Abstract
Mutations activating the KRAS GTPase or the BRAF kinase are frequent in colorectal cancer. Here, we use inducible transgenic expression of KRASG12V or BRAFV600E in intestinal organoids of mice to investigate oncogenic signal transduction in the mitogen-activated protein kinase (MAPK) cascade with cellular resolution. Using phospho-protein, reporter, and single cell transcriptome analyses, we found that BRAFV600E triggered high ERK activity and downstream gene expression in all intestinal cell types. Induction of BRAFV600E resulted in rapid epithelial disorganisation followed by organoid disintegration, providing evidence for an upper limit of tolerable MAPK activity in the intestinal epithelium. In contrast, transgenic expression of KRASG12V activated ERK to a lesser extent and in a cell type-specific pattern. Single cell RNA sequencing defined three intestinal cell types capable of activating ERK: undifferentiated crypt cells, Paneth cells and a minority population of late-stage enterocytes. Yet, in most enterocytes ERK was not activated by EGF in the medium or induction of oncogenic KRASG12V. Colorectal cancer cell lines also differed in their abilities to activate ERK in response to KRASG12V. Furthermore, we found that Wnt/β-Catenin amplified ERK phosphorylation in the normal intestine and KRASG12V could maintain proliferation only in adenomatous cells forming after β-Catenin activation. These findings may explain why activation of Wnt signalling precedes KRAS mutations in the classical CRC progression pathway. Our experiments highlight key differences between MAPK activity elicited by the BRAF or KRAS oncogenes in colorectal cancer and find unexpected functional heterogeneity in a signalling pathway with fundamental relevance for cancer therapy.
Introduction
Multiple signalling pathways, including the Wnt/β-Catenin and the mitogen-activated protein kinase (MAPK) cascades, form a signalling network in cells of the intestinal epithelium [1]. Collectively, activities within the signalling network control cellular turnover, that is, stem cell maintenance, cell proliferation, differentiation into absorptive enterocyte and secretory cell lineages, and apoptosis. Wnt/β-Catenin and MAPK activities are regionalized within the folded single-layered intestinal epithelium. Both are high in crypt bases harbouring stem cells and low in villus areas containing differentiated enterocytes.
The MAPK module transduces signals downstream of receptor tyrosine kinases, such as EGFR, and RAS family GTPases. MAPK comprises of three consecutively activated kinases, namely RAF, MEK and ERK. Upon activation, ERK can phosphorylate and activate a series of transcription factors orchestrating a complex cellular response that often is pro-proliferative [2]. In the normal intestine, EGFR-RAS-MAPK is activated by ligands from the crypt microenvironment, which are secreted by e.g. epithelial Paneth cells of the small intestine, Reg4+ secretory niche cells of the large intestine, or adjacent fibroblasts [3,4]. In colorectal cancer (CRC), MAPK activity is thought to be more cell-autonomous due to oncogenic mutations activating KRAS (in 40% of CRCs) or NRAS (5% of CRCs) [5] or BRAF (10% of CRCs) [6], or by de novo expression of EGFR ligands such as amphiregulin [7]. Activity of the RAS-MAPK signal transduction module is a main determinant of cancer development and therapy response [5,6,8].
Recent studies suggest that the relationship between MAPK-activating mutations, MAPK signal transduction and phenotypic outcome in CRC is complex: firstly, mutations in KRAS and BRAF are associated with distinct CRC development routes: KRAS, but not BRAF, mutations frequently occur as secondary events after mutations activating Wnt/β-Catenin in the conventional CRC progression sequence [9,10]. Conversely, BRAF, but less frequently KRAS, mutations precede upregulation of Wnt/β-Catenin in the alternative serrated progression route [11,12]. The observed disequilibrium between KRAS and BRAF mutations in the conventional versus serrated pathways of CRC evolution suggest the existence of functional differences, resulting in distinctive effects on clinical course and treatment efficacy [13].
Secondly, MAPK signal transduction appears to be heterogeneous in genetically identical CRC cells. Cells at the invasive front frequently exhibited more MAPK activity compared to cells in central areas of the same cancer, as judged by phosphorylation of ERK (p-ERK), and CRCs with activating KRAS mutations retained regulation of MAPK [14]. Previous studies already showed heterogeneous Wnt/β-Catenin activity in cancer specimens, suggesting a more general paradigm of graded pathway activities in CRC [15]. Furthermore, CRC cells exhibit functional differences within a cancer. Differentiation gradients in CRC appear related to differentiation trajectories of normal intestinal cells, with stem cells at the apex of the hierarchy [16–19]. CRC subtypes can share similarities with cell types of the normal crypt, such as stem cells, enterocytes or secretory cells in bulk cell analysis [20]. Finally, because of variable signal transduction and differentiation states, genetically identical CRC clones exhibit variable proliferative potential and therapeutic response [21].
