Figure 2.

ACE2 is detected in the biliary pole of moderately-to-well-differentiated HCCs with trabecular or pseudo-glandular patterns. Combined immunoperoxidase and immunofluorescence analysis of a tissue microarray containing 41 HCCs and two normal liver controls, spotted in triplicates. Representative images are shown. Immunoperoxidase signal (brown) is counterstained with hematoxylin (blue). ACE2 appears in red by immunofluorescence, other markers in green and nuclei in blue (DAPI staining). (a), (b) ACE2 colocalizes with the apical hepatocyte marker ABCC2 (a.k.a. MRP2) (arrows). (c) Tumor capillary vessels are marked with CD34. Confocal digital images were acquired with a 40X objective. Images are Z-stacks of four 500 nm focusing steps. (d) Quantification of ACE2, ABCC2, dual (ABCC2 + ACE2) staining and the ratio of (ACE2 + ABCC2) × ACE2^–1, representing the fraction of ACE2 protein expression detected as dual (ACE2 + ABCC2) staining, i.e., the fraction of overlap of ACE2 with ABCC2. Values for individual TMA spots from 32 HCCs are expressed as the fraction of DAPI staining of nuclei, to correct for natural variations in cell density in each spot. Means ± 95% confidence intervals are shown for each condition. The statistical significance between groups was calculated with Kruskal–Wallis test (p < 0.0001), followed by the post-hoc Dunn’s test as indicated. (e) Sample scan from a TMA spot showing examples for the different signals quantified in (d). (f) The fraction of overlapping (ACE2 + ABCC2) over total ACE2 signal (i.e., HCC cells expressing ACE2 at the biliary pole), is higher in HCCs carrying CTNNB1 mutations. Each data point represents the mean of two to three cores for each tumor. Statistical significance calculated by Mann–Whitney U test.