Direct targeting of the downstream mitogen-activated protein kinase (MAPK) pathway to suppress extracellular-regulated kinase (ERK) activation in KRAS and BRAF mutant colorectal cancer (CRC) has proven clinically unsuccessful, but promising results have been obtained with combination therapies including epidermal growth factor receptor (EGFR) inhibition. To elucidate the interplay between EGF signalling and ERK activation in tumours, we used patient-derived organoids (PDOs) from KRAS and BRAF mutant CRCs. PDOs resemble in vivo tumours, model treatment response and are compatible with live-cell microscopy. We established real-time, quantitative drug response assessment in PDOs with single-cell resolution, using our improved fluorescence resonance energy transfer (FRET)-based ERK biosensor EKAREN5. We show that oncogene-driven signalling is strikingly limited without EGFR activity and insufficient to sustain full proliferative potential. In PDOs and in vivo, upstream EGFR activity rigorously amplifies signal transduction efficiency in KRAS or BRAF mutant MAPK pathways. Our data provide a mechanistic understanding of the effectivity of EGFR inhibitors within combination therapies against KRAS and BRAF mutant CRC.
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Source data are provided with this paper. All other data supporting the findings of this study are available from the corresponding author on reasonable request.
Custom-written ImageJ/Fiji-scripts that were used to analyse 2D and 3D FRET data are available from the corresponding author upon request.
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We thank members of the Snippert, Riquet and Trusolino laboratories for reagents, suggestions and discussions. We thank M. Gloerich for careful reading of the manuscript. This work is part of the Oncode Institute, which is partly financed by the Dutch Cancer Society and was funded by the Gravitation Program CancerGenomics.nl from the Netherlands Organisation for Scientific Research (NWO), by a grant from the Dutch Cancer Society (KWF/UU2013-6070, H.J.G.S.), ERC starting grant (IntratumoralNiche, H.J.G.S.) and a ‘Sta op tegen Kanker’ International Translational Cancer Research grant (J.L.B.). Stand Up to Cancer is a programme administered by the AACR. Research was further supported by EOS MODEL-IDI (FWO grant no. 30826052), iBOF ATLANTIS (BOF20/IBF/039), FWO research grants (G.0E04.16N, G.0C76.18N, G.0B71.18N and G.0B96.20N), Methusalem (BOF16/MET_V/007), Foundation against Cancer (F/2016/865 and F/2020/1505), CRIG and GIGG consortia, and VIB (to P.V.). This research was supported by the Agence Nationale pour la Recherche (ANR) via the G2Progress programme (ANR-13- BSV2-0016-02, F.B.R.). F.B.R. acknowledges the Nikon BELUX partnership and funding from Oseo–Ministère de l’enseignement supérieur et de la recherche via the national contest 2013 d’aide à la création d’entreprise de technologies innovantes catégorie émergence in the context of the KiBioS spin-off project. This collaborative work was encouraged by the CNRS Groupement de recherche (GDR) 2588 ‘Microscopie et Imagerie du Vivant’ scientific community via the biosensor workgroup initiative and especially during IMOB2018. Additional funding was provided by AIRC (Associazione Italiana per la Ricerca sul Cancro) Investigator grants 20697 (A.B.) and 22802 (L.T.), AIRC 5x1000 grant no. 21091 (A.B. and L.T.), AIRC/CRUK/FC AECC Accelerator Award 22795 (L.T.), European Research Council Consolidator Grant 724748—BEAT (A.B.), H2020 grant no. 754923 COLOSSUS (L.T.), H2020 INFRAIA grant no. 731105 EDIReX (A.B.) and Fondazione Piemontese per la Ricerca sul Cancro-ONLUS, 5x1000 Ministero della Salute 2014, 2015 and 2016 (L.T.). A.B. and L.T. are members of the EurOPDX Consortium.
The authors declare no competing interests.
Peer review information Nature Cell Biology thanks the anonymous reviewers for their contribution to the peer review of this work. Peer reviewer reports are available.
