Journal: bioRxiv

Article Title: Breaking the cycle: How targeting CD44v6/MET signaling disrupts colorectal cancer cell plasticity

doi: 10.1101/2024.09.27.614761

Figure Lengend Snippet: A. Experimental procedure. Created in BioRender. Sonnentag, S. (2023) BioRender.com/h24u924 B. Blocking of CD44 using a panCD44 antibody (IM7) or IgG as control during the dedifferentiation of Lgr5 - into Lgr5 + AKPL cells (n=3 independent experiments) Values are presented as percentages *p-value<0.05. Representative pictures of organoids. Scale bar: 100 µm C. FC analysis six days after seeding of Lgr5 - cells cultured in full intestinal growth medium and treated with IM7 or IgG. Representative histogram and quantification of eGFP + cells (n=3 independent experiments). Values are presented as fold changes. *p-value<0.05 D. Inhibition of CD44v6 by a CD44v6 peptide during the dedifferentiation process of Lgr5 - AKPL cells cultured in full intestinal growth medium. Representative pictures of day three and day six after seeding. Quantitative analysis of developed organoids (Day three: n=4 independent experiments; Day six: n=3 independent experiments). Values are presented as percentages. *p-value<0.05. Scale bar: 100 µm E. Inhibition of CD44v6 by a CD44v6 peptide during the dedifferentiation process of Lgr5 - AKPL cells cultured in minimal medium. Representative pictures of day three and day six after seeding. Quantitative analysis of developed organoids (Day three: n=3 independent experiments; Day six: n=5 independent experiments). Values are presented as percentages. *p-value<0.05, **p-value<0.01. Scale bar: 100 µm F. FC analysis of D. Representative histogram and quantification of the presence of eGFP at day six on AKPL cells treated with the CD44v6 or the respective control peptide (n=3 independent experiments). Values are presented as fold changes. *p-value<0.05 G. FC analysis of E. Representative histogram and quantification of eGFP at day six on AKPL cells treated with the CD44v6 or the respective control peptide (n=3 independent experiments). Values are presented as fold changes. **p-value<0.01

Article Snippet: RMOs were cultured in murine intestinal growth medium (IntestiCult, STEMCELL Technologies Inc.) .

Techniques: Blocking Assay, Control, Cell Culture, Inhibition

Journal: bioRxiv

Article Title: Breaking the cycle: How targeting CD44v6/MET signaling disrupts colorectal cancer cell plasticity

doi: 10.1101/2024.09.27.614761

Figure Lengend Snippet: A. Gene expression weighted 2D Kernel density plot of Met RNA expression visualized on the UMAP. B. Violin plot showing the Met normalized expression across the cluster. Clusters are sorted according to the median expression (white bar). C. Gene expression weighted 2D Kernel density plot of Cd44 RNA expression visualized on the UMAP. D. Dotplot showing the percentage of cells expressing Cd44 and Met (dot size) as well as the Average RNA expression of both genes across the Louvain clusters. E. Inhibition of MET/ALK during the dedifferentiation of Lgr5 - AKPL cells via crizotinib. Representative pictures of day three and day six after seeding. Quantitative analysis of developed organoids (Day three: n=3 independent experiments; Day six: n=5 independent experiments). Values are presented as percentages. *p-value<0.05, **p-value<0.01. Scale bar: 100 µm. Representative histogram of eGFP re-expression after plasticity upon treatment with crizotinib. F. Analysis of 40-70 µm micro-organoids during plasticity in AKPL organoids cultured in full intestinal growth medium and treated with the CD44v6 or control peptide for three days (n=3 independent experiments). Values are presented as fold-changes. *p-value<0.05 G. qPCR analysis of YAP/TAZ target genes in micro-organoids after plasticity (n=4 independent experiments). Values are presented as fold changes. *p-value<0.05, **p-value< 0.01

Article Snippet: RMOs were cultured in murine intestinal growth medium (IntestiCult, STEMCELL Technologies Inc.) .

