Figure 1.
Depiction of hepatic bile duct and CCA.
(A) Upon infection of the common bile duct, C. sinensis produces ESPs, which stimulate CCA. (B) CCA and ESPs stimuli can be simulated using a microfluidic platform, culturing HuCCT1 cells (in the bile duct channel) on a COL1 hydrogel incorporated in the ECM channel. The cultured HuCCT1 cells form aggregates on COL1 in the bile duct channel and invade into the COL1 in response to ESPs stimuli.
Figure 2.
3D culture of HuCCT1 cells in a microfluidic device.
(A) The PDMS device was bonded with a glass coverslip, and microchannels were coated with PDL. The gel port was filled with pre-polymerized COL1 solution and then the device was incubated at 37°C for 30 minutes. A cell suspension (1×106 cells/ml) was injected into the bile duct channel. The cells were stacked onto the polymerized COL1 hydrogel by gravity by standing the device vertically for 2 hours. The medium was replaced with serum-free RPMI-1640 containing different concentrations of ESPs. (B) (i) Dimensions of the COL1 hydrogel and bowl-like structure that precisely fixes the number of cells on the COL1 hydrogel. (ii) Phase-contrast image of cells stacked on the collagen gel. Scale bar = 200 µm. (iii) SEM image of the fibrous structure of COL1.
Figure 3.
Concentration profiles of an ESPs-mimicking molecule in the COL1 hydrogel, media, and HuCCT1 cell aggregates.
ESPs at a concentration of 4,000 ng/ml were delivered directly into the bile duct channel (left) or indirectly to form a gradient (right). Direct application resulted in a high concentration of ESPs in the bile duct channel in 24 hours (left). In the gradient application, ESPs formed a gradient and stimulated HuCCT1 cells attached onto the COL1 ECM in 24 hours (right).
Figure 4.
Formation of a HuCCT1 cell tumor mass in the microfluidic assay system.
(A) ESPs exposure conditions: (i) direct stimulation (Dx1, Dx5), and (ii) indirect stimulation via gradient formation (▽x1, ▽x5). ×1 = 800 ng/ml; ×5 = 4,000 ng/ml. (B) Phase contrast images depicting growth of the HuCCT1 tumor mass. (C) Normalized growth of HuCCT1 cell tumors. *P<0.05 compared with the control. Significance was analyzed by Student's t-test. Error bars, ± SEM. (D) Fluorescence image of the HuCCT1 tumor mass (outlined in yellow dashed line) under untreated control (above) and Dx5 (below) conditions. Scale bars = 100 µm.
Figure 5.
Expression and localization of focal adhesion molecules.
(A) Expression of paxillin (above) and vinculin (below) in HuCCT1 cells under control and ESPs-stimulated (Dx1) conditions. Scale bars = 20 µm. (B) Quantification of localized paxillin (PAX) and vinculin (VIN) expression. *P<0.05 compared with the control. Significance was analyzed by Student's t-test. Error bars, ± SEM.
Figure 6.
3D invasion of HuCCT1 cells into COL1 ECM over 6 days.
(A) Phase-contrast image of protruding cells on day 3. (B) Summary data showing the total numbers of COL1-invading cells over 6 days. *P<0.05 and *** p<0.001 compared with the control. Significance was analyzed by Student's t-test. Error bars, ± SEM. (C) Immunofluorescence image of HuCCT1 cell penetration (red arrowheads) into COL1 under control (top) and Dx1 (bottom) conditions. Scale bars = 100 µm.
Figure 7.
Effect of ESPs on MMP mRNA and protein expression.
HuCCT1 cells were treated with 800 ng/ml ESPs or an equivalent volume of PBS, harvested after 24 hours, and analyzed for the expression of MMP1, −2, −9 and −13 mRNA and protein. (A) Expression of MMP1, −2, −9, and −13 mRNA normalized to 18 S rRNA was assessed by qRT-PCR. *P<0.05, ** p<0.01. Significance was analyzed by Student's t-test. Error bars, ± SEM. (B) Representative immunoblots of MMP1, −2, −9, and −13. The intensity of individual protein bands was determined by scanning densitometry and normalized to that of GAPDH. Data in graphs are expressed as a percentage of control values (in densitometric units).