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a, Typical mitotic EKAREV-FRET profile in HEK293 cells, including rising phase, steep increase at nuclear envelope breakdown (NEB) and sharp decline at anaphase (Supplementary Movie 1). Corresponding snapshots of above cells with H2B-mScarlet support cell-cycle phases (20 cells; 1 experiment). 1, G2; 2, NEB; 3, metaphase; 4, anaphase; 5, cytokinesis (c.k.); 6, G1. FRET-signal relative to PMA saturation (150nM). Black, FRET-ratio of YPet(yellow)/Turq2(blue) intensities. b, As a, with cell-cycle stages recognized by EKAREV(Tq) biosensor exclusion from condensed chromosomes; consistently observed (53 cells, 4 experiments). c, As a, but EKAREV(Tq) biosensor lacking nuclear localization. Observed in 28 cells, 2 experiments. d, As a, but EKAREV(TA) control biosensor that cannot be phosphorylated. Observed in 5 cells, 1 experiment. e, EKAREV(Tq) FRET signal in mitotic arrested HEK293 cells (nocodazol, 0.83μM; 2hrs) is sensitive to CDK1 inhibitor RO-3306. In mitotic cells, recognized by absence of nuclear localization (NEB) of NLS-tagged biosensor (insert images), FRET decreased upon 10µM RO-3306 (9 cells; 2 experiments with similar results), or 3x 1µM (1 cell). e’, Loss of normalized FRET signal (ΔR, %) upon RO-3306 (10µM) or MAPK pathway inhibitors (sel+SCH, 5μM each). Box-and-whisker plots: boxes represent quartile 2 and 3, horizontal line represents median, whiskers represent minimum and maximum within 1,5x interquartile range. RO-3306: n=23 cells, sel+SCH n=16 cells. f, EKAREV(Tq) FRET signal is sensitive to CDK1-inhibition in G2-phase. Synchronized cells were imaged before, during and after incubation with RO-3306 (10µM) and retrospectively analyzed if mitotic entry was observed <15 minutes after drug washout (n=23 cells). Right, similar experiment, here inhibiting MEK and ERK (n=20 cells). Graph shows mean±s.d. of baseline-normalized traces. g, As f, monitoring G2-phase in HeLa cells (n=19) co-expressing ERK-KTR-mCherry and EKAREV(Tq). ERK-KTR biosensor suffers from same undesired CDK1-sensitivity. ***, two-sided student’s T-test, p<0.0005. Scale bar, 10μm.
a, HeLa cells expressing EKAREV substrate variants Alt_ERK_Substr_1-6 (Extended Data Fig. 4b). PMA, 500nM; SCH, 10μM. Right, responses (mean±s.d.), normalized to EKAREV-GW4.0. b, HeLa cells expressing EKAREV-GW variants were arrested in mitosis (Extended Data Fig. 1e) to test CDK1/cyclinB sensitivity (RO-3306). Purple, mean of individual traces. Right, overview for several variants, mean ratio loss ± s.e.m. Best responder EKAREV-GW(Alt_substr._6) is compromised by RO-sensitivity. c, Repeat of Fig. 1e, quantifying FRET in G2- and M-phase HEK293 cells(mean ± s.d), complemented with results from third-residue-substitution variants (purple). No improvements compared to EKAREN4/EKAREN5. d, Sensor dephosphorylation kinetics, assessed by instant ERK inactivation (sel+SCH, 5μM) after initial sensor saturation (PMA). For identical experimental conditions, HEK293 stably expressing EKAREV(Tq) or EKAREN4 were mixed. H2B-mScarlet selectively marked EKAREV(Tq) (left) or EKAREN4 cells (right) (insets: scale, 25 μm). Cells analyzed individually and averaged after double normalization (baseline and PMA-plateau). e, Maximum FRET range (ΔR(%), approximated through saturation (PMA) in serum-starved HEK293 cells of widely variable expression levels. e’, Baseline and plateau ratios corresponding to cells in e. Increased FRET range of EKAREN4 likely results from elevated plateau ratios. Expression levels affect FRET range by differentially affecting baseline ratios (see slopes in a.u.). Experiment performed twice. f, As e, differential effect of expression level on FRET range is similar for ERK-insensitive control sensors EKAREV(TA) and EKAREN4(TA). g, Mean (± s.d.) ΔR per expression level category (see e). For panels a-g the n numbers represent cells and are indicated in the graph for each group. Box-and-whiskers: boxes represent quartile 2 and 3, horizontal line represents median, whiskers represent minimum and maximum within 1,5x interquartile range. Dots are outliers. P values in all relevant panels were calculated using a two-sided student’s T-test, * p<0.05; ***, p<0.0005. n.s., non-significant.
