collagen organoids  (Thermo Fisher)

Journal: Nature Biomedical Engineering

Article Title: Heterogeneity-driven phenotypic plasticity and treatment response in branched-organoid models of pancreatic ductal adenocarcinoma

doi: 10.1038/s41551-024-01273-9

Figure Lengend Snippet: a Organoid morphologies of epithelial (upper panel mouse line ID: 9591, from 3 independent experiments-exception is the concentration 2.5 mg/mL with only 2 independent replicates included) and mesenchymal (lower panel mouse line ID 16992, from 3 independent experiments-exception is the concentration 2.5 mg/mL with only 2 independent replicates included) organoids grown in various collagen concentrations from 1.0 mg/mL up to 2.5 mg/mL, Scale bars, 500 μm. b Quantification of the organoids major axis length in the different collagen concentrations (n = 612 for the epithelial and n = 563 for the mesenchymal), graph represents mean±sem. c Organoid morphologies for epithelial (upper panel mouse line ID: 9591, 3 independent experiments) and mesenchymal (lower panel mouse line ID: 16992, 3 independent experiments) organoids grown in collagen Type I gels with the addition of the ECM proteins fibronectin (FN, concentration 40 µg/mL), laminin (LM, concentration 40 µg/mL) or the combination (FN + LM), Scale bars, 500 μm. d Quantification of the organoids major axis length in the different collagen Type I matrices with the addition of FN, LM or both (n = 378 organoids for the epithelial and n = 388 organoids for the mesenchymal), graph represents mean±sem. e-f Heatmaps of most up and down-regulated genes between epithelial and mesenchymal organoids for the “Reactome ECM degradation” and the “WP MMPs”. g Confocal IF imaging of an EMT panel, first column: E-cadherin (green) /N-cadherin (red) /DAPI (blue), second column: E-cadherin (green) /Vimentin (red) /DAPI (blue), third column: E-cadherin (green) /β-catenin (red) /DAPI (blue), fourth column: ZEB-1 (green) /ZO-1 (red) /DAPI (blue) and fifth column: YAP (green) / phalloidin (white) /DAPI (blue) for epithelial (top) and mesenchymal organoids (bottom), scale bars upper panel (10x magnification)= 200 μm, lower panel-higher magnifications (60x)= 30 μm. Images on the second and fourth row are taken on a 60x objective (higher magnification) of their respective upper panel or from the similar organoid phenotypes. h Gene set enrichment analysis (GSEA) comparing epithelial and mesenchymal 3D organoid cultures, NES= normalized enrichment scores, FDR= false discovery rate. Every bar represents individual genes for the given gene set. i Morphologies of epithelial and mesenchymal organoids in primary, secondary and tertiary structures. Scale bars primary structures= 1000 μm, secondary 1000 for the epithelial and 500 μm for the mesenchymal and tertiary= 500 μm. j Extreme limiting dilution analysis (ELDA) and log plot of nonresponding wells vs. cell dosage for the primary, secondary, and tertiary structures of epithelial and mesenchymal organoids.

Article Snippet: For the 3D collagen organoids, a Collagenase Type V (Worthington) digestion was first performed for 12–15 min until the collagen matrix was no longer visible.

Techniques: Concentration Assay, Imaging

Journal: Nature Biomedical Engineering

Article Title: Heterogeneity-driven phenotypic plasticity and treatment response in branched-organoid models of pancreatic ductal adenocarcinoma

