Identification of cellular targets in human intrahepatic cholangiocarcinoma using laser microdissection and accurate mass and time tag proteomics

Abstract

Obtaining accurate protein profiles from homogeneous cell populations in heterogeneous tissues can enhance the capability to discover protein biomarkers. In this context, methodologies to access specific cellular populations and analyze their proteome with exquisite sensitivity have to be selected. We report here the results of an investigation using a combination of laser microdissection and accurate mass and time tag proteomics. The study was aimed at the precise determination of proteome alterations in intrahepatic cholangiocarcinoma ICC, a markedly heterogeneous tumor. This cancer, which is difficult to diagnose and carries a very poor prognosis, has shown an unexplained increase in incidence over the last few years. Among a pool of 574 identified proteins, we were able to report on altered abundance patterns affecting 39 proteins conforming to a variety of potential tumorigenic pathways. The reliability of the proteomics results was confirmed by Western blot and immunohistochemistry on matched samples. Most of the proteins displaying perturbed abundances had not yet been described in the setting of ICC. These include proteins involved in cell mobility and actin cytoskeleton remodeling, which may participate in the epithelial to mesenchymal transition, a process invoked in migration and invasion of cancer cells. The biological relevance of these findings was explored using a tissue microarray. An increased abundance of vimentin was thus detected in 70% of ICC and none of the controls. These results suggest that vimentin could play a role in the aggressiveness of ICC and provide a basis for the serious outcome of this cancer.

Fig. 1.

Study design. Cholangiocytes microdissected from control livers (TB–TF) and from intrahepatic cholangiocarcinomas (CA–CD) were pooled prior to sample fractionation and LC-MS/MS analysis to generate an AMT tag database (A). Individual samples were subsequently analyzed by LC-FTMS to determine differential alterations of their proteome (B).

Fig. 2.

Quantitative LC-FTMS data quality control. A, Chaorder plot for the 27 acquisitions (three for each sample) grouped by sample. B, Pearson pairwise correlation (R) matrix of peptide abundances measured in each acquisition sorted by sample.

Fig. 3.

Distributions of AbI for 1121 peptides detected in both populations for original data (A) and for permuted data (B). The permutation allowed an estimation of the variance of the AbI measure under the assumption that all samples were equivalent. The dashed lines in A delimit the 95% confidence interval (−0.24 < AbI < 0.53), which is equal to the median AbI (0.14) of the original data ±1.96 times the standard deviation (Stdev) (0.199) of the permuted data, a region where no change in abundance between the two populations was observed.

Fig. 4.

Expression analysis of vimentin, CAII, and profilin-1 in intrahepatic cholangiocarcinoma. A, cell plot of the log of peptide abundances in cancer and control samples. Outline colors correspond to peptides returned as significant by the statistical analysis (blue), peptides shared with other protein groups (pink), peptides not returned as significant by the statistical analysis (yellow), and population-specific peptides (gray). B, immunodetection of vimentin, CAII, and profilin-1. Western blots carried out as described under “Experimental Procedures” were performed using microdissected tumorous and non-tumorous cholangiocytes from subjects included in the study. C, immunohistochemical analysis of vimentin and CAII. Immunohistochemistry was performed as described under “Experimental Procedures.” Data are from a representative experiment including the tumorous and non-tumorous areas of the liver of patient CA and a control liver, TB. Vimentin is expressed in tumorous cholangiocytes from ICC (top left) and negative in non-tumorous cholangiocytes of peripheral (middle left) and perihilar (bottom left) bile ducts. Internal controls (vascular smooth muscle cells and inflammatory cells) are positive. Conversely, CAII is negative in tumorous cholangiocytes from ICC (top right) and expressed in non-tumorous cholangiocytes of peripheral (middle right) and perihilar (bottom right) bile ducts.

Fig. 5.

Vimentin expression in liver lesions by immunohistochemical staining using tissue microarray. A, representative cores of perihilar bile duct (1), intrahepatic cholangiocarcinoma (2), hilar cholangiocarcinoma (3), and hepatocellular carcinoma (4). Immunohistochemical staining was performed as described under “Experimental Procedures.” Vimentin is negative in normal cholangiocytes from perihilar bile duct, whereas inflammatory cells stained as positive internal controls (1). Vimentin is diffusely positive in the tumorous cholangiocytes from ICC (2) and totally negative in the tumorous cholangiocytes from hilar cholangiocarcinoma (3) and tumorous hepatocytes from HCC (4). B, vimentin expression in 23 intrahepatic cholangiocarcinoma, 17 hilar cholangiocarcinoma, 18 hepatocellular carcinoma, and 22 controls patients (seven perihilar areas from liver without ICC, four normal livers from amyloid neuropathy, and 11 non-tumoral counterparts of ICC). Staining was considered negative when less than 5% of the cells of interest were immunostained. *, p < 0.001 (two-tailed Fisher's exact text).