Experimental techniques with cellular resolution, ranging from fluorescent reporters [22] to single-cell transcriptome analyses [23,24] hold the promise to disentangle the relationship between oncogenes, cell differentiation states and cell signal transduction while taking into account cellular heterogeneity. Here, we ask whether oncogenic KRAS or BRAF, representing the most common alterations in the MAPK pathway in CRC, show cell-to-cell heterogeneity in their activities. For this, we use organoid cultures that can be efficiently manipulated and which maintain the cell hierarchy of the tissue in vitro [25]. In these organoids, we express transgenes encoding BRAFV600E, KRASG12V and/or stabilized β-Catenin (β-Cateninstab), along with the fluorescent marker tdTomato. We assess the immediate impact of the oncogenes on cell signal transduction, gene expression programs and phenotypic outcome using an ERK-dependent fluorescent reporter and single cell RNA sequencing. We discover strong functional differences between the BRAF and KRAS oncogenes and find that signal transduction by KRAS is cell type-specific.
Results
BRAFV600E, but not KRASG12V, induces high levels of MAPK activity and epithelial disorganisation in intestinal organoids
To study direct effects of the KRASG12V and BRAFV600E oncoproteins on cells of the intestinal epithelium, we engineered transgenic mice carrying doxycycline-inducible single copy transgenes in the Gt(ROSA26)Sor locus [26,27]. For the present study, we employed lines with transgenes encoding the red fluorescent marker tdTomato linked by a self-cleaving p2A peptide to BRAFV600E, KRASG12V, or stabilized β-Catenin (Fig. 1A), alone and in combination, and tdTomato linked to firefly luciferase (FLUC) as a control.
We initiated organoid cultures by embedding intestinal crypts from FLUC-, KRASG12V-, and BRAFV600E-inducible mice into extracellular matrix, as described before [25]. BRAFV600E induced irreversible disintegration of organoids within 1-2 days, whereas transgenic KRASG12V or the FLUC control protein were well tolerated, even after several passages, when we initiated oncoprotein production by adding doxycycline to the culture media (Fig. 1B). To examine whether the BRAF oncogene has detrimental effects on the epithelium beyond the previously reported loss of stem cells [27,28], we examined histology at ultrastructural level of the induced organoids using transmission electron microscopy (Fig. 1C). We found that control and KRASG12V-induced organoids show the expected tissue structure, that is, a single-layered polarized epithelium with continuous apical and basal surfaces as well as a brush border at the apical side. Desmosomes, providing lateral cell adhesion, were clearly visible. We frequently observed intercellular vacuoles, which have previously been attributed to osmotic stress during fixation of organoids [25]. In contrast, BRAFV600E-induced organoids displayed a continuous basal surface, whereas the apical side was grossly distorted, although it contained a brush border as evidence of polarisation. Nuclei were pleomorphic and no longer lined up basally but were scattered at different positions. Cells were still attached to each other by desmosome bridges, indicating that the ongoing epithelial disorganisation was taking place in the presence of lateral cell adhesion.
To ascertain whether the epithelial disorganisation provoked by BRAFV600E was correlated with MAPK activity, we measured phosphorylation of ERK. We found that induction of BRAFV600E, but not KRASG12V, resulted in high phosphorylation levels of ERK in intestinal organoids, as determined by capillary protein analysis (Fig. 1D). BRAFV600E-induced organoid disintegration could be counteracted by inhibition of the BRAF-downstream MEK and ERK kinases using AZD6244/Selumetinib [29] and BVD-523/Ulixertinib [30], respectively, but not by inhibition of the upstream EGFR tyrosine kinase receptor family using AZD8931/Sapitinib [31] (Fig. 1E), showing that the induced phenotype is due to excessive MAPK activity. Indeed, only 24 h of BRAFV600E induction activated almost all direct MAPK target genes [32], whereas conditional expression of KRASG12V had no obvious effect on bulk organoid transcription (Supplementary Fig. 1).