doi: 10.1038/s41551-024-01273-9

Figure Lengend Snippet: a , Schematic representation of the preparation of PDAC organoid cultures in floating collagen gels. Image created with BioRender.com. b , Confocal IF imaging of the organoid cytoskeleton (purple) and DAPI (blue) for epithelial and mesenchymal organoids. Images are maximum projections generated with Imaris. Scale bars, 200 µm (for mesenchymal), 500 µm (for epithelial organoids). c , Daily imaging of single-cell-derived organoids from the epithelial line (ID: 9591) and the mesenchymal line (ID: 16992) over the course of 13 days of development (here displaying Days 3–13). Scale bars, mesenchymal: 200 μm (Days 3–9), 500 μm (afterwards); epithelial: 200 μm (Days 3–5), 500 μm (afterwards). d , Major axis length of the organoid development of n = 1,099 epithelial (from the mouse line ID: 9591, 3 individual experiments) and n = 904 mesenchymal (from the mouse line ID: 16992, 3 individual experiments) organoids. Plot presents mean ± s.e.m. e , Confocal IF imaging of the proliferation marker Ki67 (green) and DAPI (blue) for epithelial (from the mouse line ID: 9591, 3 individual experiments) and mesenchymal (from the mouse line ID: 16992, 3 individual experiments) organoids. Scale bars, 100 µm (Day 3) and 200 µm (Day 5 onwards) for mesenchymal panel; 50 µm (Day 3) and 200 µm (Day 5 onwards) for epithelial panel. f , Confocal IF imaging of the epithelial marker E-cadherin (green), the mesenchymal marker N-cadherin (red) and DAPI (blue) for mesenchymal ( n = 3 independent mouse lines; IDs: 8028, 9091, 16992) and epithelial ( n = 3 independent mouse lines; IDs: 8442, 9591, 53631) organoids. Scale bars, 200 µm. g , Hierarchical clustering of RNA sequencing data from epithelial and mesenchymal 3D organoids derived from different Kras G12D background mice. Tumour grading, Kras mRNA levels retrieved from a previous study . Heat map of the leading-edge genes for both clusters. h , H&E staining of the primary tumours and corresponding brightfield organoid morphologies ( n = 3 mouse lines for mesenchymal, n = 3 mouse lines for epithelial). Scale bars, 200 µm (H&E), 500 µm (organoids). i , ELDA of epithelial and mesenchymal organoids (left) and plot of the log fraction of non-responding wells (without organoids) versus the number of seeded cells (right). j , k , GSEA comparing 2D mesenchymal and 3D mesenchymal organoids ( j ) and 2D epithelial and 3D epithelial organoids ( k ). Every bar represents individual genes for the given gene set.

Article Snippet: For the 3D collagen organoids, a Collagenase Type V (Worthington) digestion was first performed for 12–15 min until the collagen matrix was no longer visible.

Techniques: Imaging, Generated, Derivative Assay, Marker, RNA Sequencing, Staining

Journal: Nature Biomedical Engineering

Article Title: Heterogeneity-driven phenotypic plasticity and treatment response in branched-organoid models of pancreatic ductal adenocarcinoma

doi: 10.1038/s41551-024-01273-9

Figure Lengend Snippet: a , Morphological effect of major developmental pathways: EGFR by AG1478 (EGFR inhibitor) and EGF, Wnt/β-catenin by Wnt3a, XAV393 (Tankyrase inhibitor), iCRT14 (β-catenin-Tcf inhibitor), Notch (γ-secretase inhibitor), HGF, and Hedgehog by Ihh, Shh, GANT61 (GLI antagonist) and SANT-1 (Smo antagonist) on epithelial ( n = 1,045 organoids, line ID: 9591, 3 individual experiments) and mesenchymal organoids ( n = 873 organoids, line ID: 16992, 3 individual experiments). Treatments were performed with 10 µM AG1478, 5 ng EGF, 100 ng Wnt3a, 5 µM XAV939, 5 µM iCRT14, 5 ng HGF, 10 µM DAPT, 10 µM GANT61, 100 ng Ihh, 100 ng Shh, 2 µM Sant-1. Scale bars, 500 µm. b , GSEA of epithelial ( n = 3 mouse lines, from 3 individual experiments) and mesenchymal ( n = 3 mouse lines, from 3 individual experiments) 2D monolayers and 3D organoids for the TGFβ pathway. c , Heat maps of most up and downregulated genes between 2D monolayers and 3D cells for epithelial and mesenchymal organoids (from left to right). d , Monotreatments of Kras G12D epithelial ( n = 468 organoids, line ID: 9591, 3 individual experiments) or mesenchymal ( n = 618 organoids, line ID:16992, 3 individual experiments) organoids with 5 ng of TGFβ-1; treatment administration from Day 0, Day 7 or Day 11 after seeding, 1× StemXVivo EMT-inducing media supplement (termed STEMX) treatment from Day 0, or 5 µM TGFβ-RI inhibitor A83-01 treatment from Day 0. All organoids were imaged on Day 13. Scale bars, epithelial organoids: 200 µm (all A83-01), 500 µm (all others); mesenchymal organoids: 200 µm (all STEMX and the bottom A83-01), 500 µm (all others).

