Abstract
Isolation of hepatocytes from cirrhotic human livers and subsequent primary culture are important new tools for laboratory research and cell-based therapeutics in the study of hepatocellular carcinoma (HCC). Using such techniques, we have previously identified different subpopulations of human hepatocytes and among them one is showing a progressive transformation of hepatocytes in HCC-like cells. We have hypothesized that increasing the distance from the neoplastic lesion might affect hepatocyte function and transformation capacity. However, limited information is available in comparing the growth and proliferation of human hepatocytes obtained from different areas of the same cirrhotic liver in relation to their distance from the HCC lesion. In this study, hepatocytes from 10 patients with cirrhosis and HCC undergoing surgical resections from specimens obtained at a proximal (CP) and distal (CD) distance from the HCC lesion were isolated and placed in primary culture. CP hepatocytes (CP-Hep) were isolated between 1 to 3 cm (leaving at least 1cm margin to avoid cancer cells and/or satellite lesions), while CD hepatocytes (CD-Hep) were isolated from more than 5 cm or from the contralateral-lobe. A statistical model was built to analyze the proliferation rates of these cells and we evaluated expression of HCC markers (Glypican-3 (GPC3), αSmooth Muscle Actin (α-SMA) and PCNA). We observed a significant difference in proliferation and in-vitro growth showing that CP-Hep had a proliferation pattern and rate significantly different than CD-Hep. Based on these data, this model can provide information to predict growth of human hepatocytes in primary culture in relation to their pre-cancerous state with significant differences in the HCC markers expression. This model provides an important innovative tool for in-vitro analysis of HCC.
Highlights
We showed that cirrhotic proxima (CP)-Hepatocytes over a period of 16 weeks transformed into cells expressing high levels of markers for hepatocellular carcinoma (HCC) (Glypican-3 (GPC3) and α Smooth Muscle Actin (α-SMA) and cell proliferation nuclear antigen (PCNA)
We previously showed that hepatocytes isolated from different areas of a single cirrhotic liver with HCC present different morpho-functional characteristics over time while cultured invitro
Primary cultures of the cells obtained in closer proximity to HCC lesion seem to be already committed to become HCC and behave as precancerous hepatocytes [11]
Summary
Primary cultures of human hepatocytes (PHH) are a great resource for biomedical research and therapeutics purposes. [1,2,3,4] It is well accepted that a good portion of immortalized hepatocytes cell lines lack many typical aspects of primary cell function and are questionable when used to evaluate mechanistic or therapeutic approaches. [5] The liver plays a crucial role in drug metabolism and for this hepatocytes are used to study the metabolic fate of drugs, drug-drug interactions and toxicity. [6,7,8,9] Different protocols have been developed to isolate hepatocytes from the liver and few studies showed that hepatocytes differ when isolated from healthy and diseased tissue [4, 5]. We have recently developed a new model for the study of HCC in-vitro and we demonstrated the existence of various human hepatocyte populations in the same liver which over time will transform into the cells with different morphologic and cancerous characteristics. We showed that CP-Hepatocytes over a period of 16 weeks transformed into cells expressing high levels of markers for HCC (Glypican-3 (GPC3) and α Smooth Muscle Actin (α-SMA) and cell proliferation nuclear antigen (PCNA). They developed invasiveness characteristics and gained the ability to aggregate in spheres that are well known characteristics of cancer cells with stem properties [11,12]
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