Effects of hepatocellular carcinoma-associated fibroblasts on differentiation of monocyte-derived dendritic cells

Objective To study the effects of hepatocellular carcinoma-associated fibroblasts(hCAF) on the differentiation of monocyte(Mo)-derived dendritic cell(DC). Methods The human hCAF were obtained from hepatic carcinoma tissue by enzyme digestion method. The healthy human peripheral blood mononuclear cells(PBMC) were prepared by density gradient centrifugation and CD14⁺Mo from healthy human peripheral white blood cells were purified by density gradient centrifugation and magnetic bead separation. hCAF-CD14⁺Mo(1:10) and CD14⁺Mo were treated by granulocyte-macrophage colony-stimulation factor(GM-CSF) (20 ng/ml) and interleukin(IL)-4 (20 ng/ml) which were added in day 1 and day 4 for 6 days. And then the cells of two groups were divided into two halves: one with or without the further stimulation of lipopolysaccharides(LPS, 200 ng/ml) for 3 days respectively. Finally the cells were divided into 4 groups: hCAF-Mo, hCAF-Mo+LPS, immature DC(iDC) and mature DC(mDC) group. Morphological changes of cells in 4 groups were observed by inverted fluorescence microscope. Cells in each group were divided into two parts. The expression of CD83,CD80, CD1a of cells in one part were detected by flow cytometry. And ability of T cells proliferation in the other part was analyzed by flow cytometry after co-culturing with CFSE-labeled T cells for 5 days. Results The result of inverted microscope showed that Mo couldn't differentiate into DC by the interference of hCAF. The expressions of CD83, CD1a and CD80 were (3.2±0.7)%,(61.7±8.4)%,(30.1±0.9)% respectively in iDC group,(80.2±2.8)%, (83.15±6.0)%,(96.1±1.9)% in mDC group,(1.6±0.9)%,(1.8±0.9)%,(16.0±3.2)% in hCAF-Mo group,(9.0±1.2)%、(1.1±0.4)%、(58.4±3.6)% in hCAF-Mo+LPS group. There was significant difference of the expressions of CD1a and CD80 between hCAF-Mo and iDC groups(both in P<0.01). Due to the extremely low expression of CD83 in iDC group, there was no significant difference between hCAF-Mo and iDC groups(P>0.05). There was significant difference of the expressions of CD83,CD1a and CD80 between hCAF-Mo+LPS and mDC groups (all in P<0.01). Proliferation rate of T cells were (3.3±0.9)% in iDC group, (34.5±7.3)% in mDC group,(5.3±1.2)% in hCAF-Mo group,(7.0±1.2)% in hCAF-Mo+LPS group. Compared with iDC group, mDC could promote the proliferation of T cells effectively. And the proliferation of T cells in hCAF-Mo and hCAF-Mo+LPS group was lower than that in mDC group(P<0.01). Conclusions hCAF can suppress the differentiation of Mo-derived DC, which may play an important role in immune escape of hepatocellular carcinoma.