Single cell RNA sequencing reveals disruption of intestinal differentiation trajectories by BRAFV600E
To uncover potential cellular heterogeneity in the response to the oncogenes, we performed single cell transcriptome analyses. We induced FLUC control, BRAFV600E and KRASG12V-transgenic organoids for 24 h, prepared single cell suspensions, and stained them with a fluorescent antibody against the crypt cell marker CD44 [33], and with a fluorescent dye to eliminate dead cells. We next sorted single live and transgene-expressing organoid cells into two 96-well plates for RNA sequencing. We acquired samples of CD44-high crypt and CD44-low villus cells. In total, we obtained transcriptomes of 167 cells with >1000 detected genes per cell that were used for further analysis. Single cell transcriptomes were assigned to six interconnected clusters with help of k-means clustering and visualised in a t-SNE-based representation (Fig. 2A). Mapping of signature genes for intestinal stem cells (ISCs), proliferative TA cells, differentiated enterocytes [34] and secretory Paneth cells [35] (Fig. 2B), and the CD44 status as inferred from flow cytometry (Fig. 2C) confirmed the calculated differentiation trajectories (shown as branched grey overlay in Fig. 2A; cluster 1 was assigned starting cluster): undifferentiated CD44-high ISC and TA cell signature genes were enriched in clusters 1 and 2 while Paneth cell marker genes were highest in cluster 2, indicating the route for secretory crypt cells differentiation; expression of enterocyte signature genes increased gradually in clusters 3-5, marking the CD44-low absorptive lineage.
We next considered the distribution of cells expressing specific transgenes: FLUC control and KRASG12V-expressing cells intermingled throughout the clusters 1-5 of the normal cell differentiation trajectories. BRAFV600E-expressing cells were in contrast depleted from the central clusters 2-4, but formed the outsider cluster 6, composed entirely of BRAF-induced cells. BRAFV600E-positive cells in cluster 6 uniformly expressed high levels of ERK target genes, regardless of whether they were sorted as CD44-high or CD44-low (Fig. 2C). Cluster 6 also had high expression of Anxa10, which has been identified as a marker for BRAF-positive serrated adenoma [36](Supplementary Fig. 2). The single cell analysis thus shows that BRAFV600E imposes a specific gene expression program onto intestinal cells, independent of their prior differentiation state.
ERK-dependent reporter activity and single cell analyses identify KRASG12V-responsive intestinal cells
Since mutant KRAS is one of the most frequent oncogenes in colon cancer, we were intrigued by the absence of KRASG12V-induced MAPK activity, phenotypes, or gene expression changes in our previous assays. We hypothesized that its activity could be weak and restricted to specific cell types in the normal intestine. To visualize ERK activity with single cell resolution in organoids, we employed the Fra-1-based integrative reporter of ERK (FIRE) that translates ERK kinase activity into stability of a nuclear yellow-green venus fluorescent protein (Fig. 3A) [22]. FIRE fluorescence in organoids cultured in normal growth medium containing EGF was strong in crypt bases, whereas differentiated villus tissue was largely FIRE-negative (Fig. 2B). In the absence of EGF from the medium, ERK activity in the crypt base persisted, likely due to autocrine and paracrine signals from EGF-producing Paneth cells [4].
We next conditionally expressed the FLUC control, KRASG12V, or BRAFV600E-encoding transgenes in FIRE-transfected organoids (Fig. 3C). Transgene induction was often variable, as inferred by tdTomato fluorescence, allowing to compare individual tdTomato-positive cells with transgene-negative neighbouring tissue. tdTomato-FLUC control transgene expression had no influence on FIRE activity. In contrast, expression of KRASG12V resulted in increased FIRE signals in crypt cells, which consistently displayed stronger reporter activity compared to adjacent KRASG12V-negative cells. Surprisingly, a large majority of villus cells remained FIRE negative, despite strong KRASG12V positivity, as inferred from tdTomato fluorescence. We confirmed the differential signal transduction from KRASG12V to ERK using phospho-ERK immunohistochemistry (Fig. 3D). In line with our FIRE reporter data, p-ERK-positive cells were largely absent in the central differentiated (Ki67-negative) villus areas of organoids, despite strong tdTomato-KRASG12V positivity. Taken together, our results show that ERK activity in differentiated villus epithelial cells can neither be induced by EGF in the medium nor by induction of oncogenic KRASG12V. However, when we induced BRAFV600E, we found widespread and strong FIRE signals across the complete organoid (Fig. 3C). This suggests a strict and cell-type specific control of signal transduction by oncogenic KRAS, but not BRAF in intestinal epithelial cells.