Article Snippet: For the 3D collagen organoids, a Collagenase Type V (Worthington) digestion was first performed for 12–15 min until the collagen matrix was no longer visible.

Techniques:

Journal: Nature Biomedical Engineering

Article Title: Heterogeneity-driven phenotypic plasticity and treatment response in branched-organoid models of pancreatic ductal adenocarcinoma

doi: 10.1038/s41551-024-01273-9

Figure Lengend Snippet: a Confocal IF imaging of E-cadherin (green), Vimentin (red) and DAPI (blue) for the epithelial (top) and mesenchymal (bottom) 2D cells (n = 3 mouse E and 3 mouse M lines) after 7 days EMT induction with 5 ng TGFβ1 or 1x STEMX, Scale bars= 50 μm. b Epithelial (top, from the mouse line ID: 9591) and mesenchymal (bottom, from the mouse line ID: 16992) organoid morphologies after treatment of the 2D cells for 7 days prior to seeding into organoid cultures with: 5 ng TGFβ1, 1x STEMX or 5 µM A83-01. Scale bars, 500 μm. c Epithelial and mesenchymal organoid morphologies after treatment of the 2D cells for 20 days prior to seeding into 3D-cultures with: 5 ng TGFβ1, 1x STEMX or 5 µM A83-01, additionally the treatment renewal with 1x STEMX, 5 µM A83-01 at day 0 and at specific days for TGFβ1: day 0, day 7, day 11 was included. All scale bars are 500 μm except from the 20 Days treated with A83-01 W.O. and then treated with additional A83-01 (most right column) where scale bars are 200 μm. d Quantification of epithelial and mesenchymal organoid morphologies in their W.O. phase from epithelial origin organoids (from the mouse line ID: 9591) after 7- or 20-days pre-treatment with 5 ng TGFβ1 or 1x STEMX (7 days treatment control n = 101, TGFβ1 W.O. n = 89 and STEMX W.O. n = 114 organoids from 3 individual experiments. 20 days treatment control n = 159, TGFβ1 W.O. n = 59 and STEMX W.O. n = 80 organoids). Unpaired two-tailed parametric t-test with Welch’s correction, two-tailed. e Confocal IF imaging of E-cadherin (green), Zeb1 (red) and DAPI (blue) for the epithelial organoids after 20 days of EMT induction with: 5 ng TGFβ1, 1x STEMX as control populations and with treatment renewal using 1x STEMX at day 0 and at specific days for TGFβ1: day 0 and day 7. The two main populations (without treatment renewal) are displayed, #1 the stable mesenchymal and #2 the reverting epithelial, Scale bars, 200 μm.

Article Snippet: For the 3D collagen organoids, a Collagenase Type V (Worthington) digestion was first performed for 12–15 min until the collagen matrix was no longer visible.

Techniques: Imaging, Control, Two Tailed Test

Journal: Nature Biomedical Engineering

Article Title: Heterogeneity-driven phenotypic plasticity and treatment response in branched-organoid models of pancreatic ductal adenocarcinoma

