Aging promotes neoplastic disease through effects on the tissue microenvironment

Size of nodular cell clusters in young and old recipients

In a first study, we examined the size distribution of cell clusters originating from nodular hepatocytes transplanted in either young or old recipient livers and analyzed at 3 months post-Tx. Panel A in Figure 1 shows the average number of cells per cluster in each animal. In rats transplanted at 4 months of age, the highest mean size of clusters per animal was 21±3, while the largest single cluster comprised 27 cells. By contrast, the same figures were much higher in the group transplanted at 18 months of age, being 81±29 and 563 cells, respectively. This finding was confirmed when the mean size of clusters was compared in the two groups (6 animals each, panel B in Figure 1): 11±3 cells per cluster were detected in young recipients, while this value was 42±8 in animals transplanted at 18 months of age.

Figure 1. Size of donor-derived, pre-neoplastic hepatocyte clusters in animals transplanted at young or old age and killed 3 months after Tx (see Experimental procedures for details). Panel (A) mean cluster size (± SE) in individual animals. Panel (B) mean cluster size per group. Data are mean ± SE; **significantly different from young group:P<0.01

The growth of hepatocyte nodules in young and old recipients

In a second series of experiments, young (3-5 months old) and aged (18-20 months old) Fischer rats were similarly transplanted with nodular hepatocytes and killed 8 months later. On macroscopic examination (Table 1), no visible lesion were seen in any of 21 recipient animals transplanted at young age (Figure 2, panel A). In stark contrast, 17/18 animals transplanted at old age displayed visible hepatic nodules, ranging from 1 to 8 mm in size, and including 7 larger tumors measuring up to 3 cm in diameter (Figure 2, panel B). All large lesions expressed the DPPIV marker enzyme, indicating that they originated from the transplanted cell population (Figure 3, panels A-C). Their gross appearance and structural morphology was similar to that of primary hepatocyte nodules generated through classical protocols for the induction of carcinogenesis in rat liver [16]. They were generally paler in color compared to surrounding tissue, displayed a prominent vasculature and were composed of two-three cell-thick hepatocyte plates (Figure 3, panel D).

Figure 2. Macroscopic appearance of livers from animals transplanted with pre-neoplastic hepatocytes in at young or old age and killed 8 months after Tx. Panel (A) liver from a rat transplanted at 5 months of age and killed 8 months later: liver surface is regularly smooth and no lesions were detected. Panel (B) liver from a rat transplanted at 20 months of age and killed 8 months later: note the presence of two large nodules with prominent vasculature (arrows).

Figure 3. Panels (A) and (B) Histochemical staining for DPPIV of liver samples obtained from rats transplanted with pre-neoplastic hepatocytes at young age and killed 8 months after Tx. Small clusters of DPPIV-expressing hepatocytes (orange-rust) were discerned in all animals. Histochemical staining for DPPIV (panel C) and standard H&E staining (panel D) of a large hepatic nodule from a rat transplanted with pre-neoplastic hepatocytes at old age and killed 8 months later. Note the typical appearance of nodular hepatocytes arranged in multiple cell-thick plates. Magnification: 100x.

Markers of cell senescence in aged rat liver

In order to identify alterations in the tissue microenvironment possibly relevant to the growth of transplanted cells in the aged liver, expression of markers of cell senescence was investigated. Cell senescence has long been interpreted as a fail-safe mechanism to limit neoplastic progression of altered cells [12]. On the other hand, it is now well established that it can also contribute to the emergence of the neoplastic phenotype, possibly through secretion of a host of factors, variably referred to as senescence-associated secretory phenotype (SASP) [13] or senescence-messaging secretome (SMS) [14], and comprising cytokines, growth factors and proteases. In light of the results reported above, expression of SA-β-gal, one of the most consistent markers of cell senescence, was determined in the liver of young vs. aged rats. Typical results are shown in Figure 4. Diffuse histochemical staining for SA-β-gal was observed in liver sections obtained from 18-month old animals, while it was a rare finding in the corresponding samples from young controls.

