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. 2017 Mar 6;214(3):579–596. doi: 10.1084/jem.20162024

Figure 2.

Figure 2.

Co-cultures of mouse PSCs and pancreatic cancer organoids recapitulate properties of PDA desmoplasia. (A) Schematic illustration of the co-culture platform. (B) Representative images of mCherry-labeled PSCs (red) cultured alone or in co-culture with GFP-labeled tumor-derived organoids (green), and imaged by confocal microscopy after 4 or 7 d in bright field and by fluorescent microscopy (n = 3). Arrows point to close interactions between organoids and PSCs. Bars, 100 µm. (C) Bright field images of primary PSCs plated in Matrigel directly after isolation, and either cultured alone or co-cultured with tumor organoids for 5 d (n = 3). Bar, 100 µm. (D) Representative electron microscopy image showing the proximity between organoids and PSCs in co-culture (n = 2). Bar, 5 µm. (E) H&E staining and Masson’s trichrome (MT) staining of fixed and paraffin-embedded organoids cultured alone or in co-culture with PSCs (n = 2). Bar, 50 µm. (F) Representative bright field and IF images of collagen I deposition (red) in organoid cultured alone or in co-culture with PSCs (n = 2). Bar, 200 µm. (G) Representative electron microscopy image of banded collagen fibrils (arrow), with fibril diameters ranging between 24 and 35 nm, in the extracellular space between organoids and PSCs (n = 2). Bar, 1 µm. (H) PSC proliferation curves plotting changes in mCherry intensity over time. Results show mean ± SD of two biological replicates. **, P < 0.01, unpaired Student’s t test. (I) Organoid proliferation curves plotting changes in GFP intensity over time. Results show mean ± SD of three biological replicates. *, P < 0.05, unpaired Student’s t test. (J) Passaging of organoids in different culture conditions in the presence or absence of PSCs. Complete media, DMEM/F12 supplemented with mitogens and growth factors. Reduced media, DMEM + 5% FBS. Red dot indicates the passage number when all organoids were found dead. Green dot indicates surviving organoids when the experiment was terminated. Each dot represents one biological replicate. Bars indicate the average number of passages for each condition. (K) RNA ISH of fixed and sectioned co-cultures for αSMA (Acta2, red) and Krt18 (green) illustrating the spatial distribution of αSMAhigh PSCs in comparison to Krt18+ (green) tumor organoids (n = 2). Counterstain, DAPI. Higher magnification on the right. Bars, 50 µm.