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: 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