Figure 6.

Photomicrographs of representative H & E stained histological sections of 3-dimensional organotypic cultures of BDEsp-TDE_H10 cholangiocarcinomacells cultured for 6 days either in monoculture (A) or in co-culture with BDEsp-TDF_E4 CAFs (B & C). Cholangiocarcinoma cells were initially plated at a cell density of 2 × 10⁵ cells/type I collagen gel in both the mono- and co-cultures. The initial CAF cell plating density in the co-cultures was at 8 × 10⁵ cells/gel. The histological section shown in B was obtained in the same experiment as that for A, whereas that in C was from a separate experiment essentially repeating the culture conditions used for B. Note the dramatic increase in the number of spheroidal/”duct-like” structures formed from the cholangiocarcinoma cells in the co-cultures (B & C) over those formed under comparable culture conditions in control cholangiocarcinoma cell monoculture without CAFs (A). Also observe a much more intense eosinophilic staining of the gel matrix in B & C compared to that of A, indicative of an increase in the cellular secretion of insoluble proteins into the co-culture matrix versus that produced in the cholangiocarcinoma cell monoculture. As further depicted in B & C, only the co-cultures exhibited prominent irregular clusters of anaplastic cholangiocarcinoma cells surrounded by stromal CAFs, reflective of malignant progression and apparent invasiveness. (D) Bar graph demonstrating the mean number of spheroidal/”duct-like” structures/cm² tissue section area formed from BDEsp-TDE_H10 cholangiocarcinoma cells to significantly increase as a function of higher initial BDEsp-TDF_E4 plating densities when these ICC derived cell types were co-cultured for 6 days in rat type I collagen gel matrix. Each value represents the mean ± SD determined from microimaging measurements made on 10μm thick H & E stained histological sections (n = at least 2 sections/slide prepared from triplicate cultures for each CAF plating density). P values were determined against the 0 CAF culture condition. Gel contraction, measured as a reduction in mm gel diameter ( = mean ± SD) was determined to be significantly greater at the higher initial BDEsp-TDF_E4 cell plating densities. (E) Representative data demonstrating BDEsp-TDF_E4 CAFsto increase the cell migration/invasiveness of BDEsp-TDE_H10 cholangiocarcinoma cells in vitro when assessed in the Matrigel^™ invasion bioassay system. T= top chamber coated by Matrigel; B = bottom culture well coated with rat tail type I collagen. Invasion Index was determined according to the manufacturer’s instructions.