Novel high throughput 3D ECM remodeling assay identifies MEK as key driver of fibrotic fibroblast activity

Figures

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  Fig. 1. Multi-cluster model for quantitative assessment of TGFβ- and S1P-induced ECM remodeling. (A) Cartoon showing model design and brightfield and reflection images of the ROI after 72 h TGFβ treatment. (B) Multiple clusters of NF or AF printed in collagen at 900 μm distance and maintained in control medium (CTR) or stimulated with TGFβ or S1P for 72 h. Brightfield and reflection images are shown of a single Z-section through the clusters. Note that the pattern of white spots in the reflection images is an artefact caused by reflection from optical elements in the microscope [36]. Bar = 500 μm. The white square indicates the area depicted in (C). (C) Single Z-section reflection images for NF and AF collagen-embedded fibroblast clusters treated with or without TGFβ or S1P for 48 and 72 h. Note that the ROI (red rectangle) does not include the white dots caused by the reflection artefact. Bar = 100 μm. (D) Quantification of reflection signal from (C) taking the average reflection signal density in the ROI across the Z-stack. Graphs show the mean and SEM of two independent experiments, each performed in duplicate. Two-way ANOVA followed by Dunnett's multiple comparisons test was…

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  Fig. 2. TGFβ stimulated fibroblast clusters show cells with prominent F-actin fibers interacting with a bundle of remodeled ECM containing collagen andFN. (A) Maximum projection images showing ⍺SMA and F-actin expression in normal- (NF) or TGFβ pre-activated fibroblast (AF) clusters exposed to control medium (CTR) or TGFβ containing media. Blue, Hoechst; Green, ⍺SMA; red, F-actin. Bar = 100 μm. (B) Brightfield and maximum projection images showing FN expression in NF or AF clusters exposed to CTR or TGFβ containing media. Blue, Hoechst; Green, FN. Bar = 100 μm. (C) Brightfield, reflection, and collagen immunostaining of collagen-embedded AF clusters exposed to CTR or TGFβ containing media. Arrow indicates collagen bundle formed between two AF clusters in presence of TGFβ. The image shows a single Z-section through the clusters. Bar = 100 μm. (D) Brightfield and FN immunostaining of collagen-embedded AF clusters exposed to CTR or TGFβ containing media. The image shows a single Z-section through the clusters. Bar = 100 μm. (E) Maximum projection and single Z-section images showing F-actin and reflection for AF under CTR or TGFβ conditions. White arrows indicate F-actin fibers in protruding…

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  Fig. 3. Cytoskeletal contractility and collagen fiber cross-linking are required for TGFβ mediated ECM remodeling by fibroblast clusters and application of the model to lung fibroblasts. (A) ECM remodeling visualized by reflection microscopy between two AF clusters stimulated for 72 h with TGFβ in presence of the indicated concentrations of two LIMKi or ROCKi. CTR and TGFβ conditions without inhibitors are shown below. Bar = 100 μm. (B) Quantification of the reflection signals as shown in (A). Graphs show the mean and SEM of two independent experiments, each performed in triplicate. Two-way ANOVA followed by Dunnett's multiple comparisons test was performed; ∗∗, p < 0.01; ∗∗∗, p < 0.001 compared to the TGFβ alone condition. (C) Z-stack of reflection images showing ECM remodeling between two AF clusters under CTR or TGFβ stimulation in absence or presence of the indicated concentrations of the GSK-429286A ROCKi. (D) Cartoon illustrating imaging areas for assessing ECM remodeling and cell viability (top), and brightfield and reflection (single Z-section) images of the area between AF clusters and Hoechst (blue)/PI (red) staining (maximum projection) inside AF clusters exposed to CTR or…

