Table of Contents  

Bennaser, Bird, and Canovas: In vitro interactions between rodent hepatic stellate cells and metastatic and non-metastatic human colorectal cancer cell lines

Introduction

Colorectal carcinoma is one of the major causes of death from cancer in Western countries.1 The outcome of the disease is usually determined by the occurrence of metastatic dissemination, predominantly to the liver.2 Surgical resection, the only curative therapy for colorectal liver metastases, is possible in fewer than 25% of the patients at the time of diagnosis and the overall prognosis of non-resected colorectal liver metastases is poor.3 A detailed understanding of the biological mechanisms that regulate the establishment and development of colorectal liver metastases may lead to enhancement in non-surgical antitumour management.4

Hepatic stellate cells (HSCs) play a central role in the uptake and storage of vitamin A. During liver injury of any aetiology, HSCs undergo a response known as activation, which is the transition of quiescent cells into proliferative, fibrogenic and contractile myofibroblast-like cells.5 This is widely known to play an essential role in hepatic fibrogenesis, in that they differentiate into myofibroblasts and produce several cytokines (CKs), chemokines and matrix-degrading metalloproteinases as well as their specific inhibitors in their activated state.6,7 Alpha-smooth muscle actin [α-SMA (Abcam, Cambridge, UK)] is a marker of stellate cell activation;7 it increases during the stellate cell activation phase8 and may also serve to increase contractile potential of the cell.9 Moreover, activation of α-SMA-positive HSCs is also observed in the liver parenchyma adjacent to metastases.10 Glial fibrillary acidic protein (GFAP) is a glial-specific protein found in long processes of astrocytes11 and also seems to be expressed in quiescent HSCs (qHSCs). It disappears in normal cells after a week of culture and is not found in the activated phase of HSCs.12 Lipid droplets are the most conspicuous ultrastructural feature of fresh isolated HSCs in the intact liver13 and the loss of lipid droplets is a sign of HSC transdifferentiation into myofibroblast-like cells.14

Shimizu et al.15 showed that a highly metastatic colon carcinoma cell line (LM-H3) retained direct contact with the HSCs that surrounded the sinusoid from which the tumour masses received their nourishment. The authors also showed that peritumoral HSCs were more numerous than HSCs in the surrounding normal parenchyma, implying their local proliferation or migration.15 Furthermore, in a separate study by Illemann et al.,16 the authors showed that, in the desmoplastic growth pattern, the liver cells at the desmoplasia–liver parenchyma interface collapse and disappear as a result of the tumour expansion. In addition, the reticulin fibres, which are made up of various types of collagen, are left behind and generate a collagen-rich fibrous capsule. Furthermore, in the desmoplastic growth of colorectal liver metastases, all three components of the urokinase plasminogen activator (uPA) extracellular protease [urokinase plasminogen activator (uPAR), plasminogen activator inhibitor-1 (PAI-1) and uPA mRNA] showed intense expression whereas little or no upregulation was detected in the pushing growth pattern.16 In other words, liver metastases with desmoplastic growth break down the extracellular matrix and may be a crucial to the ability of metastatic cells to grow and invade the liver.16

In hepatocellular carcinoma and colorectal liver metastases, most investigations are concerned with the activated HSCs that are responsible for the formation of cancer stroma and fibrotic capsule.10,17 In addition, in biliary malignancy and hepatocellular carcinoma, activated stellate cells contribute to the accumulation of tumour stroma.18,19 These results confirm findings from animal studies suggesting that activated stellate cells play a role in hepatic metastases.20,21 In addition, cell culture studies have demonstrated paracrine activation of stellate cells by tumour cells.22,23

In this study, we have characterized qHSCs and compared the expression pattern of α-SMA, Ki-67 and CK18 cleavage products in qHSCs that have been cultured alone and in co-culture with metastatic and non-metastatic colon cancer cell lines and condition media. This has shown changes both in expression patterns of these markers and in rates of proliferation and apoptosis in stellate cells.

Materials and methods

Isolation and culture of quiescent hepatic stellate cells

Quiescent HSCs were isolated from the liver of a male Wistar rat, as reported in a previous study.13 The HSC isolation and purification was based on the enzymatic digestion of the rat liver by collagenase followed by the centrifugation of crude cell suspension through a density gradient containing 28.7% (w/v) Histodenz (Sigma-Aldrich, St. Louis, MO, USA).