Since FIRE fluorescence was detected in cells responsive to KRASG12V, we next used the reporter to assist selection of cells for single cell sequencing. Our aim was to define cell types with high ERK activity, either in response to KRASG12V or as part of the normal cell hierarchy. For this, we induced organoids with the integrated ERK reporter for KRASG12V or FLUC, prepared single cell suspensions and sorted cells by FACS into 96-well plates for transcriptome analysis (Supplementary Fig. 3). We focussed on single cells with high transgene (tdTomato) signal and either strong or weak FIRE (venus) fluorescence (Fig. 4A). In total, we obtained 197 single cell transcriptomes. K-means clustering into 8 groups and t-SNE-based visualisation (Fig. 4B, C) revealed the cell type distribution. We found gene expression patterns characteristic for immature and secretory cells in clusters 1-4. Cluster 1 was enriched for ISC and TA marker genes, whereas clusters 2-4 were defined by Paneth cell signature genes (Fig 4D; Supplementary Fig. 4). Cluster 2 was enriched for the Paneth cell markers Lyz1 encoding Lysozyme [37] and several genes encoding Defensins, while other cluster-defining genes such as Mptx1 and Agr2 in cluster 4 hint at a high degree of Paneth cell heterogeneity. Clusters 5-8 formed a differentiation trajectory for absorptive cells, with Ifabp1 as the top defining genes for clusters 5-7 (Supplementary Fig. 4)
Using this information, we assessed the distribution of transcriptomes derived from KRASG12V-induced FIRE-high cells (Fig. 4C, D). These were confined to distinct aggregates encompassing the ISC/TA cell zone of cluster 1, as well as transcriptomes inhabiting the outer right rim of the tSNE representation that we above assigned to be derived from late-stage enterocytes and Paneth cells. Immunofluorescence microscopy using the Paneth cell marker Lysozyme confirmed high FIRE activity in this cell type after KRASG12V induction (Supplementary Fig. 5). In contrast, a central area of the tSNE plot encompassing the largest clusters 5 and 6 of bulk enterocytes was almost devoid of KRASG12-producing FIRE-high cells but harboured many KRASG12V/FIRE-low cells, confirming that enterocytes cannot activate ERK, even when expressing oncogenic KRASG12V.
Inspection of the MAPK-driven ERK target signature in the clustered transcriptomes revealed a complex picture (Fig.4D): KRASG12V/FIRE-high cell transcriptomes of cluster 1 displayed low expression of ERK signature genes, probably due to quickly diminishing ERK activity and transcription in committed TA cells that transiently remain positive for FIRE, due to its integrative fluorescence reporting characteristics [22]. In contrast, multiple FIRE-positive Paneth cells of clusters 2-4 and enterocytes at the edge of clusters 7 and 8 displayed had ERK activity, as inferred from ERK target gene expression. We hypothesize that the burst of ERK activity in late-stage enterocytes is directly or indirectly related to the apoptotic process at the end of their life span. Indeed, a previous publication has identified a role for ERK signals in cells neighbouring an apoptotic enterocyte [38]. It remains to be tested whether the burst of ERK activity in some late-stage enterocytes is amplified by KRASG12V; we found that isolated FIRE-positive cells were present in organoid villus domains regardless of KRASG12V induction (compare cells marked with asterisks in Figs. 3B and 3C). Taken together, our single cell RNA and reporter analyses show that crypt cells, encompassing ISC/TA cells and Paneth cells, can activate ERK as a response to oncogenic KRASG12V. In contrast, enterocytes were unable to activate ERK despite EGF in the medium or expression of KRASG12V.
β-Catenin interacts with KRASG12V in regulating ERK phosphorylation and EGFR signal autonomy
We asked whether Wnt/β-Catenin activity may enable the cellular ERK response, because ERK-responsive crypt cell types, as defined above, are known to have high Wnt/β-Catenin activity, whereas unresponsive enterocytes have low Wnt/β -Catenin activity [39] (see also Wnt target gene activity in Fig. 4D). Furthermore, activating KRAS mutations usually follow mutations activating the Wnt/β-Catenin pathway in conventional CRC progression [9]. We first analysed ERK phosphorylation in intestines of induced transgenic mice carrying single or combined transgenes encoding stabilized β-Catenin (β-Cateninstab) and KRASG12V (Fig. 5A). We detected increased and regional ERK phosphorylation in the proliferative zone, but not in differentiated enterocytes, following KRASG12V activation, which agrees with our reporter and transcriptome analyses. The increased ERK phosphorylation did not extend into the crypt bases, likely due to lower levels of transgene induction (see tdTomato staining in Fig. 5A, lower panels). Again, some enterocytes at the tip of the villus were strongly p-ERK-positive regardless of KRASG12V induction, confirming our findings of isolated p-ERK-positive late-stage enterocytes in the previous assays. Interestingly, we also detected increased p-ERK levels in the proliferative zone of transgenic mice producing β-Cateninstab. However, only in mice with a double KRASG12V-β-Cateninstab-encoding transgene, we observed many p-ERK-positive enterocytes throughout the villi. In contrast, we found that ERK phosphorylation depended on Wnt/β-Catenin activity in organoids, since removal of the Wnt co-ligand R-Spondin from cultures reduced p-ERK levels within 24h (Fig. 5B).