a, FRET-range versus sensor expression level, as in Extended Data Fig. 2e (EKAREN4, n=80 cells; EKAREN5, n=75 cells). a’, baseline and plateau ratios corresponding to cells plotted in a. b, Means (± s.d.) of ΔR from a, calculated in three expression level categories (as in Extended Data Fig. 2g). c, Dephosphorylation kinetics of EKAREN5 were directly compared with EKAREV(Tq) (as Extended Data Fig. 2d). Retrospective unmixing was based on clustering plateau amplitudes (PMA) (see Fig. 2a). Experiment performed once. d, As in c, comparing phosphorylation and dephosphorylation kinetics of EKAREN5 with ~33-fold expression level difference. Co-seeded high and low expressors were simultaneously monitored. Experiment performed three times. e-h, Various automated analyses on autonomous ERK fluctuations of HeLa cells (dataset of Fig. 2f,g), registered simultaneously by ERK-KTR-mCherry and either of EKAREV/EKAREN FRET sensors. EKAREV, n=15 single-cell traces; EKAREN4, n=10 single-cell traces; EKAREN5, n=17 single-cell traces. e, Automated peak counting per individual cell. f, Temporal matching of rising phases in KTR versus FRET signals. g, Temporal matching of falling phases in KTR versus FRET signals. h, Counted ‘inflection’ points per trace, that is points where ERK changes accelerate or decelerate(see Methods). i, Correlation between EKAREN5-FRET and ppERK staining (mean nuclear signal). After various ERK manipulations, HeLa-EKAREN5 cells were FRET-imaged and fixed instantly after acquisition, yielding various ERK activity states between complete inhibition (MEKi+ERKi) and pathway saturation (>7 min EGF). Grey line, regression analysis (y=ax+b). Traces are mean ratios ± s.d. For panels a-i the n numbers represent cells and are indicated in the graph for each group. Box-and-whisker plots: boxes represent quartile 2 and 3, horizontal line represents median, whiskers represent minimum and maximum within 1,5x interquartile range. Dots, outliers. Scale bar, 50μm. Two-sided student’s T-tests: *, p<0.05; **, p<0.005; n.s., non-significant.
a, Silent mutations introduced into Turquoise2 (insert) to minimize sequence homology with the YPet fluorophore in the same construct. Red ‘x’ marks silent mutations. Blue, residues discriminating Turquoise2 from parental eCFP (Goedhart et al.30). Green, residues rendering Turquoise2 prone to dimerize with YPet, in analogy to EKAREV design (Komatsu et al., 201128). Dark green, the V224L mutation was added to further enhance dimerization and, hence, FRET efficiency in ON-state (Vinkenborg et al., 200732). b, Alternative ERK substrate sequences were derived from ERK targets RSK1 (human) and ELK1 (human) using the Kinexus website and compared to parental EKAREV-GW-4.0 (GW = GateWay) with CDC25C substrate sequence. c, Overview of generated and tested point mutant variants of EKAREV. Red, central Threonine, target of ERK phosphorylation. Blue, the Lysine at position +4 mimics the general CDK1-consensus site, mutated to Proline (K->P). Purple, the Lysine at position +6 mimics the CDK1-consensus site and mutated to bulky Trp (K->W) to create steric hindrance with cyclinB. Underlined, ERK docking domain FQFP. Purple boxed characters, rational attempts to further eliminate CDK1-sensitivity with third amino acid replacements. V422T was aimed at favoring ERK over CDK1 consensus site; L427W was aimed at further augmenting sterical hinderance of cyclinB interaction; L427E was aimed at impeding cyclinB interaction through electrostatic repulsion. The Asp (D) at position+3 was left unchanged for its reported importance for the Pin1 affinity (Komatsu et al., 201128). d, Summary of available EKAREN4 and EKAREN5 plasmid constructs (including variable targeting motifs and Thr-Ala control versions), as well as adapted version of pInducer20 (Meerbrey et al., 201145) to initiate expression of HRASN17 and P2A-coupled reporter fluorophore mKate2-NLS. Constructs were deposited at Addgene.
a, PDO model expressing the non-phosphorylatable (hence ERK-insensitive) control sensor EKAREV(TA) was FRET-imaged to assess non-biological geometry effects on raw signals in 3D organoid FRET microscopy. YFP/CFP ratios can differ between organoids situated either far away or close to the objective (distance difference ~150 μm). Experiment performed once, this direct comparison representing general observations. b, Performing FRET acquisition, Turq2 and YPet emissions were determined from all cells of a bulky organoid (>200 cells) and plotted against their z-coordinates. YFP/CFP ratios increase subtly with increasing depth within the organoid, likely due to differential scattering-induced loss of fluorescence between the two fluorophores. Experiment performed twice with same outcome.