The cell pellet was suspended and cultured in Dulbecco's modified Eagle medium (DMEM) containing 10% fetal calf serum (FCS), 5 ml penicillin/streptomycin, 5 ml fungizone® (Life Technologies Limited, Paisley, UK) and 5 ml glutamine and then counted with a haemocytometer. Mean yields of 6 × 106 mean HSCs were obtained per animal. The purity of the HSC preparations obtained from the density centrifugation ranged from 75% to 80%, as shown by vitamin A autofluorescence and fat droplet staining. The viability of the cell preparations was > 80%, as estimated by trypan blue exclusion.

Vitamin A autofluorescence, staining of the lipids and immunofluorescent detection of glial fibrillary acidic protein

To identify freshly isolated rat HSCs, vitamin A autofluorescence was detected by seeding the freshly isolated HSCs at a density of 20 000 cells per ml of DMEM supplemented with 10% FCS, 5 ml penicillin/streptomycin, 5 ml fungisone and 5 ml glutamine in a 48-well plate. The cells were then incubated for 2 hours at 37°C in a 5% carbon dioxide (CO2) atmosphere and 100% humidity. Subsequently, the HSCs were examined in a dark room under a fluorescent microscope (Leica AF 6000 LX microscope system v. 2.1.1, Leica Microsystems GmbH, Wetzlar, Germany) at approximately 330 nm. Then, in order to detect lipid droplets, the HSCs were incubated for 3 hours in a 48-well plate in which the cells were fixed with 10% formol saline for 10 minutes and then washed in distilled water. The cells were incubated with Oil red O for 15 minutes at room temperature and then rinsed in distilled water. Lipid droplets were then visualized under the light microscope and appeared as red vesicles. Ultimately, the fresh isolated HSCs were also identified with the mouse monoclonal primary antibody against GFAP (Abcam, Cambridge, UK). Astrocyte primary cells were seeded in an additional two-chamber slide for GFAP expression control. One chamber was used as a positive control (treated by a primary antibody) and the other as a negative control. The slides were then visualized under the fluorescence microscope.

Immunofluorescent detection of alpha-smooth muscle actin

To determine the duration of qHSC transdifferentiation, α-SMA was sequentially stained on days 1, 3 and 5. The cells were then plated in a chamber slide with a cover at a density of 2 × 104 cells/ml for each chamber. This chamber slide was labelled with details of the α-SMA and the day of staining. The LX-2 cell line was seeded in a further two-chamber slide for α-SMA expression control. One chamber was used as a positive control (treated by primary antibody) and the other as a negative control. Stellate cells with controls were grown in a 10% FCS-containing medium and incubated then stained on days 1, 3 and 5. The cells were then washed in 1 × phosphate-buffered saline (PBS) and fixed with 4% paraformaldehyde in PBS for 15 minutes. After fixation, the cells were washed twice in ice-cold PBS and then permeabilized with PBS containing 0.25% Triton X-100 (Sigma-Aldrich, St. Louis, MO, USA) for 10 minutes. They were then washed three times in PBS (for 5 minutes each time) and incubated with blocking solution (1% bovine serum albumin in PBS with Tween-20) for 30 minutes. Mouse monoclonal primary antibodies for α-SMA (diluted to a 1:100 ratio in blocking solution) were incubated with the cells overnight at 4 C. After three washes in PBS, the cells were incubated with fluorescein-labelled anti-mouse immunoglobulin G (IgG) (Abcam, Cambridge, UK) (diluted to a 1 : 4000 ratio in blocking buffer) for 1 hour. The cells were washed three times in PBS and then stained with 4′,6-diamidino-2-phenylindole (DAPI) for 1 minute and then rinsed with PBS. The cells were then viewed through a fluorescence microscope. The lipid droplets staining of qHSCs using Oil red O was also used in a sequential manner to determine how long the cells remain quiescent throughout days 1, 3 and 5.

Tumour cells, primary astrocytes and the LX-2 cell line

Two types of tumour cells were used, a highly metastatic colon carcinoma cell line, HT-29, and a non-metastatic colorectal carcinoma, Colo-741. Primary astrocytes were provided by Professor Mimoun Azzouz of the Medical School at the University of Sheffield, UK. The LX-2 cell line, which was the activated phase of human HSCs, was provided by Professor Scott Friedman of the Mount Sinai School of Medicine in New York, NY, USA.

