Targeting mutant RAS in patient-derived colorectal cancer organoids by combinatorial drug screening
- Center for Molecular Medicine, University Medical Center Utrecht, Netherlands
- Cancer Genomics Netherlands, Netherlands
- University Medical Center Utrecht, The Netherlands
- Netherlands Cancer Institute, The Netherlands
Figures
Figure 1 with 1 supplement
Drug responses of patient-derived CRC organoids with and without mutant KRAS.
(A) Dose-response curves of patient-derived CRC organoids P8T (KRASWT; APC and TP53 mutant) and P26T (KRASG12V; APC and TP53 mutant) treated with the dual EGFR/HER2 inhibitor afatinib, MEK inhibitor …
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Figure 1—source data 1
ImageJ/Fiji macro script: ‘Organoid movie macro’.
Converts XYZT confocal data sets into analyzable 2D-movies, consisting four quadrants: depth coding, maximum projection in ‘glow’, transmitted light image and a merge between transmitted light and fluorescence. All supplementary movies were generated using this method (Figures 1 and 3 show 2 of 4 quadrants only).
- https://doi.org/10.7554/eLife.18489.004
Figure 1—figure supplement 1
Stills from representative time-lapse imaging (three days) of CRC organoids P8T and P26T treated with vehicle (DMSO) or a combination of targeted inhibitors afatinib (33 nM) and selumetinib (200 nM) (see also Video 2).
In every panel, upper images show color-coded depth of maximum-projected z-stacks of H2B-mNeonGreen fluorescent organoids. Lower images: corresponding transmitted light images. Time interval: 15 …
Figure 2 with 1 supplement
In vivo drug response of xenotransplanted CRC organoids.
(A) P26T CRC organoids were subcutaneously transplanted in immunodeficient mice. Once tumors have grown to a volume of 300 mm3, animals were treated for 28 days with vehicle, afatinib (12,5 mg/kg; …
Figure 2—figure supplement 1
Secondary organoid cultures were derived from xenografted P26T tumors (organoid-derived xenograft, ODX) of mice that have been treated with vehicle, afatinib (12,5 mg/kg; five days on, two days off), selumetinib (20 mg/kg; five days on, two days off) or both.
Dose response curves were determined from these secondary post-xenograft cultures, as well as from the parental P26T organoid culture. Regardless of the in vivo applied drug treatment, the drug …
Figure 3 with 4 supplements
CRISPR genome editing in CRC organoids reveals effect of KRASG12D on drug response .
(A) Schematic representation of the CRISPR/Cas9-induced homologous recombination strategy to introduce the KRASG12D mutation in the KRASWT patient-derived CRC organoid P18T. Green bar: start codon. …
Figure 3—figure supplement 1
Original heat map of viability and heat map of calculated scores for p18T and p18T-KRASG12D.
Positive Bliss scores (red hues) indicate combinations where the effect is greater than expected based on additive effects.
Figure 3—figure supplement 2
Quantifying life and death during real-time imaging of drug response.
(A) Time-lapse XYZT acquisitions were visualized using color-coded depth projections and mitotic and apoptotic analysis were manually marked. (B) Increases (mitoses) and decreases (apoptosis) in …
Figure 3—figure supplement 3
Drug response of CRC organoids as examined by Western blot.
Combined Pan-HER/ MEK inhibition results in reduction of ERK phosphorylation in KRASWT and KRASG12D CRC organoids. Organoids were treated for 24 hr with MEK inhibitors selumetinib (1 μM), trametinib …
Figure 3—figure supplement 4
Stills from representative time-lapse imaging (three days) of CRC organoids P18T and P18T-KRASG12D treated with vehicle (DMSO) or a combination of targeted inhibitors afatinib (33 nM) and selumetinib (200 nM) (see also Video 2).
In every panel, upper images show color-coded depth of maximum-projected z-stacks of H2B-mNeonGreen fluorescent organoids. Lower images: corresponding transmitted light images. Time interval: 15 …
Figure 4 with 2 supplements
Differential drug sensitivities upon combination therapies including EGFR inhibition.
(A) Heat map of dose-response measurements (cell viability) in CRC organoids P18T (top panel) and P18T-KRASG12D (bottom panel). Organoids were treated (72 hr) with vehicle (DMSO) or inhibitors …
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Figure 4—source data 1
Dose-response curves for patient-derived tumor organoids P18T and P18T KRASG12D as indicated.