a, As in Fig. 3b, here using selumetinib at 50 nM concentration. Shown are 33 single-cell analyses from two p9T organoids. Right, waterfall plot summarizing cellular recovery from ERK inhibition (see also Fig. 3b). b, Exact copy of Fig. 3b, using selumetinib at 200 nM concentration. Shown are 59 single-cell analyses from three p9T organoids. Right, waterfall plot summarizing the cellular recovery from ERK inhibition. c, As in Fig. 3b, here using selumetinib at 1 μM concentration. Shown are 42 single-cell analyses from three p9T organoids. Right, waterfall plot summarizing the recovery from ERK inhibition. d, Three example FRET traces to illustrate the power of time-resolved signal transduction analysis. Trace colours correspond to bars indicated with coloured arrows in waterfall plot of c. Top, cell displaying onset of recovery, interrupted by super-inhibition. Middle, selumetinib-induced inhibition is followed by a sustained phase without apparent recovery. Bottom, inhibitory effect is more prolonged, explaining negative outcome for Recovery (= ‘Recov’ – ‘Inh’). Grey, green and purple colours correspond with bars in waterfall plot of Extended Data Fig. 6b.
Extended Data Fig. 7 EGFR plays a central role in generating pulsatile ERK dynamics in PDO-KRASG12V.
a, Fluctuating ERK dynamics in PDO-KRASG12V, abolished by afatinib at 50nM concentration. Shown traces are representative for 14 single-cell analyses from 3 PDOs. Experiment performed twice. b, As a, loss of ERK activity oscillations, observed in PDO-KRASG12V upon administration of pan-HER inhibitors lapatinib (1 μM, 21 cells), dacometinib (500 nM, 12 cells), or EGFR-specific inhibitors erlotinib (500 nM, 17 cells) and gefinitib (500 nM, 18 cells). Two independent experiments performed. ~5 organoids per condition. c, As a, loss of ERK oscillations, observed in PDO-KRASG12V upon anti-EGFR antibody cetuximab (500ng/ml). Residual ERK activity was sensitive to trametinib (MEKi). Experiment performed once; 30 cells analyzed. d, Oscillating ERK dynamics persist in PDO-KRASG12V despite HER2-inhibitor CP-724714 (5 μM). Three traces representative for 24 single-cell analyses, from one experiment with 4 organoids. e, FRET-trace demonstrating that afatinib (200nM) instantly interrupts rising phase of pulsatile ERK (arrow). Representative for >20 observations in various PDO-lines. f, Shp2-inhibitor SHP099 (5μM) abrogates autonomous ERK activity in PDO-KRASG12C, but not BRAFV600E(#4). Shown are representative multi-cellular z-plane analyses. Experiment performed once; 3 organoids. f’ BRAFV600E(#4) is similarly unresponsive to drugs targeting Src (KX2, 500 nM), FAK (PND-1186, 500 nM) and cKit/ PDGFR/ Bcr-Abl (dovitinib, 100 nM; imatinib, 1.0 μM; masitinib, 100 nM; pazopanib, 250 nM). Shown are 2x-normalized ratios (mean±s.d.). n numbers represent PDOs and are indicated in the graph per group. Experiment performed once. g, Adapted pInducer20 for doxycyclin-inducible expression of HRASN17 and P2A-coupled reporter mKate2-NLS (TRE2, Tet-Responsive Element). Western blot demonstrating doxycyclin-mediated induction of HRASN17 expression (general anti-RAS antibody) in BRAFV600E(#3)(EKAREN5+pInducer). S.E., short exposure; L.E., long exposure. Vinculin as loading control. Experiment performed once. h, Western blot analyses on indicated PDOs illustrating pan-HER inhibition on components of the linear EGFR-MAPK-pathway. Pharmacological treatments as in Fig. 4e. Experiment performed once.
