Fig. 3.

Exosome secretion mediates chemoresistance in the biomimetic co-culture system. A) Exosome inhibitor drugs slowed organoid growth and abolished chemoresistance in the co-culture model. Upper panel, representative microscope image of 4-day organoid-CAF co-culture treated with vehicle (DMSO), climbazole (20 μM) or Imipramine (10 μM) and corresponding quantitative analysis of resulting organoid sizes. Lower panel, representative microscope image of organoid-CAF 48hr-gemcitabine treated co-culture along with or without climbazole (20 μM) or Imipramine (10 μM) and corresponding quantitative analysis of resulting organoid sizes. B) Representative Western blot image showing RAB27A knockdown in CAFs 1 week after lentiviral infection. C) RAB27A knockdown in both CAF and tumor organoids are required to decrease chemoresistance in the co-culture system. Quantitative analysis of organoid size in co-cultures in corresponding knockdown conditions. D) Conditioned media from non-transfected co-culture cells rescued chemosensitivity by RAB27A knockdown. Quantitative analysis of organoid sizes in co-cultures after gemcitabine treatment with or without an addition of conditioned media from a regular 4-day co-culture. E) Left panel, representative immuno-fluorescent staining of proliferation marker Cyclin D1 (red) and nuclear staining DAPI (blue) from a total of 4-day plus 2-day gemcitabine or gemcitabine plus conditioned media treated co-culture in corresponding conditions. Right panel, quantification of cyclin D1 positive ratio in organoids from corresponding co-culture conditions after gemcitabine treatment. For A, C and E, One-Way ANOVA with Dunnett’s multiple comparison test was performed. For D, two-tailed student’s test was performed. ***p < 0.001, ****p < 0.0001.Error bars represent standard deviation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)