We next assessed KRASG12V-dependent proliferation and ERK phosphorylation in spheroid cells that have undergone a switch to an adenoma-like phenotype after long-term activation of stabilized β-Catenin and removal of the Wnt co-ligand R-Spondin (Fig. 5C-E). These cells largely phenocopy adenoma of the conventional CRC progression pathway, i.e. an early stage in tumour evolution after activation of the Wnt/β-Catenin pathway, but before mutational MAPK activation [40]. We found that adenomatous spheroids proliferate in the absence of EGF in the culture medium over several passages, regardless of whether they express a transgene encoding β-Cateninstab, or a transgene encoding both β-Cateninstab and KRASG12V. To test for self-sufficiency of spheroid proliferation, we used the EGFR inhibitor Gefitinib [41] to suppress residual autocrine or paracrine EGFR signals. Under these conditions, the growth of β-Cateninstab-induced spheroid cultures stalled, while combined β-Cateninstab- and KRASG12V-cultures maintained proliferation (Fig. 5B, C). This observation contrasted with organoids derived from normal intestinal tissue (that is, without long-term induction a β-Cateninstab transgene), which were highly EGF dependent and could not employ KRASG12V to sustain proliferation (Supplementary Fig. 6). In contrast to our finding of increased ERK phosphorylation after β-Catenin activation in normal tissue, p-ERK levels were lower in adenomatous spheroids compared to normal tissue organoids (Fig. 5E). These experiments indicate that functional interactions between the Wnt/β-Catenin and MAPK pathways in the intestine may be different in normal and adenomatous tissue.
Dosage- and cell type-dependence of ERK phosphorylation in response to KRASG12V in CRC cell lines
Our data indicate that signals transduced from KRAS to ERK are context dependent and thus may be subject to adaptation during CRC progression. To test this hypothesis, we compared ERK phosphorylation in response to KRASG12V or BRAFV600E in two CRC cell lines that have no mutations in KRAS, NRAS or BRAF. For this, SW48 and Caco2 cells were transfected with inducible expression vectors for tdTomato-KRASG12V, -BRAFV600E, or -FLUC control, as previously used in the organoid cultures. 24h after induction, cells were fixed and oncogene levels, as extrapolated from tdTomato, and phosphorylation of ERK were measured via flow cytometry (Fig. 6A). We found that even low levels of BRAFV600E strongly increased ERK phosphorylation in both cell lines. In contrast, the ability of KRASG12V to induce ERK phosphorylation was markedly different (Fig. 6B). In Caco2 cells, ERK phosphorylation increased step-wisely with KRASG12V-associated fluorescence, whereas in SW48 even high levels of transgenic tdTomato-KRASG12V were unable to induce strong ERK phosphorylation.
Discussion
By combining reporter assays and single cell RNA sequencing in transgenic intestinal organoids, we demonstrate that phosphorylation of ERK, the terminal kinase of the MAPK module, is regulated in a differential and cell typic-specific manner in response to KRASG12V or BRAFV600E. Both oncogenes therefore also differed in their abilities to induce cancer-associated phenotypes, such as loss of epithelial integrity and cell proliferation.
Importantly, we found that the ability of KRASG12V to activate ERK was cell type-specific within the intestinal epithelium and divergent between CRC cell lines. This unexpected heterogeneity in MAPK signal transduction extends our understanding of how mutated KRAS, the most prevalent oncogene in CRC, exerts its effects. Importantly, local differences of ERK activity have recently been found in clinical specimens of CRC, including in KRAS-mutant CRC [15]. ERK levels were generally higher in cancer cells adjacent to stromal cells at the invasive front, and lower in more central areas of the cancer specimen, in line with modulation of ERK activity by cues from the microenvironment. Our results agree with the view of a dynamic MAPK pathway in the presence of oncogenic KRAS. However, we find here that ERK activity was turned off in differentiated enterocytes in organoid culture, even in the presence of extracellular EGF. Therefore, ERK activity in the presence of oncogenic KRAS can be regulated by both, external signals received from the microenvironment and intrinsic cellular differentiation states.
We found that Paneth cells and enterocytes represent main differentiated cell types of the intestinal epithelium with marked differences in their abilities to activate ERK following oncogenic activation of KRAS. It is of note that the former reside in the crypt compartment with high Wnt/β-Catenin activity, while the latter inhabit the villus with low β-Catenin activity. We therefore suggest that Wnt/β-Catenin is among the signals in the microenvironment that could regulate ERK activity in the intestine and in CRC. Indeed, a functional role for Wnt/β-Catenin in the activation of ERK in the intestinal epithelium has been proposed before [43].