a, Single-cell analyses from data set presented in Fig. 7b. After 72 hrs afatinib (1 μM), EKAREN5 showed constant, non-oscillatory basal ERK signal. Rare activity spikes in few cells were observed in PDO-NRASQ61H and BRAFV600E(#3). b, Single-cell drug response analysis after 8 days of afatinib treatment (1μM). ERK dynamics in EGFR-inhibited PDO-KRASG12C (63 cells in 6 organoids) and PDO-NRASQ61H (70 cells in 5 organoids) are constant, non-oscillatory in nature. Depletion of the continuous oscillatory dynamics unmasked a hidden pattern of (EGFR-independent) rare activity spikes in few cells (one per ~30 hr (19 pulses observed in 571 hours of single-cell signaling evaluation). Never were two pulses observed in one single-cell derivation. Plots show all single cell analyses from multiple organoids or from a single representative organoid. Dotted line: to indicate that the two pulses are from separate cells.
a, Growth assay performed as in Fig. 7c,d, here monitoring the BRAFV600E mutant PDOs #1, #2 and #3 under treatment with drugs from clinical trial by Kopetz et al. (NEJM, 20197). Cetuximab, 1000 ng/ml; binimetinib (MEK-inh), 100 nM; encorafenib (BRAF-inh), 300 nM. Mean number of objects per time point: BRAFV600E(#1), n=84; BRAFV600E(#2), n=62; BRAFV600E(#3), n=78. For the exact n-numbers of all presented points, see Source Data file. Data are represented as mean size ± s.e.m. Growth medium was supplemented with 200x reduced EGF concentration (0.25 ng/ml) to minimize competition between cetuximab and EGF ligand. Experiment performed once. b, In same PDO lines, ERK responses were recorded using EKAREN5-FRET. Data are represented as mean value ± s.d. Quantification scheme based on double calibrated multi-cellular z-plane analyses (n numbers represent PDOs and are indicated in the graph for each group). ERK levels were maximally reduced in presence of triple combination. Box-and-whisker plots: boxes represent quartile 2 and 3, horizontal line represents median, whiskers represent minimum and maximum within 1,5x interquartile range. Dots are outliers.
Supplementary discussion of the characterization and applications of ERK biosensors. This discussion summarizes and analyses observations made during the optimization of this ERK biosensor and discusses the application of FRET biosensors in 3D organoids.
Driver mutations present in the panel of CRC PDOs. Overview of driver mutations present in the panel of KRASG12X and BRAFV600E mutant CRC organoids used in this study. Left, CRC PDOs drawn from ref. 23. Right, CRC PDOs drawn from Foundation Hubrecht Organoid Technology (HUB).
EKAREV(Tq) ERK biosensor shows large ratio peak during G2 and M phase. Analysis of EKAREV(Tq) FRET in HEK293 cell during G2- and M-phase. The time-lapse movie corresponds to Extended Data Fig. 1a. From left to right: co-expressed H2B-mScarlet marks the mitotic stages, sensor fluorescence (Turquoise2), FRET ratio movie, normalized ratio curve (YFP/CFP) corresponding to the drawn ROIs. PMA was added to induce sensor saturation (time point t127).
Simultaneous readout of pulsatile ERK dynamics using EKAREN5-FRET and ERK-KTR-mCherry. Top, EKAREN5-FRET. From left to right: sensor fluorescence, FRET ratio, normalized ratio curve (YFP/CFP) corresponding to the drawn ROIs. Bottom, KTR analysis in same cell. From left to right: FRET sensor (used to demarcate the nucleus), KTR sensor (shown ROI defines cytosolic compartment), normalized ratio curve (cytosol/nucleus). Note the variety of peak amplitudes.
Single-cell analysis of selumetinib response in PDO-KRASG12V. Single-cell analyis of one of the two neighbor cells shown in Fig. 3b. From left to right: sensor fluorescence (Turquoise2), FRET ratio movie, normalized ratio curve (YFP/CFP) corresponding to the drawn ROIs.
Single-cell analysis of afatinib response in PDO-BRAFV600E(#3). Single-cell analyis shown in Fig. 4b′. From left to right: sensor fluorescence (Turquoise2), FRET ratio movie, normalized ratio curve (YFP/CFP) corresponding to the drawn ROIs. Before addition of afatinib (200nM), all cells show oscillatory ERK activity behavior.
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Unprocessed western blots.
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Unprocessed western blots.
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Cite this article
Ponsioen, B., Post, J.B., Buissant des Amorie, J.R. et al. Quantifying single-cell ERK dynamics in colorectal cancer organoids reveals EGFR as an amplifier of oncogenic MAPK pathway signalling. Nat Cell Biol 23, 377–390 (2021). https://doi.org/10.1038/s41556-021-00654-5
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