dotted line) is applied to discard mutations that are introduced after the single-cell sort.
Extended Data Fig. 7 Drug sensitivity assay of NRASQ61K mutant organoids.
(a) qRT-PCR analysis of ETV4, ETV5, DUSP4, DUSP5 and CCND1 mRNA levels. These transcripts were compared between NRAS wildtype parental clone (WT) and NRAS-Q61K clone cultured in the medium with or without afatinib (Afa+/-). n = 2 clonal organoid lines. (b) Dose-response curves showing the sensitivity of NRAS-WT and NRAS-Q61K clones to inhibitors of the EGFR (Gefitinib), MEK (Trametinib), mTOR/PI3Kα (Everolimus/Alpelisib) and CDKs (Dinaciclib). Y-axis represent the viability of the samples while X-axis represent the drug concentration (power of 10 µM). Mean and SD (error bars) of n = 3 technical replicates for n = 2 independent organoid clones for each genetic background. (c) Representative bar graphs showing the sensitivity of NRAS-WT and NRAS-Q61K organoids to different inhibitors, calculated as area under the curve (AUC). n = 2 clonal organoid lines. (d) Heatmaps reporting the viability of the drug screening performed on NRAS-WT and NRAS-Q61K organoids as normalized to vehicle treated organoids. Color mapping range from low viability (red) to high viability (green).
Extended Data Fig. 8 Introduction of KRASG12D mutation on triple-pathway mutant organoids.
(a) Triple-pathway mutant organoids were transfected with Cas9, sgRNA and the oligonucleotide previously designed for KRASG12D mutation. KRASG12D mutants were selected in the medium lacking EGF, with the EGFR inhibitor gefitinib (-WRNE+Nut3+Gef). Small single organoids survived and grew (Inset). Representative pictures from n = 3 clonal organoid lines. Scale bars, 500 µm. (b) Picked up surviving KRASG12D mutant organoid (representative pictures from n = 3 clonal organoid lines). Scale bars, 100 µm. (c) Single survived organoids were expanded and passaged in the medium lacking EGF, with the EGFR inhibitor gefitinib (-WRNE+Nut3+Gef). Representative pictures from n = 3 independent replicates. Scale bars, 500 µm. (d) Sequence analysis of the targeted KRAS exon. Oncogenic GGT > GAT mutation is indicated in green; silent mutations are in blue; protospacer adjacent motif (PAM) is underlined in red.
Extended Data Fig. 9 Growth speed of AKPS mutant organoids and the quadruple-pathway mutant organoids.
(a) the quadruple-pathway mutant organoids (NRASQ61K) and AKPS mutant organoids grew in complete medium (WRNE). Representative pictures from n = 3 clonal organoid lines. Scale bars, 100 µm. (b) Quantification in cell numbers of the quadruple-pathway mutant organoids (NRASQ61K) and AKPS mutant organoids Mean and SD (error bars) of n = 3 clonal organoid lines.
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Mizutani, T., Boretto, M., Lim, S. et al. Recapitulating the adenoma–carcinoma sequence by selection of four spontaneous oncogenic mutations in mismatch-repair-deficient human colon organoids. Nat Cancer 5, 1852–1867 (2024). https://doi.org/10.1038/s43018-024-00841-x
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DOI: https://doi.org/10.1038/s43018-024-00841-x
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