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Fig. 6 | Journal of Experimental & Clinical Cancer Research

Fig. 6

From: Subtypes in pancreatic ductal adenocarcinoma based on niche factor dependency show distinct drug treatment responses

Fig. 6

CAF-derived RSPO3 contributes to the growth of well differentiated PDAC with high dependency on RSPO. A qRT-PCR analysis of the mRNA expressions of RSPO1–4 in CAFs under different conditions (2D-culture with serum medium, 2D SM; 3D-culture with serum medium, 3D SM; 3D-culture with PDO conditioned serum medium, 3D PDO-CM). Results are shown relative to the expression in CAFs 2D-cultured with serum medium after normalization by GAPDH mRNA expression (***P < 0.001). B Representative phase contrast images of Grade1 PDOs (585) cultured in niche medium with the indicated concentration of recombinant RSPO1 or RSPO3. C Dose–response curve of Grade1 PDOs to RSPO1 and RSPO3. Results were normalized using the proliferation of PDOs with RSPO1 at 1000 ng/ml as 100%. D Representative images of immunohistochemical staining for RSPO3 and αSMA in serial sections of human Grade1 PDAC tissue. Scale bars, 100 µm. E Transfection of RSPO3-targeting shRNA (sh-RSPO3#1 and sh-RSPO3#2) decreased RSPO3 mRNA expression in CAFs compared with negative control shRNA (sh-ctrl) as confirmed by qRT-PCR analysis (***P < 0.001). F Schema of 3D direct co-culture models using PDOs and shRNA-transfected CAFs. G Representative fluorescence images of Grade1 and Grade3 PDOs directly co-cultured with shRNA-transfected CAFs. H Quantification of the total area of PDOs direct co-cultured with shRNA-transfected CAFs (**P < 0.01)

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