Techniques: Gene Expression, RNA Expression, Expressing, Inhibition, Cell Culture, Control

Journal: bioRxiv

Article Title: Fluorescence Lifetime Imaging Microscopy (FLIM) visualizes internalization and biological impact of nanoplastics in live intestinal organoids

doi: 10.1101/2025.01.27.635069

Figure Lengend Snippet: A: Representative 3D confocal images of WGA-Alexa Fluor 488 and phalloidin–Texas Red (F-actin labeling) co-stained organoids with apical-out (AO), apical-basal (AB) and basal-out (BO) topology. Scale bar is 100 µm. B: Large field mosaic scan of PFA-fixed pig intestinal organoids after 18 h of polarity reversion, co-stained with fluorescent WGA and phalloidin. Left: representative images of AO, AB and BO organoids, indicated on mosaic image ( right ). Scale bar is 100 µm. C: Comparison of the yield of AO, AB and BO topology, estimated with WGA staining and F-actin labeling, respectively (AO organoids: 93.1% / 95.5%, BO organoids 0.53% / 0.55%, analyzed from 4 mosaic scanned images, see table ST3). D: Total quantification of topology, observed with WGA and Nile Red labeling for polarity reverted organoids: AO with lipid droplets (AO LDs) and AO without lipid droplets (AO no-LDs), AB and BO, quantified from mosaic scanned image. E: Lipid droplets display a characteristic distribution in AO organoids, contrasting with BO. F: Co-staining with WGA and Nile Red reveals organoid structure in apical-basal organoid (AB). Scale bar is 50 µm.

Article Snippet: Lipidure TM -CM5206 (AMS.52000034GB1G, Amsbio, UK), Matrigel growth factor-reduced (734-0269, VWR, Belgium), human intestinal organoid growth medium (STEMCELL technologies, 06010, Belgium), mouse intestinal organoid growth medium (STEMCELL technologies, 06005, Belgium), DMEM high glucose GlutaMax TM Supplement media (61965026, Gibco, Belgium), 0.5 M EDTA solution (15575020, Invitrogen, Belgium), 24-well Tissue Culture plates (734-2325, VWR, Belgium), sodium valproate (P4543, Sigma-Aldrich,Ireland), CHIR99021 (SML1046, Sigma-Aldrich,Ireland), PBS (18912-014, Gibco, Belgium).

Techniques: Labeling, Staining, Comparison

Journal: bioRxiv

Article Title: Fluorescence Lifetime Imaging Microscopy (FLIM) visualizes internalization and biological impact of nanoplastics in live intestinal organoids

doi: 10.1101/2025.01.27.635069

Figure Lengend Snippet: A: Scheme of experimental workflow. B: Representative confocal fluorescence images and corresponding phasor plots of the pig small intestinal organoid incubated with NP A-D (10 µg/ mL, 18 h), co-stained with Nile Red. NP A, B and D displayed signals on both fluorescent intensity images and phasor plots, whereas type C showed no signal, similar to control. C: Different types of uptake of NP type D uptake (10 µg/mL NPD, 20% laser power) into organoids, co-stained with WGA, with respect to the topology and size. Left: partial NP D uptake in AO, showing high pixel signal on a phasor plot. Middle: organoid size-dependent NP D uptake in AO. ROI 1: enhanced NP D pixel signal in a small AO, ROI 2: reduced NP D pixel signal in larger organoid. Right: Homogeneous NP D distribution in BO with strong signal on a phasor plot. D: Topology-dependent uptake of NP D (magenta) in apical-basal organoid (AB), co-stained with WGA (green) and Nile Red (yellow). Left: 3D reconstruction shows distinct NP D distribution in AO and BO regions. Right: representative fluorescence image of AB organoid taken from 3D reconstruction. AO and BO regions defined by WGA and Nile Red signals, with corresponding NP type D phasor FLIM plots of these regions (bottom right).

