Digital quantitation of HCC-associated stem cell markers and protein quality control factors using tissue arrays of human liver sections

Abstract

The most common type of liver cancer, hepatocellular carcinoma (HCC), affects over 500,000 people in the world. In the present study, liver tumor resections were used to prepare tissue arrays to examine the intensity of fluorescence of IHC stained stem cell markers in liver tissue from malignant HCC tumors and accompanying surrounding non-tumor liver. We hypothesized that a correlation exists between the fluorescence intensity of IHC stained HCC and surrounding non-tumor liver compared to liver tissue from a completely normal liver. 120 liver resection specimens (including four normal controls) were placed on a single slide to make a tissue array. They were examined by digitally quantifying the intensity of fluorescence using immuno-histochemically stained stem cell markers and protein quality control proteins. The stem cell markers were OCT3/4, Nanog, CD133, pEZH2, CD49F and SOX2. The protein quality control proteins were FAT10, UBA-6 and ubiquitin. The data collected was used to compare normal liver tissue with HCCs and parent liver tissue resected surgically using antibodies to stem cell markers and quality control protein markers. The measurements of the stem cell marker CD133 indicated an increase of fluorescence intensity for both the parent liver tissue and the HCC liver tissues. The other stem cell markers changed as follows: Nanog and OCT3/4 were decreased in both the HCCs and the parent livers; PEZH2 was reduced in the HCCs; SOX2 was increased in the parent livers compared to the controls; and CD49f was decreased in HCCs only. Protein quality control markers FAT10 and ubiquitin were downregulated in both the HCCs and the adjacent non-tumor tissue compared to the controls. UBA6 was increased in both the HCCs and the parent livers, and the levels were higher in the HCCs compared to the parent livers.

Keywords: Hepatocellular carcinoma (HCC); Morphometric analysis; Protein quality control pathways; Stem cells.

Figure 1. HCC Tissue Array Map

A. Map of tissue array composed of 120 liver resection specimens.

Figure 2. Low power (4x) fluorescent image of tissue array stained for Nanog

Figure 3. UBA-6 was up regulated in T (tumor) and B (parent liver) resections compared to NL (normal liver)

Intensity profiling of the stained tissue array under fluorescence microscopy (40× objective) demonstrated greater presence of UBA-6, as indicated by the higher peaks in the T and B screen snips shown as compared to the NL intensity peaks. Compared to normal liver control resections, T and B exhibited an average higher intensity. The results are shown as Mean ± S.E.

Figure 4. Presence of Nanog decreased in T and B resections

Fluorescence intensity profiling of stained tissue array indicated lower intensity peaks in T and B resections compared to normal liver tissue. Compared to normal liver control resections, T and B exhibited an average lower fluorescence intensity; the results are displayed as Mean ± S.E.

Figure 5. FAT10 was down regulated in T and B resections

Fluorescence intensity profiling of the stained tissue array demonstrated a decrease in FAT10 in T and B resections, shown by the lower intensity peaks as compared to normal liver control resections. T and B showed an average lower intensity; the results are displayed as Mean ± S.E.

Figure 6. OCT¾ decreased in T and B resections

Fluorescence intensity profiling of the stained tissue array demonstrated lower fluorescence intensity than the normal liver resections. The results displayed (Mean ± S.E, P<#VALUE) exhibit lower intensity than the control resections.

Figure 7. T and B resections showed a decrease in Ubiquitin

Fluorescence intensity profiling of the stained tissue array presented lower red intensity peaks than normal resections. The average intensities for T and B were found lower than control resections. The results are shown as Mean ± S.E.

Figure 8. T resections indicated a small decrease in pEZH2

Fluorescence intensity profiling indicated smaller peaks of fluorescence intensity; no significant change between B and NL was observed. The average fluorescence intensities indicate no significant difference between B and NL resections; the results, shown as Mean ± S.E, only indicate a small decrease in T resections.

Figure 9. SOX2 did not vary between the T, B, and NL resections

Fluorescence intensity profiling of T and B resections demonstrated no large change in fluorescence. The average intensities, shown as Mean ± S.E, indicate very little change between T, B, and NL resections.

Figure 10. CD133 was up regulated in B and T resections

Fluorescence intensity profiling revealed higher intensity peaks in B and T resections. The average intensities indicate a significant increase in B and T resections. Results shown as Mean ± S.E.

Figure 11. CD49F decreased in T and B resections

Fluorescence intensity profiling indicates smaller peaks in T and B resections. The resulting average intensities, shown as Mean ± S.E, indicate a decrease in T resections and a slight decrease in B resections.