Figure 4. Panels (A) and (B) Expression of SA-β-gal in the liver of animals transplanted with pre-neoplastic hepatocytes in at young or old age and killed 3 months after Tx. The senescence marker was diffusely expressed in aged animals (panel B), while it was a rare finding in liver samples from young rats (panel A). Panel (C) Western Blot analysis of p21, p27 and p53 in liver samples from young and old recipients.

On the other hand, no significant changes were seen between young and aged animals in the hepatic expression of other markers that have been associated with cell senescence, such as the cell cycle inhibitors p21 and p27; however, aged livers displayed higher levels of p53, a central player in growth arrest following DNA damage [15].

We next measured the levels of expression of IL6, a pro-inflammatory cytokine and a main component of SASP which has also been implicated in liver regeneration and repair [16]. Interestingly, both mRNA and IL6 protein were found to be increased in the liver of aged rats compared to young controls (Figure 5). In light of these results, we further investigated expression of the downstream effector of IL6, signal transducer and activator of transcription 3 (STAT3) in the liver of aged animals transplanted with nodular hepatocytes. However, only rare cells, scattered throughout the liver, showed nuclear staining for STAT3, with no specificity for clusters of donor-derived nodular cells.

Figure 5. Panels (A) and (B) Expression of IL6 in the liver of animals transplanted with pre-neoplastic hepatocytes in at young or old age and killed 3 months after Tx. Both the protein (panel A) and the corresponding mRNA (panel B) were expressed at higher levels in the liver of aged animals. Panels (C) and (D) serial sections showing histochemical staining for DPPIV⁺ (panel C) and immuno-histochemical staining for STAT3 (panel D) in clusters of transplanted nodular hepatocytes. Only rare STAT3+ nuclei were detected in the aged rat liver (panel E). 100x magnification.

Animals

All animals were maintained on daily cycles of alternating 12 h light/darkness with food and water available ad libitum. They were fed Purina Rodent Lab Chow diet (Ditta Mucedola, Italy) throughout the experiments and received humane care according to the criteria outlined in the National Institutes of Health Publication 86-23, revised 1985. Animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of the University of Cagliari. In order to distinguish donor-derived from recipient cells in the liver, the dipeptidyl-peptidase type IV-deficient (DPPIV-) rat model was used [38]. A colony of DPPIV- F344 rats has been established in our laboratory, at the Department of Biomedical Sciences, University of Cagliari. DPPIV^- animals were used as recipients, while donor rats were syngeneic F344, DPPIV⁺ and were purchased from Charles River, Milan, Italy.

Induction of liver nodules in donor rats and isolation of hepatocytes

Hepatocyte nodules were induced according to a well-characterized experimental model in the rat [19], as previously described [8]. Briefly, two-month old male Fischer 344 rats, expressing DPPIV enzyme activity, were injected with a single dose of diethylnitrosamine (DENA, 200 mg/kg. b.w., i.p., Sigma-Aldrich Chemical Co., St. Louis, MO) followed, 3 weeks later, by exposure to a modified version of the Solt and Farber protocol [39], to stimulate the growth of hepatocyte foci and nodules. Such protocol consisted of three consecutive daily doses of 2-acetylaminofluorene (20 mg/kg b.w., given by gavage tube, from Sigma-Aldrich) followed, on the fourth day, by a single administration of CCl₄ (0.2 ml/kg b.w., by gavage, mixed in corn oil, 1:1 v:v). Six months after the initial treatment livers were perfused according to a standard 2-step collagenase perfusion technique [40,41]. Typically, 3 to 5 large (5-10mm in size) persistent nodules are present in the liver at this time point using the above experimental protocol. When left in situ, a subgroup of these nodules (an average of 1 or 2 per animal), will progress to cancer within about 1 year [19]. Large (>5 mm) nodules were physically separated from surrounding tissue and isolated cells were suspended in PBS and prepared for transplantation experiments. Prior to transplantation, cell suspension was filtered through a nylon mesh with a pore diameter of 100μ, in order to eliminate any large cell clumps. Cell viability, determined by trypan blue dye exclusion, was ~85% in the nodular cell preparation.

