Design of microfluidic chip for tumor spheroid-fibroblast co-culture.

<p>Structure and organization of a microfluidic chip used for 3D co-culture of human colorectal cancer cells (HT-29) and normal colorectal fibroblasts (CCD-18Co). One chip contained 4 units and one unit consisted of 7 channels for either cell loading or media fill. Channel designation for co-culture: cancer cells and fibroblast cells were loaded in channel 4 and 2, respectively, and other channels (1 and 3) were used for media fill. A cell loading channel is shown with detailed structure and dimension (left-bottom).</p

Effect of co-culture on growth and size distribution of spheroids.

<p>Cells were grown in collagen-supported microfluidic channels with or without fibroblast co-cultures and number and size distribution was determined at day 5. (A) Growth of HT-29 spheroids in size and number (scale bar = 200 μm). (B) Comparison of mean diameter of HT-29 spheroids over 5 days. (C) Comparison of size distribution of tumor spheroids on day 5. Diameter of spheroids was calculated using bright field images and Image J program. Cell aggregates of diameter larger than 50 μm were considered as spheroids. Student t-test as well as χ² test were used for the statistical significance. Data are expressed as the mean ± SE of 3 replicates. * p<0.05, **p<0.01.</p

Activation of fibroblasts under co-culture with 3D tumor spheroids.

<p>(A) Fluorescence images of fibroblasts stained for F-actin and α-SMA showing differential expression levels under mono- and co-culture conditions. (B) Increased migration ability of fibroblasts towards 3D tumor compartment. A picture for a representative regions showing fibroblast migrated out of the designated channel and comparison of migration distance. The migration distance of fibroblasts was measured between the nucleus of fibroblast in medium channel and end of fibroblast culture channel. Cells were grown for 6 days before all measurements. Optical sections were acquired at 3 μm intervals and stacked into a z-projection from which fluorescence intensity was calculated. Student t-test was used to test the statistical significance. Data are expressed as the mean ± SE of 3 replicates. (Scale bars 200 μm, 100 μm). * p<0.05.</p

Differential protein expressions in HT-29 tumor spheroids with or without fibroblast co-cultures.

<p>When co-cultured with fibroblasts, TS showed up-regulation of 7 angiogenesis-related proteins with 1.5-fold or greater changes (A) and down-regulation of 5 apoptosis-related proteins with greater than 30% changes (B). HT-29 TSs were grown for 6 days with or without fibroblasts in microfluidic channels and harvested for analysis using Proteome Profiler™ (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0159013#sec002" target="_blank">Materials and Methods</a> for details).</p

Additional file 2: Figure S2. of Microfluidic co-culture of pancreatic tumor spheroids with stellate cells as a novel 3D model for investigation of stroma-mediated cell motility and drug resistance

Expression of cytokines in PSCs. (a) PSCs were grown for 5 days with or without PANC-1 spheroids in microchannel plate and harvested for proteome analysis using Proteome Profiler™. PSCs, pancreatic stellate cells; TS, tumor spheroids. (TIFF 2828 kb

Additional file 3: Figure S3. of Microfluidic co-culture of pancreatic tumor spheroids with stellate cells as a novel 3D model for investigation of stroma-mediated cell motility and drug resistance

Differential expression of EMT-related markers in different tumor cell spheroids. Immunofluorescence staining of E-cadherin and vimentin was performed in PANC-1 and HT-29 spheroids cultured for 5 days in microfluidic channels, and on paraffin sections of Huh-7 spheroids cultured for 5 days in ULA 96 well plates. For PANC-1 and HT-29 spheroids (red), confocal optical sections were acquired at 2 μm intervals and stacked into a z-projection (see Methods for details). Counter stain, DAPI (blue). Scale bars, 20 μm and 100 μm. EMT, epithelial-mesenchymal transition; TS, tumor spheroids. (TIF 667 kb

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

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

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

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