doi: 10.1038/s41551-024-01273-9

Figure Lengend Snippet: a , UMAP plots from single-cell RNA sequencing of epithelial and mesenchymal 2D bulk populations. b , UMAP plots from single-cell RNA sequencing of epithelial 2D bulk populations (cell line ID: 9591, n = 16,747 cells) and mesenchymal 2D bulk populations (cell line ID: 16992, n = 9,190 cells). Conserved EMP, Cancer EMP according to the signature gene sets and EMT hallmark scores are presented in violin plots, the y -axis represents the AUCell scores for the specific pathways. c , Major morphologies found in epithelial organoid lines ( n = 3 mouse lines, IDs: 8442, 9591, 53631). Colour coding implies the hierarchical relation of the super families. Scale bars, 500 µm. d , Manual clustering of the total number of organoids (line ID: 8442 n = 438, ID: 9591 n = 864, ID: 53631 n = 718 organoids). e , Major morphologies found in mesenchymal organoid lines ( n = 3 mouse lines, ID: 8028, 9091, 16992). Colour coding implies the hierarchical relation of the super families. Scale bars, 500 µm. f , Manual clustering of the total number of organoids (line ID: 8028 n = 392, ID: 9091 n = 562 and ID: 16992 n = 900 organoids). g , Schematic representation of the workflow to develop MOrPHeMap; image created with BioRender.com. h , K -means clustering of the image-derived features of unseen data set of n = 1,579 organoids (from 6 mouse lines, 3-E IDs: 8442, 9591, 53631 and 3-M IDs: 8028, 9091, 16992) revealed 8 distinct morphological clusters. i , NES of the EMT hallmark from the 3 epithelial and 3 mesenchymal mouse lines grown in 3D collagen gels. j , k , Individual cell-line morphological heterogeneity as visualized by density overlays superimposed on the imaged-derived clusters. The overlays indicate which cell lines correspond to which cluster. j , t -SNE plots of the organoids clustering from the epithelial lines (IDs: 8442, 9591, 53631). k , t -SNE plots of the organoids clustering from the mesenchymal lines (IDs: 8028, 9091, 16992). Br. mesenchymal, branched mesenchymal; Br. mes.-thin, branched mesenchymal thin.

Article Snippet: For the 3D collagen organoids, a Collagenase Type V (Worthington) digestion was first performed for 12–15 min until the collagen matrix was no longer visible.

Techniques: RNA Sequencing, Derivative Assay

Journal: Nature Biomedical Engineering

Article Title: Heterogeneity-driven phenotypic plasticity and treatment response in branched-organoid models of pancreatic ductal adenocarcinoma

doi: 10.1038/s41551-024-01273-9

Figure Lengend Snippet: a Characteristic organoid morphologies derived from four different transcriptional clusters as previously described (C2a, C2b, C2c, C1). Scale bars= 500 μm. b t-SNE plot clustering showing the individual organoid lines from Fig. . c t-SNE plot clustering for the C2a and C2c organoids (n = 2015 organoids) superimposed on the Extended Data Fig. 4b. d Organoid phenotypes (n = 588 organoids) from epithelial and mesenchymal type of organoids derived from various mouse models (with different driver mutations): Ptf1a Cre/+ ; Pi3kca +H1047R/+ (n = 6 mouse lines, note that the line E248 is not forming 3D organoids), Pdx1 Cre/+ ; Kras G12D/+ ; TP53 ΔHO (n = 6 mouse lines), Ptf1a Cre/+ ; Kras G12D/+ ; Cdkn2a ΔHO (n = 6 mouse lines). Scale bars= 500 μm. e Schematic representation of the morphological clone isolation and expansion. Image created with BioRender.com. f Major 3D morphological families that were used for further characterisation. Scale bars= 500 μm. g Extreme Limiting Dilution Analysis (ELDA) of epithelial and mesenchymal organoid phenotypes. h Log plot of nonresponding wells vs cell dosage for the epithelial (left) and mesenchymal (right) 3D organoid phenotypes. i Proliferation rate as % compared to the bulk population for the epithelial (from the mouse line ID: 9591 from 3 individual experiments) and mesenchymal (from the mouse line ID: 16992 from 3 individual experiments) 2D clones. Unpaired two-tailed parametric t-test with Welch’s correction, two-tailed. Both graphs represent mean±sem. j Heatmap of signature genes from E and M organoid phenotypes.

Article Snippet: For the 3D collagen organoids, a Collagenase Type V (Worthington) digestion was first performed for 12–15 min until the collagen matrix was no longer visible.