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  Fig. 4. TGFβ mediated ECM remodeling by AF clusters is irreversible. (A) Reflection (single Z-section) images of the area between AF clusters and Hoechst (blue)/PI (red) staining (maximum projection) and brightfield images inside AF clusters exposed to TGFβ for 72 h followed by exposure to CTR media in absence or presence of the indicated concentrations of the GSK-429286A ROCKi or the βAPN LOXi. Bar = 100 μm. (B) Quantification of reflection signals and Hoechst/PI staining as shown in (A). Graphs show the mean and SEM of two independent experiments, each performed in triplicate. Two-way ANOVA followed by Dunnett's multiple comparisons test was performed; ∗, p < 0.05; ∗∗∗, p < 0.001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

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  Fig. 5. Application of the developed model to screening of candidate anti-fibrotic compounds – ROCKi as example. (A) Cartoon showing setup for screening of candidate anti-fibrotic drugs: Fibroblasts are pre-activated with TGFβ for 72 h. Subsequently, two clusters of pre-activated fibroblasts (AF) are printed at 900 μm distance into each well of a 96-well plate prefilled with collagen gel. Cultures are exposed to a range of compound concentrations for 90 min, followed by the addition of TGFβ. Wells exposed only to TGFβ serve as positive control and wells exposed to control medium (CTR) serve as the negative control. After 72 h, Hoechst and Propidium Iodide (PI) are added to the wells. The efficacy and cytotoxicity of the compounds are then evaluated using confocal reflection microscopy and confocal fluorescence microscopy. The cartoon of the flask and 96-well plate was obtained from Servier Medical Art (https://smart.servier.com/), which is licensed under a Creative Commons Attribution 4.0 Unported License (https://creativecommons.org/licenses/by/4.0/). (B) Compounds used in this study with their highest concentration tested based on 1000 × IC50(cell free assay) where possible. ∗Indicates…

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  Fig. 6. Application of the developed model to screening of candidate anti-fibrotic compounds –unanticipated effect of TβRi. (A) Heatmap showing the effect of the indicated concentration ranges of compounds on the reflection microscopy signal between TGFβ-stimulated AF clusters (blue; scale ranging from 0 to 150; higher values indicate higher ECM remodeling) and on the proportion of PI-positive cells within the clusters (red; scale ranging from 0 to 1; higher values correspond to higher cytotoxicity). Highest concentration for each compound indicated as '1′, followed by subsequent 1:3 dilutions. Left Y-axis marks three biological replicates analyzed. (B) Reflection (single Z-section) images of the area between AF clusters and Hoechst (blue)/PI (red) staining (maximum projection) inside AF clusters exposed for 72 h to TGFβ containing media in absence or presence of the indicated concentrations of the two indicated TβRi. Bar = 100 μm. Graphs show quantification of reflection signals and Hoechst/PI staining. Mean and SEM of two independent experiments, each performed in triplicate. Two-way ANOVA followed by Dunnett's multiple comparisons test was performed; ∗, p < 0.05; ∗∗, p < 0.01 compared…

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  Fig. 7. MEK activation is essential for ECM remodeling induced by TGFβ or by high concentration TβRi. (A) CAGA12-dynGFP signal in 2D cultures of NF treated with CTR medium or TGFβ for 72 h in absence or presence of the indicated concentrations of the LY2109761 TβRi. Blue, nucleus; Green, CAGA12-dynGFP. Bar = 100 μm. (B) Reflection (single Z-section) images of the area between AF clusters (bottom) and Hoechst (blue)/CAGA12-dynGFP (green) staining (maximum projection) inside AF clusters exposed for 72 h to the indicated combinations of TGFβ and compounds. Bar = 100 μm. (C) Quantification of reflection signals and GFP intensity from (B). Graphs show the mean and SEM of two independent experiments, each performed in triplicate. Two-way ANOVA followed by Dunnett's multiple comparisons test was performed; ns, not significant; ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001. (D) Western blot analysis of total- and phospho-ERK in 2D cultures of AF treated with CTR or TGFβ-containing medium in presence or absence of the indicated concentrations of the LY2109761 TβRi. GAPDH serves as loading control. Graphs show the mean and SD of two independent experiments. Two-way ANOVA followed by Dunnett's multiple…