We found that KRASG12V cooperated with oncogenic β-Catenin in the induction of ERK activity, and that ERK activity in the normal intestine was reduced after removal of the Wnt/β-Catenin co-activator R-Spondin. Spheroid cells resembling intestinal adenoma were rendered EGF-independent by KRASG12V induction, in contrast to normal mouse intestinal epithelium, where KRASG12V was not sufficient to support proliferation. These findings could explain why oncogenic KRAS cannot initiate tumour development in the intestine, but nevertheless is the most frequent oncogenic event immediately after activation of Wnt/β-Catenin in the conventional tumour progression route leading to CRC [9,10]. In this model, oncogenic KRAS provides sufficient pro-proliferative ERK activity only after β-Catenin activation, such as by the loss of the tumour suppressor APC.
In our experiments, signal transduction from BRAF to ERK was independent of cellular context. Extending previous studies [27,28], we found that high levels of MAPK induced by oncogenic BRAFV600E are not tolerated in the intestinal epithelium. This is in contrast to CRC and cell lines, where BRAFV600E amplifications exist and are selected for by MEK inhibition [44,45]. It thus appears that the corridor for acceptable MAPK activity is tuneable during CRC progression and under selective pressure exerted, for instance, by targeted therapy. Therefore, our findings are reminiscent of the “just right” signalling model that has been proposed to explain step -wise increases of β-Catenin activity in CRC progression [46].
We found that Caco2 cells phosphorylated ERK stepwisely when induced with increasing doses of KRASG12V, while no such response was observed in SW48 cells. This suggests that subgroups of CRC could differ in their regulation of ERK activity, even when harbouring the same oncogene in the MAPK pathway. This finding could explain part of the heterogeneity observed in response to anti-EGFR-therapy in the clinic, even after stratification of patients for mutations in KRAS, NRAS and BRAF [13]. Furthermore, we found that the cellular differentiation state and activity of Wnt/β-Catenin could modulate ERK phosphorylation and the ERK-dependent proliferative response in intestinal cells. Our findings thus suggest that controlling cellular differentiation or targeting morphogenetic signalling pathways such as Wnt/β-Catenin could represent a means to inhibit oncogenic MAPK activity in subgroups of KRAS/BRAF-wildtype and KRAS-mutant, but not in BRAF-mutant CRCs.
Materials and Methods
Generation of transgenic mice
Transgene cassettes were constructed by linking tdTomato to human BRAF(V600E), KRAS(G12V) and/or murine stabilized mutant Ctnnb1 (S33A, S37A, T41A, S45A) or firefly luciferase via 2A peptides, and subsequent cloning of these gene combinations into a doxycycline-inducible expression cassette flanked by heterologous loxP sites, and integrated into a previously modified Gt(ROSA)26Sor locus of F1 hybrid B6/129S6 embryonic stem cells by Cre recombinase-mediated cassette exchange, as described previously [26]. Transgenic animal production was approved by Berlin authorities LAGeSo (G0185/09, G0143/14).
Cell and Organoid culture and protein analysis
Organoid cultures were initiated and propagated as described before [25], using 48-well plates with 15 μl droplets of Matrigel (Corning) per well overlaid with 300 μl crypt culture medium containing EGF (50 ng/ml), Noggin (100 ng/ml) and R-Spondin1 (functionally tested from R-Spondin-conditioned medium; CCM-REN). Transgenes were induced by addition of 2 μg/ml doxycycline to the medium. For spheroid cultures, R-Spondin was removed after induction of a transgene endoding β-Cateninstab alone or in combination with KRASG12V. During passaging, spheroids were dissociated with TrypLE (Gibco) for 3 min and Rho kinase inhibitor Y27632 (10 μM) was added to the culture medium to prevent anoikis. The following inhibitors (SelleckChem) were employed: AZD6244 (10 μM), BVD-523 (3 μM), AZD8931 (50 nm), Gefitinib (1 μm).
Organoid and spheroid survival was scored as follows: cultures were passaged, inhibitors and doxycycline were applied to the culture medium directly after passaging. Individual wells were imaged using the z-stack function of Biozero observation and analyser software (Keyence) on day 1 and 4 (organoids) or day 1 and 6 (spheroids), and full focus-reconstructed images were used for quantification.
For viral transfection, a protocol from reference [48] was employed, with modifications: organoids were cultured in the presence of Y27632 and the GSK3β inhibitor CHIR99021 for two days. Next, organoids were disaggregated into single cells using TrypLE (Gibco) for 5min. at 37°C. Cell suspensions were spin-oculated in an ultra-low adhesion round bottom 96-well plate with the virus at 300 g for 45min. Subsequently, cells were resuspended in Matrigel, and cultured for 2 days in CCM-REN supplemented with Y27632 and CHIR99021. Subsequently, medium was replaced by CCM-REN containing 2 μg/ml Puromycin to select for transfected cells. As viral transfection initially resulted in organoid pools that were heterogeneous for FIRE reporter activity, single FIRE positive organoids were manually selected and propagated before experimental analysis.