Article Snippet: Lipidure TM -CM5206 (AMS.52000034GB1G, Amsbio, UK), Matrigel growth factor-reduced (734-0269, VWR, Belgium), human intestinal organoid growth medium (STEMCELL technologies, 06010, Belgium), mouse intestinal organoid growth medium (STEMCELL technologies, 06005, Belgium), DMEM high glucose GlutaMax TM Supplement media (61965026, Gibco, Belgium), 0.5 M EDTA solution (15575020, Invitrogen, Belgium), 24-well Tissue Culture plates (734-2325, VWR, Belgium), sodium valproate (P4543, Sigma-Aldrich,Ireland), CHIR99021 (SML1046, Sigma-Aldrich,Ireland), PBS (18912-014, Gibco, Belgium).

Techniques: Fluorescence, Incubation, Staining, Control

Journal: bioRxiv

Article Title: Fluorescence Lifetime Imaging Microscopy (FLIM) visualizes internalization and biological impact of nanoplastics in live intestinal organoids

doi: 10.1101/2025.01.27.635069

Figure Lengend Snippet: A, B: Representative images of NP D uptake (10 µg/mL, 18 h) in pig intestinal organoids in respect to their apical-basal topology with high loading in both AO and BO (A) and high loading in BO and zero at AO (B). Scale bar is 50 µm. C, D: Comparison of NP D uptake in AO vs. BO organoids as a function of loading concentration (0-1 µg/mL, left panel and 1-50 µg/mL, right panel) on a widefield fluorescence microscope (intensity-based approach, C), and confocal FLIM microscope (phasor FLIM event counting approach, D). FLIM events were counted from the phasor plots reconstructed in the napari phasor plugin from the exported list of G and S coordinates. Results of one of the two independent experimental replicates are shown. Both C and D data were produced from the same samples. The box charts represent 25, median and 75 percentiles with dots corresponding to individual intensity or event count square ROI square ROI normalized values. Attribution of organoids to AO and BO-topology groups was done based on WGA staining. BO group also includes AB organoids. Statistical comparison between AO and BO groups over a range of loading concentrations was performed by Mann-Whitney test (lines represent detected statistical difference, p <0.05).

Article Snippet: Lipidure TM -CM5206 (AMS.52000034GB1G, Amsbio, UK), Matrigel growth factor-reduced (734-0269, VWR, Belgium), human intestinal organoid growth medium (STEMCELL technologies, 06010, Belgium), mouse intestinal organoid growth medium (STEMCELL technologies, 06005, Belgium), DMEM high glucose GlutaMax TM Supplement media (61965026, Gibco, Belgium), 0.5 M EDTA solution (15575020, Invitrogen, Belgium), 24-well Tissue Culture plates (734-2325, VWR, Belgium), sodium valproate (P4543, Sigma-Aldrich,Ireland), CHIR99021 (SML1046, Sigma-Aldrich,Ireland), PBS (18912-014, Gibco, Belgium).

Techniques: Comparison, Concentration Assay, Fluorescence, Microscopy, Produced, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Breaking the cycle: How targeting CD44v6/MET signaling disrupts colorectal cancer cell plasticity