A number of biological replicates for each dose-response curve are indicated between parenthesis (first monotherapy/ second monotherapy/ combination therapy).
- https://doi.org/10.7554/eLife.18489.016
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Figure 4—source data 2
Dose-response curves for patient-derived tumor organoids P18T and P18T KRASG12D as indicated.
A number of biological replicates for each dose-response curve are indicated between parenthesis (first monotherapy/ second monotherapy/ combination therapy).
- https://doi.org/10.7554/eLife.18489.017
Figure 4—figure supplement 1
Upper panel: Heat map of all IC50 values (Log10-scale) for P18T and P18T-KRASG12D, determined from dataset shown in Figure 4A (and Figure 4—source data 1 and Supplementary file 1).
IC50 values are color-coded: blue for highest and red for lowest efficacies (see scale bar below). IC50 values account for both drugs when added in combination, except combinations that include …
Figure 4—figure supplement 2
Drug combinations on P18T and P18T-KRASG12D organoids targeting EGFR-RAS-ERK and PI3K-AKT pathways.
(A) Heat map of dose-response measurements (cell viability) in CRC organoids P18T (top panel) and P18T-KRAS (bottom panel). Organoids were treated (72 hr) with vehicle (DMSO) or targeted inhibitors …
Figure 5 with 2 supplements
Comparable drug response profiles in normal and tumorigenic background.
(A) Heat map of dose-response measurements of cell viability in normal colon organoids (top panel) and in normal colon organoids with an oncogenic KRAS mutation (bottom panel) after 72 hr drug …
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Figure 5—source data 1
Dose-response curves for normal and normal KRASG12D organoids as indicated.
Number of biological replicates for each dose-response curve are indicated between parenthesis (first monotherapy/ second monotherapy/ combination therapy).
- https://doi.org/10.7554/eLife.18489.021
Figure 5—figure supplement 1
Comparison of normal organoids and normal organoids with an introduced oncogenic G12D mutation within the endogenous KRAS locus.
No overall phenotypic differences are observed based on morphology and proliferative activity.
Figure 5—figure supplement 2
Upper panel: Heat map of all IC50 values (Log10-scale) for normal colon organoids with and without mutant KRAS, determined from dataset shown in Figure 5A (and Figure 5—source data 1 and Supplementary file 1).
IC50 values are color-coded: blue for highest and red for lowest efficacies (see scale bar below). IC50 values account for both drugs when added in combination, except combinations that include …
Figure 6 with 1 supplement
Screening multiple human CRC organoids confirm RAS mutational status for outcome EGFR inhibition.
(A) Dose-response curves of 11 different patient-derived CRC organoids and one engineered CRC organoid (P18T-KRASG12D) treated for 72 hr with single targeted inhibitors or combinations thereof, …
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Figure 6—source data 1
Dose-response curves for panel of patient-derived tumor organoids as indicated.
A number of biological replicates for each dose-response curve are indicated between parenthesis (first monotherapy/ second monotherapy/ combination therapy).
- https://doi.org/10.7554/eLife.18489.025
Figure 6—figure supplement 1
Top panel: Heat map of all IC50 values (Log10-scale) for multiple drug responses in CRC organoids with and without mutant RAS signaling, determined from datasets shown in Figure 6—source data 1 and Supplementary file 1.
IC50 values are color-coded: blue for highest and red for lowest efficacies (see scale bar below). IC50 values account for both drugs when added in combination. Bottom panel: Heat map of all IC50 …
Figure 7 with 1 supplement
Therapy surviving cancer cells reignite proliferation after release of targeted inhibition.
(A) Scheme of image-processing workflow. Multiple z-stacks were acquired in a tile-scan mode. H2B-mNeonGreen and bright field images were recorded of >10 organoids (left panel) over multiple days. …
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Figure 7—source data 1
ImageJ/Fiji macro script: ‘Macro Drug&release experiment’.
Guides the user through XYZ stacks of organoids, acquired at various time points (days apart). Helps to find back individual organoids and, per z-slice, lets the user indicate dead H2B particles by manual drawing. All output data are summarized in excel output file. For more detail, see Materials and methods section.
- https://doi.org/10.7554/eLife.18489.028
Figure 7—figure supplement 1
The custom-made image analysis software for quantifications in Figure 7 is extensively described in the Materials and methods Section.