Techniques: Derivative Assay, Isolation, Clone Assay, Two Tailed Test

Journal: Nature Biomedical Engineering

Article Title: Heterogeneity-driven phenotypic plasticity and treatment response in branched-organoid models of pancreatic ductal adenocarcinoma

doi: 10.1038/s41551-024-01273-9

Figure Lengend Snippet: a , Scoring of the individual organoid phenotypes isolated from the E-mouse line ID: 9591 and the M-mouse line ID: 16992 for PDAC subtype-specific signatures . Graph represents mean ± s.e.m., each dot represents an independent experiment. b , Violin plots of scRNA-seq from the parental epithelial and mesenchymal cells scored for the organoid phenotype signatures. The y -axis represents the AUCell scores for the specific signatures. c , UMAP scoring the individual organoid phenotype signatures to a human PDAC data set . d , Schematic representation of the in vivo orthotopic transplantation and the subsequent analysis, including histopathological analysis, whole-tissue clearing and IF staining, and organoid line isolation/characterization; image created with BioRender.com. e , H&E staining of orthotopically transplanted organoids. Scale bars, 60 µm. f – h , 3D in vivo growth patterns of PDAC organoids. f , 3D views of PDAC organoid grafts stained for pan-Keratin and Vimentin. All scale bars, 100 μm. g , High magnifications of pan-Keratin from the PDAC grafts in f , demonstrating different growth patterns of the various organoid lines. All scale bars, 20 μm. h , 3D segmentations of coherent tumour cell strands from different organoid grafts. All scale bars, 30 μm. i , Manual phenotype analysis of organoids post implantation ( n = 41 lines, n = 1,171 organoids from the epithelial lines and n = 633 organoids from the mesenchymal lines). Bar plot represents the mean ± s.e.m. of the average number of organoid phenotypes (%). j , Truncated violin plots of the total number of metastatic nodules in the liver from epithelial (left) and mesenchymal (right) transplanted organoid phenotypes. k , Truncated violin plots of the total number of metastatic nodules in the lung from epithelial (left) and mesenchymal (right) transplanted organoid phenotypes. Br. mesenchymal, branched mesenchymal; sign., signature.

Article Snippet: For the 3D collagen organoids, a Collagenase Type V (Worthington) digestion was first performed for 12–15 min until the collagen matrix was no longer visible.

Techniques: Isolation, In Vivo, Transplantation Assay, Staining

Journal: Nature Biomedical Engineering

Article Title: Heterogeneity-driven phenotypic plasticity and treatment response in branched-organoid models of pancreatic ductal adenocarcinoma

doi: 10.1038/s41551-024-01273-9

Figure Lengend Snippet: a Images of epithelial and mesenchymal tumours after transplantation of distinct organoid phenotypes. Scale bars= 1cm. b-c n = 41 mice, for the epithelial transplanted organoids n = 5 mice/phenotype (tree-like n = 4 mice) and for the mesenchymal transplanted organoids n = 4 mice/phenotype (firework n = 5 mice). b Pancreas tumour weight (g) for epithelial and mesenchymal organoid phenotypes derived tumours. Graphs represent mean±sem. c Tumour grading for epithelial and mesenchymal organoid phenotypes derived tumours. Graphs represent mean±sem. d In vivo marker expression of different transplanted organoid phenotypes. 3D views of in vivo PDAC organoid grafts stained for E-cadherin and Hnf1β. Note absent nuclear Hnf1β staining in mesenchymal organoid lines. All scale bars= 20 μm. e Characteristic organoid morphologies from cell lines isolated post-implantation (epithelial on top, mesenchymal bottom). Scale bars= 500 μm. f Summary table of the tumour engraftment and liver/lung metastatic colonization efficacy from epithelial and mesenchymal transplanted organoids in mice. BM: branched mesenchymal. g HEs of liver metastasis from epithelial (top) and mesenchymal (bottom) organoid phenotypes transplanted in mice. Scale bars= 200 μm. h HEs of lung metastasis from epithelial (top) and mesenchymal (bottom) organoid phenotypes transplanted in mice. Scale bars= 200 μm.

Article Snippet: For the 3D collagen organoids, a Collagenase Type V (Worthington) digestion was first performed for 12–15 min until the collagen matrix was no longer visible.

