Fig. 7: Autophagy inhibition blocks paligenosis and induces apoptosis in GC PDOs. | Nature Communications

Fig. 7: Autophagy inhibition blocks paligenosis and induces apoptosis in GC PDOs.

From: Single-cell sequencing of ascites fluid illustrates heterogeneity and therapy-induced evolution during gastric cancer peritoneal metastasis

Fig. 7

a Heatmap showing the intercellular interaction by CellPhoneDB analysis. Color represents the number of significant ligand-receptor pairs among different cell subtypes. DC, dendritic cell; Macro, macrophage; Mono, monocyte. b, c Venn diagram showing the number of overlapping signature genes between differentially expressed genes (DEGs) of C2-tumor cell, poor prognostic DEGs of The Cancer Genome Atlas Stomach Adenocarcinoma (TCGA-STAD) database, and autophagy-related gene sets (b) and mTORC1-related gene sets (c). d Immunofluorescence staining for MARCKS (red, upper panel) and TXNIP (red, lower panel), early paligenosis markers DDIT4 (green) and ATF3 (green), late paligenosis markers KI67 (green) and pS6 (green), progenitor-related marker SOX9 (green), and nuclei marker DAPI (blue) in 15th generation patients-derived organoids from ascites. Scale bar, 100 μm. The experiment was repeated with 4 independent experiments, with similar results. e Fifteenth generation organoids generated as clones from single cells dissociated from 14th generation organoids after addition of inhibitors or vehicle when single cell suspensions were replated. Red arrows indicate application of autophagy and mTORC1 inhibitors promoting organoids death. Scale bar, 400 μm. The experiment was repeated with four independent experiments, with similar results. f Quantification of the size of 15th generation organoids as in (e) after 7-day treatment with autophagy or mTORC1 inhibitors (n = 4 independent experiments). Each datapoint represents the mean of mean values of organoids in all wells. Every well included the means of 25+ counts. Data are presented as mean values ± SEM (error bars); p-values are calculated by one-way ANOVA with Tukey post hoc test. g Immunofluorescence staining for apoptosis marker CC3 (green), proliferation marker KI67 (pink), and nuclei marker DAPI (blue) in 15th generation organoids as in (e) after 7-days treatment with autophagy or mTORC1 inhibitors. Scale bar, 100 μm. h The ratio of CC3/KI67 positive cells as in (g) (n = 4 independent experiments). Data are presented as mean values ± SEM (error bars); p-values are calculated by one-way ANOVA with Tukey post hoc test.

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