Contribution of laser microdissection-based technology to proteomic analysis in hepatocellular carcinoma developing on cirrhosis

Abstract

Hepatocellular carcinoma (HCC) is a major cause of cancer worldwide. Proteomic studies provide opportunities to uncover targets for the diagnosis and treatment of this disease. However, in HCC developing in a setting of cirrhosis, the detection of proteome alterations may be hampered by the increased cellular heterogeneity of tissue when analysing global liver homogenates. The aim of this study was to evaluate whether the identification of proteome alterations in these HCC cases was improved when the differential protein profile between tumour and non-tumour areas of liver was determined using hepatocytes isolated by laser microdissection (LM). Differential profiles established with LM-hepatocytes and liver section homogenates using 2-DE and MS exhibited noticeable differences: 30% of the protein spots with deregulated expression in tumorous LM-samples did not display any modification in homogenates; conversely 15% of proteins altered in tumorous homogenates were not impaired in LM-hepatocytes. These alterations resulted from the presence in cirrhotic liver of fibrotic stroma which displayed a protein pattern different from that determined in LM-hepatocytes. In conclusion, our data demonstrate the interest of LM in distinguishing between fibrotic and hepatocyte proteome alterations and thus the benefit of LM to proteome studies of HCC developing in a context of cirrhosis.

Figure 1. The effects of hematoxylin/eosin staining and laser microdissection on 2-DE profiles

A- 2-DE profiles were performed using unprocessed, stained tissue slides and cells microdissected from stained slides after optimisation of the staining procedure. B- Scatter plots showing the correlation between the relative volumes (means of 3 gels) of protein spots determined using 2-DE and carried out with unprocessed and stained tissue slides (left) and stained tissue slides and microdissected cells (right). The correlation coefficients calculated were determined according to Spearman.

Figure 2. Comparison of differential protein expression profiles established using LM-procured hepatocytes and total liver homogenate

A- Left: Differential protein profile determined during 2-DE performed with LM-procured hepatocytes: 146 protein spots were differentially expressed (white) and 640 were not altered (black) in tumorous hepatocytes. Right: the 146 protein spots deregulated in tumorous hepatocytes were analysed in global homogenate samples: 95 were similarly deregulated (white), 8 were deregulated in an opposite way (grey) and 43 were not altered (black). B- Left: Differential protein profile determined during 2-DE performed with global homogenates: 119 protein spots were differentially expressed (white) and 667 were not altered (black) in tumorous homogenate. Right: the 119 protein spots deregulated in tumorous global homogenates were analysed in LM-hepatocytes samples: 95 were similarly deregulated (white), 8 were deregulated in an opposite way (grey) and 16 were not altered (black)

Figure 3. Comparative analysis of relative 2-DE spot volumes in LM-hepatocytes (black bars) and total liver homogenate (white bars) experiments

Representative examples of spots with deregulated differently in tumorous (T) and non tumorous (NT) counterparts. Data are expressed as means ± SEM of three experiments. Statistical analysis was performed using the Mann-Whitney U-test and Student’s t-test. NS: non-significant

Figure 4. Western blot analysis of MxA, Annexin A1, mitochondrial HMGCoA synthase and GRP75 in both homogenate and LM-hepatocyte samples

Western blots were performed as described in the Material and Methods using paired tumour (T) and non-tumour (NT) pooled samples from the three patients included in 2-DE experiments.

Figure 5. Analysis of Arginase 1 and mitochondrial HMG CoA synthase in global homogenates, stromal cells and hepatocytes selected by microdissection in the non-tumorous area

A. Non tumorous liver section stained with H&E shows the abundance of fibrotic stroma (F) in the cirrhotic liver. Hepatocyte nodules are mentioned as H. B- Fibrotic stroma and hepatocytes were microdissected in the non tumorous area of liver from two patients. Levels of Arginase 1 and mitochondrial HMG CoA synthase expression were analysed as described in the Materials and Methods in fibrotic stroma (F), hepatocytes (H) and complete homogenates (C) for each patient.

Figure 6. Range of T/NT expression ratios of the spots similarly deregulated in tumorous LM-hepatocytes and total liver homogenates

On the absciss are the 95 spots similarly deregulated in tumorous samples. On the ordinates, (A) T/NT expression ratios of protein spots deregulated in tumorous LM-hepatocytes and (B) T/NT ratios of protein spots deregulated in tumorous homogenates. Expression ratios of each spot determined under both conditions were compared using the match-paired Wilcoxon rank test. Statistical analysis showed that T/NT ratios were significantly increased when measured in LM-hepatocytes (p<0.001).