doi: 10.1101/2024.09.27.614761

Figure Lengend Snippet: A. Experimental procedure. Created in BioRender. Sonnentag, S. (2023) BioRender.com/h24u924 B. Blocking of CD44 using a panCD44 antibody (IM7) or IgG as control during the dedifferentiation of Lgr5 - into Lgr5 + AKPL cells (n=3 independent experiments) Values are presented as percentages *p-value<0.05. Representative pictures of organoids. Scale bar: 100 µm C. FC analysis six days after seeding of Lgr5 - cells cultured in full intestinal growth medium and treated with IM7 or IgG. Representative histogram and quantification of eGFP + cells (n=3 independent experiments). Values are presented as fold changes. *p-value<0.05 D. Inhibition of CD44v6 by a CD44v6 peptide during the dedifferentiation process of Lgr5 - AKPL cells cultured in full intestinal growth medium. Representative pictures of day three and day six after seeding. Quantitative analysis of developed organoids (Day three: n=4 independent experiments; Day six: n=3 independent experiments). Values are presented as percentages. *p-value<0.05. Scale bar: 100 µm E. Inhibition of CD44v6 by a CD44v6 peptide during the dedifferentiation process of Lgr5 - AKPL cells cultured in minimal medium. Representative pictures of day three and day six after seeding. Quantitative analysis of developed organoids (Day three: n=3 independent experiments; Day six: n=5 independent experiments). Values are presented as percentages. *p-value<0.05, **p-value<0.01. Scale bar: 100 µm F. FC analysis of D. Representative histogram and quantification of the presence of eGFP at day six on AKPL cells treated with the CD44v6 or the respective control peptide (n=3 independent experiments). Values are presented as fold changes. *p-value<0.05 G. FC analysis of E. Representative histogram and quantification of eGFP at day six on AKPL cells treated with the CD44v6 or the respective control peptide (n=3 independent experiments). Values are presented as fold changes. **p-value<0.01

Article Snippet: AKPL organoid were obtained from Jacco van Rheenen and cultured either in full murine intestinal growth medium (IntestiCult, STEMCELL Technologies Inc.) or under minimal medium conditions (Advanced DMEM/F12 (GibcoTM, Thermo Fisher Scientific), 4 mM Glutamax (GibcoTM, Thermo Fisher Scientific), B27 (2%; GibcoTM, Thermo Fisher Scientific), N-acetylcysteine (1.25 mM; Sigma) and Noggin (1%; PeproTech)).

Techniques: Blocking Assay, Control, Cell Culture, Inhibition

Journal: bioRxiv

Article Title: Breaking the cycle: How targeting CD44v6/MET signaling disrupts colorectal cancer cell plasticity

doi: 10.1101/2024.09.27.614761

Figure Lengend Snippet: A. Gene expression weighted 2D Kernel density plot of Met RNA expression visualized on the UMAP. B. Violin plot showing the Met normalized expression across the cluster. Clusters are sorted according to the median expression (white bar). C. Gene expression weighted 2D Kernel density plot of Cd44 RNA expression visualized on the UMAP. D. Dotplot showing the percentage of cells expressing Cd44 and Met (dot size) as well as the Average RNA expression of both genes across the Louvain clusters. E. Inhibition of MET/ALK during the dedifferentiation of Lgr5 - AKPL cells via crizotinib. Representative pictures of day three and day six after seeding. Quantitative analysis of developed organoids (Day three: n=3 independent experiments; Day six: n=5 independent experiments). Values are presented as percentages. *p-value<0.05, **p-value<0.01. Scale bar: 100 µm. Representative histogram of eGFP re-expression after plasticity upon treatment with crizotinib. F. Analysis of 40-70 µm micro-organoids during plasticity in AKPL organoids cultured in full intestinal growth medium and treated with the CD44v6 or control peptide for three days (n=3 independent experiments). Values are presented as fold-changes. *p-value<0.05 G. qPCR analysis of YAP/TAZ target genes in micro-organoids after plasticity (n=4 independent experiments). Values are presented as fold changes. *p-value<0.05, **p-value< 0.01

Article Snippet: AKPL organoid were obtained from Jacco van Rheenen and cultured either in full murine intestinal growth medium (IntestiCult, STEMCELL Technologies Inc.) or under minimal medium conditions (Advanced DMEM/F12 (GibcoTM, Thermo Fisher Scientific), 4 mM Glutamax (GibcoTM, Thermo Fisher Scientific), B27 (2%; GibcoTM, Thermo Fisher Scientific), N-acetylcysteine (1.25 mM; Sigma) and Noggin (1%; PeproTech)).

Techniques: Gene Expression, RNA Expression, Expressing, Inhibition, Cell Culture, Control