Protein sample preparation and quantification of pERK was performed as previously described [47] using a WES capillary system (12–230 kD Master kit α-Rabbit–HRP; PS-MK01; Protein Simple) and the antibody pERK/2(T202/Y204) (1:50; #9101, Cell Signal). Raw pERK values were normalized to vinculin (1:30; #4650; Cell Signal).
Immunohistochemistry was done on paraformaldehyde fixed and paraffin-embedded tissues. Organoids were fixed in 4% paraformaldehyde for 30 minutes, while intestines were fixed over night at room temperature. Subsequently, tissues were dehydrated in a graded ethanol series, followed by xylene. Tissues were paraffine-embedded, sectioned at 4μm and mounted on Superfrost Plus slides (Thermo Fisher Scientific). Sections were deparaffinised, rehydrated, bleached for 10 min. in 3% H2O2. Antigens were retrieved using 10mM Na-citrate, pH 6 for 20 minutes at boiling temperature. The following antibodies were used: Phospho-ERK (T202/Y204; #4370 Cell Signal); anti-RFP (1:200; #600-401-379 Rockland). ImmPRESS secondary antibody and NovaRED substrate kits (Vector Labs) were used for signal detection, according to manufacturer’s protocols.
SW48 and Caco2 CRC cells were cultured in L-15 and DMEM, respectively, supplemented with 10% fetal bovine serum. Cells were transfected using Lipofectamin 3000 Transfection Reagent (Thermo Fischer) with vectors encoding BRAFV600E, KRASG12V or FLUC linked to tdTomato and the pTet-on Advanced Vector (Clontech). Cells were starved in medium containing 0.1%fetal bovine serum and induced with 2 μg/ml doxycycline 48h after transfection. 24 h later, cells were harvested using TrypLE, washed, rested for 30 min at 37°C in starvation medium and fixed in 4% PFA for 15 min. at 37°C. Fixed cells were washed in PBS/1% BSA, permeabilised in MeOH at -20°C overnight, and immunostained with Alexa Fluor 488 mouse anti-ERK1/2 (pT202/pY204) antibody (1:10; 612592, BD Bioscience) for 30 min.
Immunofluorescence and microscopy
For immunofluorescence imaging, organoids were washed with PBS and fixed in-well with 4% paraformaldehyde (PFA) for 30 min at 37°C. Fixation was stopped with PBS containing 100 nm Glycine. Cells were blocked and permeabilised with blocking buffer (PBS containing 1% BSA, 0.2 % Triton X100, 0.05 % Tween-20) for at least 2.5 h at room temperature. Samples were incubated for 36 h at 4°C with primary antibodies against lysozyme (1:250; ab108508, Abcam) or Ki-67 (1:100, PA5-16785, Invitrogen) diluted in blocking buffer. After washing with IF-buffer (PBS containing 0.1% BSA, 0.2 % Triton X100, 0.05 % Tween-20), samples were incubated for 24h at 4°C with secondary antibody Alexa Fluor 647 anti-rabbit (1:500, #4414, Molecular Probes) diluted in IF-buffer. Samples were counterstained for 5 min at room temperature using 0.5 μg/ml DAPI. After washing with IF-buffer, stained cultures were released from the Matrigel and collected in PBS. Samples were washed, resuspended in remaining PBS and mounted on slides using Vectashield Antifade Mounting Medium (H-1000, Vector).
Immunofluorescence and FIRE reporter images were taken with a Leica TSC SPE confocal microscope using an ACS 20x oil-immersion objective, solid-state lasers (405, 488, 532 and 635 nm) as sources of excitation and LAS X operating software (Leica). Light microscopy images of cultures were taken with a Biozero microscope using a Plan Apo 4x NA 0.20 objective and Biozero observation and analyser software (Keyence).
For transmission electron microscopy, organoids were induced for 24 h, removed from Matrigel and fixed in a buffer containing 2% paraformaldehyde and 2.5% glutaraldehyde at 4°C. Regions of approximately 100-150 μm2 showing representative sections through organoids were imaged on a 120 kV Tecnai Spirit transmission electron microscope (FEI) equipped with a F416 CMOS camera (TVIPS). Micrographs were recorded automatically at a final magnification of 4400x (2.49 nm pixel size at object scale) and -10 μm defocus using Leginon [49] and then stitched using TrakEM2 [50].