Techniques: Transplantation Assay, Derivative Assay, In Vivo, Marker, Expressing, Staining, Isolation

Journal: Nature Biomedical Engineering

Article Title: Heterogeneity-driven phenotypic plasticity and treatment response in branched-organoid models of pancreatic ductal adenocarcinoma

doi: 10.1038/s41551-024-01273-9

Figure Lengend Snippet: Targeted therapy treatment and transcriptomic analysis of individual organoid phenotypes isolated from the E-mouse line ID: 9591 and the M-mouse line ID: 16992. a , Pie chart of the library design ( n = 102 drugs) with the drug approval status: preclinical, phase 1, 2, 3, 4 and FDA approved. b , Pie chart of the specific targeted pathways by the 102 drugs. c , Schematic summary of the drug-treatment workflow; image created with BioRender.com. d , Heat maps of the z -score for specific drugs from the 102-drug screening of the 2D epithelial (4 phenotype clones and the bulk population from the mouse line ID: 9591, n = 2 individual experiments) and mesenchymal (3 phenotype clones and the bulk population from the mouse line ID: 16992, n = 2 individual experiments) cells. e , Brightfield imaging of bulk organoid morphologies post treatment with selective drugs using the IC 50 values of the most sensitive 2D clones in 3D. Scale bars, 200 µm (mesenchymal treated with JIB-04 ), 500 µm (all others). f , Manual phenotypic analysis of epithelial bulk ( n = 397 organoids from 3 individual experiments) and mesenchymal bulk ( n = 246 organoids from 3 individual experiments) organoid populations after the selective treatment. Bar plot represents the average number of organoid phenotypes (%). g , Brightfield images of epithelial control ( n = 334 organoids, 3 individual experiments) and combinatory treatment with AZD5153+Poziotinib ( n = 321 organoids, 3 individual experiments) organoids from the bulk population, TEBBO, cystic branched, thick branched and tree-like phenotypes. Scale bars, 500 µm. h , Major axis length (µm) of bulk epithelial organoids as control ( n = 51 organoids from 3 individual experiments) and organoids treated with AZD5153+Poziotinib ( n = 49 organoids from 3 individual experiments). Graph represents mean ± s.e.m., unpaired two-tailed parametric t -test with Welch’s correction, two-tailed. i , O-SFUs per gel of control and AZD5153+Poziotinib-treated (in pink) epithelial organoid phenotypes; graph represents mean ± s.e.m. of 3 individual experiments. j , Manual phenotypic analysis of control and AZD5153+Poziotinib-treated bulk organoid populations of bulk epithelial organoids ( n = 107 organoids). Pie chart represents the average number of organoid phenotypes (%). k , PCA analysis of the bulk RNA sequencing from control and AZD5153+Pozitionib-treated bulk, TEBBO, cystic branched, thick branched and tree-like organoids. Each dot represents the mean of 3 individual experiments. Dashed circles highlight the organoid phenotypes under the combinatory treatments. l , Dot plot of the GSEA comparing the epithelial control (from all phenotypes) and AZD5153+Poziotinib-treated (from all phenotypes) epithelial organoids. P adj , Benjamini–Hochberg adjusted P values. m , Mesenchymal control and RO5126766-treated organoid morphologies for the bulk population, branched mesenchymal, firework and star-like phenotypes. Scale bars, 500 µm. n , Major axis length (µm) of bulk mesenchymal organoids as control ( n = 93 organoids from 3 individual experiments) and after treatment with RO5126766 ( n = 67 organoids from 3 individual experiments). Graph represents mean ± s.e.m., unpaired two-tailed parametric t -test with Welch’s correction, two-tailed. o , PCA analysis of the bulk RNA sequencing from control and bulk, branched mesenchymal, firework and star-like organoids treated with Birinapant, Poziotinib, Saracatinib or RO5126766. In addition, we included an earlier time point for the star-like organoids (Day 10). Dashed circles highlight the organoid phenotype transitions under specific treatments. Each dot represents the mean of 3 individual experiments (except for the branched mesenchymal+RO5126766 with 1 replicate). p , Dot plot of GSEA comparing the mesenchymal control (from all phenotypes) with the RO5126766-treated (from all phenotypes) mesenchymal organoids. q , Graphical summary of the distinct epithelial and mesenchymal organoid phenotypes and how mono or combinational treatments can either plastically switch morphologies or reveal persister phenotypes. Br. mesenchymal, branched mesenchymal.

