582 that serves to deﬁne these focal lesions, i.e. their altered growth pattern. In fact, a nodule, an adenoma, a polyp or a papilloma are deﬁned as discrete lesions displaying a morphology distinct from that of the normal surrounding counterpart. While this phenotypic feature has long been neglected and implicitly considered as a mere by-product of clonal growth, it is now emerging as a fundamental property of pre-cancerous lesions with speciﬁc pathoge- netic relevance to the process (Chen et al. 2009; Doratiotto et al. 2009; Schmeichel et al. 1998). Over the past several years, we have developed a model of orthotopic hepatocyte transplantation wherein the selective clonal expansion of both normal and nodular hepatocytes can be achieved and analysed over time in an in vivo system (Laconi and Laconi 2002). This transplan- tation model is based on a pre-conditioning regimen, i.e. exposure of the recipient animal to retrorsine (RS), a nat- urally occurring alkaloid that induces a persistent block on the endogenous hepatocyte cell cycle (Laconi et al. 1999). It was observed that both normal and nodular hepatocytes undergo clonal proliferation when injected into the liver of RS-treated hosts. However, the biological outcome is rad- ically different in either case. In fact, transplanted normal hepatocytes integrate in the host liver and gradually repopulate the entire organ with a seemingly normal his- tology (Laconi et al. 2001b). In contrast, when hepatocytes isolated from liver nodules are injected into RS-treated animals, they also proliferate but fail to integrate in the host parenchyma and rather undergo expansive growth, re- forming liver nodules that rapidly progress to hepatocel- lular carcinoma (Laconi et al. 2001a). Thus, this system offers the unique opportunity to compare two clonally expanding cell populations, normal and nodular hepatocytes, in vivo and possibly to dissect the biochemical and/or molecular alterations critically relevant to their dif- ferent phenotypic behaviour (Marongiu et al. 2008). In the present study, we analysed the growth pattern of transplanted normal and nodular hepatocytes vis-a`-vis the expression of proteins involved in cell polarity, cell migration, cell-to-cell and cell-to-extracellular matrix adhesion, and communica- tion, including connexin 32 (Cx32), E-cadherin, and matrix metalloproteinase-2 (MMP-2). We observed that the altered growth pattern of transplanted nodular hepatocytes is con- sistently associated with decreased expression of Cx32, E-cadherin, and MMP-2. Furthermore, it was unequivocally found that these alterations are inherent to nodular lesions and are unrelated to growth per se. Materials and methods All animals were maintained on daily cycles of alternating 12 h light/darkness with food and water available 123 Histochem Cell Biol (2011) 135:581–591 ad libitum. They were fed Purina Rodent Lab Chow diet (Ditta Mucedola, Italy) throughout the experiment and received humane care in accordance with the regulations and criteria of the Animal Care Unit of the University of Cagliari. Induction of liver nodules in donor rats and isolation of nodular and normal hepatocytes Hepatocyte nodules were induced according to a well- characterized experimental model in the rat, as previously described (Laconi et al. 2001b) and nodule-bearing livers were perfused according to a standard two-step collagenase perfusion technique (Laconi et al. 2001a; Seglen 1976). Large ([5 mm in diameter) nodules were physically sep- arated from surrounding tissue and cells isolated from nodular tissue were suspended in Williams E culture medium (Sigma, cat No. W-4125) and prepared for trans- plantation experiments. Cell viability, determined by try- pan blue dye exclusion, was 80–85%. Normal hepatocytes were isolated from a normal young adult syngeneic Fischer 344 donor rat following a similar two-step collagenase perfusion technique. The isolated cell fraction used for transplantation studies was judged to be *95% hepato- cytes by morphological analysis and cell viability was consistently [85%, as determined by trypan blue dye exclusion. Aliquots of these cells were also used for PCR analysis of MMP-2 RNA expression (see the following paragraphs). Treatment of recipient rats and transplantation of hepatocytes In order to follow the fate of donor hepatocytes in the recipient liver, the dipeptidyl-peptidase type IV-deﬁcient (DPPIV-) rat model was used (Thompson et al. 1991). In this system, cells isolated from a Fischer 344 rat expressing DPPIV (DPPIV?) are transplanted into a syngeneic DPPIV- recipient, such that cells derived from donors may be detected in the host liver through simple (immuno-) histochemical techniques. A colony of DPPIV- F344 rats has been established in the Department of Biomedical Sciences and Technology, University of Cagliari. DPPIV- recipient rats were treated according to the RS-based pro- tocol developed in our laboratories (Laconi and Laconi 2002). Brieﬂy, 24 DPPIV- rats, with a body mass of 80–100 g, were given two injections of RS (from Sigma), 30 mg/kg each, i.p., 2 weeks apart. Two weeks after the last injection, animals were divided into two groups (A and B, of 12 animals each) and were infused with either cells isolated from DPPIV? hepatocyte nodules (groups A,) or