Flow cytometry and Fluorescence-activated cell sorting
Flow cytometry of α-pERK-stained CRC cells resuspended in PBS/1%BSA cells was done using an Accuri cytometer (BD). Cells were gated for populations displaying different tdTomato fluorescence values (negative, low, medium and high), which correlates with transgene expression. For each population, the mean α-pERK fluorescence values were determined and normalised to the tdTomato negative fraction of the corresponding FLUC control experiment.
For fluorescence-activated cell sorting of organoid cells, single cell suspensions from induced organoids were prepared by digestion with TrypLE (Thermo Fisher Scientific) in the presence of 2 mM EDTA and 200 u/ml DNAse I. Digestion was monitored by visual inspection, and stopped by crypt culture medium containing 0.2% bovine serum albumin. Cell suspensions were filtered through 30 μm Celltrix filters and stained with an anti-CD44-antibody conjugated to Allophycocyanin (APC; clone IM7, BioLegend) and the Green or Near-IR Live/Dead Fixable Dead Cell Stain Kits (Life Tech) for subsequent exclusion of dead cells. Single cells were sorted into the 96-well plates of the Precise WTA Kit (BD) with pre-dispensed library chemistry using a BD FACSAriaII SORP (BD) and a gating strategy as displayed in Supplementary Figure 3. Cells were sorted into quadrants of plates to minimize batch effects between plates. For later analysis of CD44 positivity of the subsets, sorts were performed as index sorts.
RNA sequencing and bioinformatic analyses
For single cell RNA sequencing, the Precise WTA Kit (BD) was used, according to the manufacturers’ instructions. Sequences were produced using NextSeq and/or HiSeq chemistry (Illumina). Cluster generation on NextSeq 500 followed the instructions of the manufacturer, at a final loading concentration of 2 pM on a High-Output-Flowcell. 1% PhiX was added as quality control, at least 4×107 paired reads per pool were gained during a Paired-End-75 run. Library-pools running on the HiSeq4000-system were prepared according to Illumina recommendations, loaded with 200 pM and sequenced during a Paired-End-75 run. Again, 1% PhiX was added as quality control, and at least 4×107 read-pairs per pool were targeted.
Single cell RNA-sequencing data was pre-processed using the BD Precise Whole Transcriptome Assay Analysis Pipeline v2.0 [51]. Quality control was performed using scater [52]. Read counts were normalised using the trimmed mean of median values (TMM) approach provided with edgeR [53]. Normalised read counts were used for k-means clustering and t-SNE visualisation. Differentiation trajectories in t-SNE plots were determined using slingshot [54], with intestinal stem cell cluster 1 as predefined origin. Differentially expressed genes were called on log-transformed raw counts using a hurdle model provided with R package MAST [55]. Top-10 signature genes per cluster were identified by comparing average gene expressions within cluster to average gene expressions across all other clusters. For bulk cell RNA sequencing, organoids were induced for 24 h with 2 μg/ml doxycycline in CCM-REN medium and subsequently dissociated, as described before [47]. RNA-seq reads were aligned to the mouse genome GRCm38 using STAR aligner with GENCODE annotation vM11. Differentially expressed genes were called using DESeq2. scRNA-seq and bulk RNA-seq data are available in the GEO repository under accession numbers GSE115242 and GSE115234, respectively.
Statistics
Error bars in figures denote standard deviations. p-values are calculated form two-tailed unpaired t-tests in GraphPad prism. *, ** and *** denote p-values <0.05, <0.01 and <0.001, respectively.
Funding
The work was in part funded by Deutsche Forschungsgemeinschaft (Grant MO2783/2-1 to MM), Berlin School of Integrative Oncology (starter grant to NB and MM), and the German Cancer Consortium DKTK (single cell grant to NB and MM).
Author contributions
RB, PR, CG, SiS, NM, BF, IAES conducted, analysed, and interpreted experiments; FU performed bioinformatic analyses; PR, TM, BGH, CS, NB, MM conceived, designed, interpreted experiments and/or supervised parts of the study; MM wrote the manuscript.
Conflict of interest
The authors declare that they have no conflict of interest.
Acknowledgements
The authors gratefully acknowledge excellent technical assistance by Gaby Blaess and Sonja Banko (MPIMG, Berlin) for mouse genotyping and mouse care, respectively. The authors are also gratefully acknowledge the help of lab students Ekaterina Eroshok and Maximilian Anders (Molecular Medicine Masters’ programme, Charité – Universitätsmedizin Berlin) with immunohistochemistry in the early phase of this project. We received the FIRE plasmid as a kind gift from John Albeck, UC Davis.