Article Snippet: For the 3D collagen organoids, a Collagenase Type V (Worthington) digestion was first performed for 12–15 min until the collagen matrix was no longer visible.

Techniques: Isolation, Drug discovery, Clone Assay, Imaging, Control, Two Tailed Test, RNA Sequencing

Journal: Nature Biomedical Engineering

Article Title: Heterogeneity-driven phenotypic plasticity and treatment response in branched-organoid models of pancreatic ductal adenocarcinoma

doi: 10.1038/s41551-024-01273-9

Figure Lengend Snippet: All epithelial organoid phenotypes in Fig. are derived from the mouse line ID: 9591 and the mesenchymal from the mouse line ID: 16992. a Heatmap of AUC values from epithelial (left) and mesenchymal (right) clones. b Representative organoid morphologies of epithelial 3D morphological clones: TEBBO, cystic branched, thick branched, and tree-like (from left to right) under control (DMSO treated) conditions or treated with the most sensitive IC 50 values of: AZD5153, Birinapant, JNJ-64619178, KU60019, ML264, Poziotinib and Trametinib. All scale bars= 500 μm except for the TEBBO and thick branched phenotypes treated with KU60019, scale bars= 200 μm. c Representative organoid morphologies of mesenchymal 3D morphological clones: branched mesenchymal, firework-like and star-like (from left to right) under control conditions or treated with the most sensitive IC 50 values of: Abexinostat, Adavosertib, Birinapant, JIB-04, Poziotinib, RO5126766, Saracatinib and Trametinib. All scale bars= 500 μm except for the firework phenotypes treated with Abexinostat, Adavosertib or JIB-04, scale bars= 200 μm. d Average Organoid structure formation units O-SFU/gel for the epithelial 3D morphological clones under the abovementioned (Extended Data Fig. 6b) treatment conditions. Graph represents mean±sem, from 3 individual experiments. e Average Organoid structure formation units O-SFU/gel for the mesenchymal 3D morphological clones under the abovementioned (Extended Data Fig. 6c) treatment conditions. Graph represents mean±sem, from 3 individual experiments. f Major axis length (in μm) of star-like control at Day 13 (n = 42 organoids), star-like control at Day 10 (n = 68 organoids), and firework organoids at Day 13 after treatment with Poziotinib (n = 53 organoids) or Saracatinib (n = 47 organoids). Graph represents mean±sem, unpaired two-tailed parametric t-test with Welch’s correction, two-tailed. g Dot plot of the GSEA from the mesenchymal star-like organoid phenotype control vs. Birinapant treated. h Dot plot of the GSEA from the mesenchymal firework organoid phenotype control vs. Poziotinib treated. i Dot plot of the GSEA from the mesenchymal firework organoid phenotype control vs. Saracatinib treated. j Dot plot of the GSEA from the mesenchymal star-like (Day 10) organoid phenotype control vs. the firework organoids treated with Saracatinib (star-like acquired phenotype, at Day 13). g-j NES= normalized enrichment score, padj=Benjamini-Hochberg adjusted p-values.

Article Snippet: For the 3D collagen organoids, a Collagenase Type V (Worthington) digestion was first performed for 12–15 min until the collagen matrix was no longer visible.

Techniques: Derivative Assay, Clone Assay, Control, Two Tailed Test

Journal: Nature Biomedical Engineering

Article Title: Heterogeneity-driven phenotypic plasticity and treatment response in branched-organoid models of pancreatic ductal adenocarcinoma

doi: 10.1038/s41551-024-01273-9

Figure Lengend Snippet: a Schematic representation of organoid generation directly from in vivo or 3D in vitro cultures (Matrigel), image created with BioRender.com. b Epithelial (top) and mesenchymal (bottom) collagen gel grown organoids. Corresponding HEs of the primary tumours. Confocal IF imaging of E-cadherin (green), Vimentin (red), DAPI (blue). Scale bars= 500 μm (bright field), 200 μm (HEs), 200 μm (IF).

Article Snippet: For the 3D collagen organoids, a Collagenase Type V (Worthington) digestion was first performed for 12–15 min until the collagen matrix was no longer visible.

Techniques: In Vivo, In Vitro, Imaging