Imaging mass spectrometry-based proteomic profiling of mixed liver carcinomas (hepatocholangiocarcinoma)

Background: Hepatocholangiocarcinomas (H-ChCs) are mixed primary tumors of the liver, defined by the presence of hepatocellular carcinoma (HCC) and cholangiocarcinoma (CC) components that may display stem cell features. Accordingly, H-ChC represents the most illustrative heterogeneous liver malignancy. Its tumoral heterogeneity hampers molecular analysis that may provide various signatures according to the area analyzed. The aim of this study was to assess directly on tissue the peptide profiles of the different morphological patterns constitutive of H-ChC by Imaging Mass Spectrometry (IMS), without preliminary step of molecular extraction or labeling. Method: FFPE TMAs built from surgical cases of H-ChC, HCC, and CC were sectioned and processed for dewaxing, antigen retrieval, and in-situ enzymatic digestion with trypsin (0,025µg/µL). An organic matrix solution (CHCA, 10mg/mL) was deposited onto digested tissues using a TM-Sprayer (HTX Technologies, USA). Mass spectral measurements were carried out using a rapifleX MALDI Tissuetyper (Bruker Daltonik, DE) at 50 μm spatial resolution. Data were analyzed by FlexImaging (Bruker Daltonik, DE) and SCiLS Lab (SCiLS, DE) software. Results: More than 200 ion peptides were obtained from the different tumor patterns constitutive of H-ChC as well as from CC and HCC. The comparison of the relative intensities of each m/z ion peptide between the three tumor types (H-ChC, HCC, CC) revealed 19 discriminant peaks exhibiting a statistical significance. They showed a receiver operating curve (ROC) superior to 0.7 in the identification of H-ChCs when applied on cases and controls. Comparative analysis of H-ChC cases showed high heterogeneity from case to case, within case from different areas of the tumor, and within case from a single tumor area. Conclusion: This study demonstrates that IMS is a powerful tool for providing specific peptide signatures from highly heterogeneous FFPE H-ChCs at the molecular level and both at the inter- and intra-tumoral scale. The identification of the proteins specifically associated with the different tumor components is currently ongoing.