MALDI Imaging, a Powerful Multiplex Approach to Decipher Intratumoral Heterogeneity: Combined Hepato-Cholangiocarcinomas as Proof of Concept

SIMPLE SUMMARY: To provide a comprehensive molecular characterization of combined hepatocholangiocarcinomas (cHCC-CCA), the MALDI (Matrix Assisted Laser Desorption Ionization) imaging approach was used to acquire high-resolution spatialized molecular profiles directly from fixed tissue sections without a previous labeling step. The heterogeneity of cHCC-CCA was explored through the analysis of whole tumor slides by comparing peptidic profiles and histological and immunophenotypical images. MALDI imaging analysis was able to identify both tumor components (HCC and iCCA) and also small tumor areas not seen at the microscopic level. In addition, the morphological distribution of tryptic peptides of the different immunophenotypic markers was analyzed in silico. Their distribution allowed us to identify areas initially unlabeled by immunohistochemistry (IHC). This study demonstrates that an in silico MALDI imaging approach may improve conventional histological analysis and allow the development of new markers. ABSTRACT: Combined hepato-cholangiocarcinomas (cHCC-CCA) belong to the spectrum of primary liver carcinomas, which include hepatocellular carcinomas (HCC) and intrahepatic cholangiocarcinomas (iCCA) at both ends of the spectrum. Mainly due to the high intratumor heterogeneity of cHCC-CCA, its diagnosis and pathological description remain challenging. Taking advantage of in situ non-targeted molecular mapping provided by MALDI (Matrix Assisted Laser Desorption Ionization) imaging, we sought to develop a multiscale and multiparametric morphological approach, integrating molecular and conventional pathological analysis. MALDI imaging was applied to five representative cases of resected cHCC-CCA. Principal component analysis and segmentations with MALDI imaging techniques identified areas related to either iCCA or HCC and also hidden tumor areas not visible microscopically. In addition, the overlap between MALDI segmentation and immunostaining provided a comprehensive description of cHCC-CCA tumor heterogeneity by identifying transitional and micro-metastatic areas. Moreover, a list of peptides derived from in silico digestion was obtained for each immunohistochemical marker and was matched within the peptide peak list acquired by MALDI. Comparison of immunostaining images with ions from in silico digestion revealed an accurate identification of iCCA and HCC areas. Our study provides further evidence on the performance of MALDI imaging in exploring intratumor heterogeneity and offering virtual multiplex immunostaining through a single acquisition.