Hepatocyte transplantation

Young (3-5 months old) or aged (18-20 months old) Fisher 344 rats of the DPPIV^- strain were used as recipients. They were injected with 5x10⁵ cells freshly isolated from DPPIV⁺ hepatocyte nodules, via a mesenteric vein [8]. Animals from various groups were sacrificed at different time points during the experiment, as indicated in the Results section. Liver samples were fixed in 10% buffered formaldehyde or snap frozen. Histochemical determination of DPPIV enzyme activity was performed as described [8]. At least ten random sections were cut from each liver lobe of each animal and stained for DPPIV enzyme activity. Sections were then analyzed under the microscope and the number of hepatocytes for each DPPIV+ cluster was evaluated. Data from two separate experiments are reported. Data represent means±S.E. Statistical analysis was performed using the Student t test.

Staining for senescence associated β-galactosidase activity

Detection of senescence associated β-galactosidase (SA-β-Gal) activity was performed as described [42]. Immediately before staining, X-Gal stock solution was prepared by dissolving 20mg/ml X-Gal (Invitrogen, Carlsbed, CA) in dimetylformamide. SA-β-Gal staining solution was prepared as follows: 1 mg/ml of X-Gal stock solution were dissolved in 40 mM citric acid in sodium phosphate, pH 6.0/5 mM potassium ferrocyanide/5 mM potassium ferricyanide/150 mM NaCl/2 mM MgCl2. Frozen sections of 10-µm thickness were fixed for 5’ in 4% formaldehyde/0.5% glutaraldehyde at 4°C, washed in PBS and incubated in fresh SA-β-Gal staining solution for 16h at 37°C. No special blocking step is required to perform the staining. Sections were counterstained with Hematoxylin.

Histochemistry and immunohistochemistry

To follow the fate of transplanted cells, histochemical detection of DPP-IV positive clusters was performed on 5µm frozen sections as previously described [38].

Immunohistochemical staining for p-STAT 3 was performed on serial frozen sections. Slides were fixed with 3% formalin, blocked for 30’ and incubated with the primary antibody (Cell Signalling Technology, Danvers, MA) overnight at 4°C. Detection of specific signal was accomplished using an HRP/AEC detection IHC Kit (Abcam, Cambridge, UK).

Western blot

Liver tissue samples were homogenized in RIPA lysis buffer containing Protease Inhibitors, then centrifuged at 12000 rpm for 30’ at 4°C. Protein concentration in supernatants was measured using the BCA method [42]. Samples (20µg protein) were prepared in Laemmli buffer, boiled at 95°C for 5’ then loaded into SDS-PAGE precast gels (Biorad, Hercules, CA) and run under denaturing conditions. Proteins were transferred onto nitrocellulose membranes (Amersham, UK), blocked with 5% non-fat milk for 1 h, then incubated with primary antibody for p21, p27 and p53 (Santa Cruz, Santa Cruz, CA), IL6, beta actin and gapdh (Abcam) overnight at 4°C. Membranes were washed and incubated for 2 h with the appropriate secondary antibody conjugated with HRP. Protein bands were detected using a chemoluminescent substrate (Biorad) and imaged onto Kodak film.

RNA isolation, RT-PCR and Real-Time qPCR

Total RNA was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufacturer’s protocol. RNA integrity and purity were confirmed by 1% agarose gel electrophoresis and OD260/OD280 nm absorption ratio >1.8. Two grams of DNase-I treated RNA of each sample were reverse-transcribed by PCR using Promega reagents. The resulting cDNA was analyzed by quantitative real-time PCR using specific TaqMan assays and TaqMan Gene Expression Master Mix on an StepOne System (all from Applied Biosystems, Carlbad, CA). The rat specific assays were: IL-6 (Rn01410330_m1); β2-microglobulin (Rn00560865_m1). For both assays the thermal profile was as follows: 50˚C for 2 minutes, 95˚C for 10 minutes, 45 cycles at 95˚C for 15 seconds and 60˚C for 1 minute. Fold change was calculated by the 2-ΔΔCT method [43].