Fig 1.
Physiological features of the human bile duct infected with C. sinensis and experimental design for assessing invasion by CCA cells in a clonorchiasis-associated tumor microenvironment.
(A) A common hepatic bile duct cancer (hilar cholangiocarcinoma) and C. sinensis infestation of a human liver (left). Formation of a tumor gland in the bile duct and chemical stimulation by ESPs (brown triangles) from C. sinensis (right). (B) The schematic figure (box with black border) is a COL1 hydrogel scaffold region for cell culture. Phase-contrast image showing green fluorescence protein-expressing HuCCT1 CCA cells in a complex tumor microenvironment (box with dotted line in A and B) consisting of H69 normal cholangiocyte and the directional application of ESPs.
Fig 2.
Features of H69 cells in 3D culture and development of a quiescent 3D biliary ductal plate.
(A) Cholangiogenesis by H69 cells in 3D culture. Red dotted lines indicate initial formation of a ductal plate and arrows indicate 3-dimensionally sprouting primitive ductal structures. (B) An orthogonal confocal microscopic image of a mature duct structure enveloped by H69 cells sprouted into the COL1 scaffold (asterisks) visualized from above (x-y axis) in the center image and the z (height axis) in the right and below. A z-stack image shows the 3D structure of neoductule formation by sprouts of H69 cells. (C) H69 cells sprouting in media of differing compositions; complete (Complete), FBS-free (FBS (-)) and FBS-free/EGF-depleted (FBS (-)/EGF (-)). (D) Quantification of H69 cell sprouting into COL1 hydrogel (area). (E) Expression of Ki-67 (green) in H69 cells in complete (above) and FBS-free/EGF-depleted (below) media. (F) Expression and localization of integrin α6 (green) in H69 cells on the COL1 hydrogel scaffold. Scale bars = 100 μm (A, B, C, E and F). DAPI and phalloidin staining highlight nucleus in blue and actin cytoskeleton in red, respectively (B, E and F). *P < 0.05 and **P < 0.01 versus complete medium. Student’s t-test was used to analyze significance. Error bars = ± SEM.
Fig 3.
H69 cell sprouting into COL1 hydrogels with differing mechanical properties.
(A) Sprouting of H69 cells under various ECM conditions; general control (2.0 mg/mL of COL1, pH 7.4), higher pH (2.0 mg/mL, pH 11), and higher concentration (2.5 mg/mL, pH 7.4). (B) Quantification of cell sprouting of H69 cells into COL1 hydrogels (area). Compared to the general control, there were no significant differences between groups. Scale bars = 100 μm (A). Student’s t-test was used to analyze significance. Error bars = ± SEM.
Fig 4.
Behavioral changes in H69 cells in quiescent culture by the pathogenic effect of ESP treatment and the diffusion profile of ESPs in a COL1 hydrogel.
(A) Our model predicts that ESP concentrations varied in the region of the biliary ductal plate (red dotted lines) between 1.5 μg/mL to 3.0 μg/mL and 1.7 μg/mL to 0.5 μg/mL after direct and gradient application respectively. Concentration profiles and heat-map images show the ESP concentration along the cell-seeded channel, COL1 hydrogel scaffold and opposite channel. (B) H69 cells sprouting induced by ESP treatment conditions; non-treated control (NC), direct treatment (Direct) and treatment with a gradient (Gradient). Dotted lines (red) and arrows (red) indicate the biliary ductal plate and H69 cell sprouting, respectively. (C) Quantification of the area of H69 cell sprouting into the COL1 hydrogel (area). There was no significant difference between the direct and gradient ESP applications and the NC. Scale bars = 100 μm (B). Student’s t-test was used to analyze significance. Error bars = ± SEM.
Fig 5.
Increased migration and invasion of CCA cells after ESP treatment of the biliary tumor microenvironment.
(A) H69 and HuCCT1 cells shown as a bright field image with green fluorescence overlay. Initial positions of H69 (unstained) and HuCCT1 cells (green) after serial cell seeding (left, Day 1) and subsequent invasion of HuCCT1 cells through the H69 cell layer in the microfluidic device (right, Day 3). (B) Migration of HuCCT1 cells toward the COL1 hydrogel scaffold on day 3. (C) Single cell invasion by HuCCT1 cells (red circle) into the COL1 hydrogel scaffold on day 3. (D) Quantified migration of HuCCT1 cells (penetrated area of the COL1 ECM) and invasion by HuCCT1 cells (counted number of individually invading cells). Green fluorescence indicates HuCCT1-GFP (A, B and C). Scale bars = 100 μm (A, B and C). #P = 0.058, *P < 0.05 versus non-treated control. Student’s t-test was used to analyze significance. Error bars = ± SEM.
Fig 6.
Expression of IL-6 and TGF-β1 in ESP-treated H69 and HuCCT cells.
(A) H69 cells were treated with 800 ng/mL of ESPs for 0 (NC, non-treated control), 12, 24, and 48 hours, and the production of IL-6 or TGF-β1 in each culture supernatant was determined by ELISA. (B) HuCCT1cells were treated with 800 ng/mL of ESPs for 24 hours, and the medium was then replaced by culture supernatant from one of four groups of H69 siRNA transfectants exposed to ESPs for 48 hours. Cells were incubated further for 48 hours, and the production of IL-6 or TGF-β1 in each culture supernatant was determined by ELISA. The concentration of each cytokine was calculated from IL-6 and TGF-β1 standard curves. *P < 0.05, **P < 0.01 versus non-treated control. Student’s t-test was used to analyze significance. Error bars = ±SEM.
Fig 7.
Expression of E- and N-cadherin in ESP-treated HuCCT1 and H69 cells.
HuCCT1 (A) or H69 (B) cells were treated with 800 ng/mL of ESPs and harvested after 0–48 hours for immunoblot analysis of E- and N-cadherin expression. (C) The 24-hour ESP-treated medium of HuCCT1 cells was replaced by the culture supernatants from each four different H69 siRNA transfectants as described in the materials and methods section. After a 48-hour-incubation, total soluble proteins were immunoblotted using polyclonal antibodies against E- and N-cadherin. Protein bands were quantified densitometrically and normalized to the density of the GAPDH band. The ratio of E- or N-cadherin to GAPDH in each group is presented as its fold-change relative to the non-treated 0–hour control (NC). *P < 0.05, **P < 0.01 and ***P < 0.001 with control. Student’s t-test was used to analyze significance. Error bars = ± SEM.