In short: alive nuclei were discriminated from dead fragments by particle recognition and manual drawing based on their size and morphology. Due to long culture periods (>10 days), we decided to …
Figure 8 with 1 supplement
Cell cycle arrest upon dual inhibition of EGFR-MEK-ERK pathway.
(A) Representative cell cycle analysis of P18T-KRASG12D and P26T by flow cytometry (n = 2). DNA was stained with DAPI and DNA-synthesis was detected using a 3 hr EdU pulse to clearly discriminate …
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Figure 8—source data 1
ImageJ/Fiji macro script: ‘Score Events macro’.
Guides the user through the analysis of the event-rich organoid movies (e.g. as generated with the Organoid movie macro), by numbering and drawing indicated events (mitosis, apoptosis) in the movie and generating an overview excel file. Graphs in Figure 8D and Figure 3—figure supplement 2 were generated using this method.
- https://doi.org/10.7554/eLife.18489.031
Figure 8—figure supplement 1
Average growth speeds of the organoids were determined by linear fitting of the traces shown in Figure 8D.
The two time frames roughly correspond to the first half and the second half of the experiment. Directly after drug removal, afatinib- and selumetinib-treated organoids show a significant reduction …
Figure 9 with 3 supplements
Robust inhibition of the EGFR-RAS-ERK pathway sensitizes for navitoclax-induced cell death.
(A) Dose-response curves of patient-derived CRC organoids P18T-KRASG12D treated with the dual EGFR/HER2 inhibitor afatinib, MEK inhibitor selumetinib, BCL2/BCLXL inhibitor navitoclax or a …
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Figure 9—source data 1
ImageJ/Fiji macro script: 'Macro PI versus H2B'.
Analyzes H2B-mNeon expressing organoids, that have been labelled with propidium iodide to mark dead cells/fragments. Measure for death induction is the ratio between PI-positive pixels (dead) and H2B-positive pixels (total). Thresholding values for both channels can be tweaked. Thresholded images (z-stacks) are stored and all readouts are summarized in excel data file. For more detail, see Materials and methods section.
- https://doi.org/10.7554/eLife.18489.035
Figure 9—figure supplement 1
Dose-response curves of patient-derived KRAS mutant CRC organoids P9T and P26T treated with the dual EGFR/HER2 inhibitor afatinib, MEK inhibitor selumetinib, BCL2/BCLXL inhibitor navitoclax or a combination thereof.
Cell viability was measured by an ATP-based assay after 72 hr of drug treatment. Inhibition of the EGFR-RAS-ERK pathway using high concentrations for both afatinib and selumetinib (1 µM) strongly …
Figure 9—figure supplement 2
Dose-response curves of patient-derived CRC organoids P18T-KRAS treated with different combination therapies against the EGFR-RAS-ERK pathway with addition of BCL2/BCLXL inhibitor navitoclax and the corresponding mono and dual therapy controls.
Top panel: EGFR-RAS-ERK pathway inhibition with dual EGFR/HER2 inhibitor afatinib and MEK inhibitor trametinib. Lower panel: EGFR-RAS-ERK pathway inhibition with MEK inhibitor selumetinib and ERK …
Figure 9—figure supplement 3
Drug response of P18T-KRASG12D and P26T CRC organoids examined by Western bot after 24 hr.
Most effective reduction of p-ERK is detected when the organoids were treated with high concentrations of inhibitors afatinib (1 μM) and selumetinib (1 μM) and not at lower concentrations (65 nM of …
Videos
Video 1
Real-time imaging of cellular drug responses in tumor organoids using high concentrations targeted inhibitors.
https://doi.org/10.7554/eLife.18489.006
Video 2
Real-time imaging of cellular drug responses in tumor organoids using low concentrations targeted inhibitors.
https://doi.org/10.7554/eLife.18489.007
Video 3
Real-time imaging of cellular behavior in tumor organoids surviving treatment with afatinib and selumetinib.
P18T-KRASG12D and P26T organoids were treated for 72 hr with afatinib (1 μM) and selumetinib (1 μM), similarly to Figures 1 and 3 and Video 1. After the subsequent washout of the drugs, organoids …
Additional files
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Supplementary file 1
Dose-response data (%ATP normalized to DMSO) for all patient-derived tumor organoids as described in Figure 4—source data 1, Figure 4—source data 2, Figure 5—source data 1 and Figure 6—source data 1.
- https://doi.org/10.7554/eLife.18489.039
