Schematic overview of pancreatic tumor spheroids (TS) generated in concave microwells.

<p>Pancreatic cancer cells cultured in agarose-coated 96 well plates produced only loose aggregates (A). The TS formation process in a BSA-coated concave microwell plate (B). The TS structure, compared with an <i>in vivo</i> tumor, showed a close resemblance to the <i>in vivo</i> condition. Cells in the TS retained the characteristics of <i>in vivo</i> tumors under 3D culture conditions.</p

Analysis of pancreatic cancer stem cell markers.

<p>Expression patterns of stem cell markers such as CD44, CD24, and ESA were compared between 2D (A) and 3D cultures (B). Percentages of the cell population expressing CD44, CD24, and ESA in Panc-1 cells cultured under 2D and 3D conditions are summarized in the table.</p

Morphology and histological examination of tumor spheroids (TS) cultured in concave microwell 600 or in 96 well plates.

<p>Representative images of H&E stained paraffin sections or toluidine blue stained semi-thin sections, SEM and TEM images of HT-29 (A), Panc-1 (B), Aspc-1 (C), and Capan-2 (D) spheroids cultured in concave microwell 600 plates for 5 and 13 days. Aggregates of Panc-1, Aspc-1, and Capan-2 cells formed in agarose-coated 96 well plates shown as an H&E stained paraffin section or bright field images (E). Arrow: desmosome; dotted lines: gap junctions; arrow head: tight junction; cross: lipid droplet; I: invagination structure; N: necrotic regions. The scale bars indicate 100 μm, 50 μm, 2 μm and 500 μm, in H&E or toluidine blue stained, SEM, TEM and bright-field images, respectively.</p

Expression of proteins associated with tumor growth and drug resistance.

<p>Spheroids were cultured for 5-β1, CTGF, and MT1-MMP (A) and for ECM proteins such as collagen type I, fibronectin, and laminin (B). (Scale bar  = 20 μm).</p

Penetration of DOX into Panc-1 spheroids.

<p>Penetration was evaluated by imaging DOX autofluorescence in sections of spheroids. Spheroids were cultured in a concave microwell 600 plate for 5 μM for 12 h. Fluorescence intensity across sections was measured and expressed as the mean ± SE of 5 replicates. (Scale bar  = 100 μm) * and **, p<0.01 and p<0.001, respectively.</p

The miRNA expression profiles were compared between 2D and 3D cultures.

<p>A heatmap of differentially expressed miRNAs generated by unsupervised hierarchical cluster analysis (A). Differential expression levels of miRNAs between 2D and 3D cultures were confirmed using qRT-PCR for several genes selected from microarray data. Among the differentially expressed miRNAs, up-regulated miRNAs including miR-34b-5p, miR-578, miR-1304, and miR-324-5p and down-regulated miRNAs including miR-7-5p, and miR-34b-3p were analyzed using primers for mature mRNAs (B). Data are expressed as the mean ± SE of triplicates. * p<0.05, n = 3.</p

Differentially expressed miRNAs in Panc-1 cell cultured under 2D and 3D conditions. Fold change indicates 3D intensity/2D intensity.

<p>Differentially expressed miRNAs in Panc-1 cell cultured under 2D and 3D conditions. Fold change indicates 3D intensity/2D intensity.</p

Concave PDMS microwell plate for culture and growth of pancreatic tumor spheroids (TS).

<p>Shape and dimension of concave microwell plate 600 (A). Changes in size of three different pancreatic spheroids cultured in microwell 600 (B). Size distribution of Panc-1 spheroids cultured in three different sized-microwell plate during 13 days of culture (C). Data are expressed as mean ± SE of a minimum of 10 spheroids cultured in one microwell plate.</p

Cytotoxicity/viability assays for Panc-1 spheroids.

<p>Changes in Ki-67 expression were measured following exposure to DOX at the indicated concentrations for 12 h (A). Changes in MitoSOX were measured following exposure to GEM at the indicated concentration for 12 h (B). MTS (C) and APH assay (D) after exposure to GEM for 72 h. Data are expressed as the mean ± SE of 6 replicates. (Scale bar  = 100 μm) * p<0.001.</p

DUSP22-induced cell death.

<p>(A) Cell viability was determined as described in Materials and Methods. HEK 293 cells were transfected with FLAG-DUSP22 (WT or C88S mutant, 1 μg) expression plasmid. Data represent means ± SEM of six observations from three independent experiments. The expression levels of DUSP22 were determined by immunoblotting with an anti-FLAG antibody. IB, immunoblot. *<i>p</i> < 0.01 versus the control sample by Student’s <i>t</i>-test. (B) HCT 116 cells were transfected with 2 μg of FLAG-DUSP22 WT or FLAG-DUSP22 C88S expression plasmid. After 48 h of transfection, cell lysates were subjected to immunoblotting with indicated antibodies. Fifty microgram of cell lysate was loaded in each lane and the blot was probed with antibodies specific to p-JNK, cleaved PARP, or cleaved caspase-3. Twenty microgram of cell lysate was used for detection of JNK and FLAG. HCT 116 cells were transfected with (C) and (E) FLAG-DUSP22 WT (0, 0.5, 1, 2, 3, 4 μg) or (D) and (F) FLAG-DUSP22 C88S (0, 0.5, 1, 2, 3, 4 μg) for 48 h and then incubated for 1 h in the absence or presence of H₂O₂. Total cell lysates were subjected to immunoblot analyses with appropriate antibodies. Right panel: Relative fold of cleaved PARP levels after normalization to corresponding tubulin levels. All data are representative of three independent experiments.</p