Hepatic stellate cell co-culture with colon cancer cell lines of HT-29 and/or Colo-741

A MitoTracker probe (Invitrogen, Paisley, UK) was used to label the tumour cell lines to distinguish them from the primary HSCs. In a comparative approach, α-SMA expression was investigated in the HSC co-culture with (1) a metastatic colorectal adenocarcinoma cell line, HT-29 and (2) a non-metastatic colon carcinoma cell line, Colo-741. Using immunocytochemistry, the α-SMA was measured on days 1, 2, 3, 5, 7 and 10 in a sequential manner. The labelled tumour cells were plated and incubated with the HSCs in a four-well chamber slide with cover 2 hours after the HSCs began the culture. The tumour cells were plated at a density of 1500 cells/ml and the primary HSCs at a density of 7000 cells/ml. Primary qHSCs were used as a control and the cells were viewed through a fluorescence microscope. Actin expressing cells were counted in five typical fields of view per well. Photographs were then imported to the Image J program, v. 1.45 (developed at the National Institutes of Health, Bethesda, MD, USA) and the cells expressing actin were counted. The cell numbers were expressed as a percentage per field of view and the results reported as a mean ± standard error of the mean (SEM) of triplicates.

Proliferation assessment of hepatic stellate cells co-cultured with colon cancer cell lines conditioned medium using Ki67 essay

Ki67 was used to clarify the proliferation effects of the conditioned medium of tumour cell lines on qHSCs on day 1 of the co-culture experiment. The HT-29 tumour cell line was used as a positive control because it was proliferating, while the qHSCs were used as a negative control because the qHSCs at day 1 were not proliferating.

Hepatic stellate cells were plated and incubated in a four-well chamber slide with cover (Nunc™, Fisher Scientific, Leicestershire, UK) at a density of 7000 cell/ml/well in DMEM supplemented with 10% FCS. The incubated stellate cells were co-cultured with HT-29 cell lines or Colo-741 cell lines conditioned medium after 3 hours of seeding overnight. By adapting immunocytochemistry, Ki67 was investigated on day 1 of the co-culture. The cells were washed in 1× PBS and then fixed in 1:1 methanol–acetone for 10 minutes. After fixation, the endogenous peroxidise activity was quenched by incubating the chamber slides with 3% H2O2 for 30 minutes. The peroxidise was washed off with distilled water for 5 minutes and the slides were incubated with diluted horse serum to a 2% final concentration for 20 minutes.

The blocking serum was drained off and the primary antibody monoclonal mouse anti-human (Dako Denmark A/S, Glostrup, Denmark) (diluted to a 1:100 ratio) was prepared in a tris-buffered saline with Tween-20 (TBST) solution and incubated for an hour at room temperature. After three washes with TBST for 5 minutes each, the cells were incubated for 30 minutes with diluted biotinylated secondary antibody (to a 1:200 ratio in 2% serum TBST). An avidin–biotin complex (ABC) reagent was prepared at the same time with the secondary antibody, as it has to stand for 30 minutes before use. The cells were washed twice in PBS (for 5 minutes each time) and then incubated with ABC reagent for 30 minutes at room temperature. Peroxidase substrate 3,3′-diaminobenzidine (DAB) solution was made up in 5 ml distilled water by adding two drops of buffer, four drops of DAB and two drops of peroxide, which were prepared immediately before use. The solution was incubated with the cells until the desired stain intensity had developed, usually within 10 minutes, and then rinsed in tap water. After that, the slides were counterstained in haematoxylin for 30 seconds and rinsed in tap water. Finally, the slides were dehydrated and cleared by immersion in 70%, 95% and 100% ethanol, for 3 minutes at each concentration, followed by xylene 1 for 3 minutes and then mounting xylene 3 for 3 minutes also. The slides were kept wet during the DePex mount under a fume hood by leaving them in xylene and then viewed using a light microscope.

Immunocytochemistry detection the cleavage of cytokeratin-18 in the co-cultured hepatic stellate cells

In order to determine CK18 cleavage product in HSCs co-culture with the (1) metastatic colorectal adenocarcinoma cell line tumour, HT-29, and (2) non-metastatic colon carcinoma cell line tumour, Colo-741, for 3 days co-culture, M30 CytoDEATH™ monoclonal antibody (Roche Diagnostics Ltd., West Sussex, UK) was used to detect the apoptotic HSCs.

The HSCs were plated and incubated in an eight-well chamber slide with cover at a density of 7000 cells/ml and co-cultured with HT-29 or Colo-741 labelled with a MitoTraker red CMXRos at a density of 1500 cells/ml for 3 days. The HSC co-culture was divided into positive and negative treatment groups. The cells were then washed in 1 × PBS and fixed in ice-cold methanol at −20 C for 30 minutes. After fixation, the cells were washed in washing buffer (PBS containing 0.1% Tween-20) twice, for 5 minutes each time. The washing buffer was then removed and the primary antibodies (M30 CytoDEATH™) diluted to a 1:50 ratio in blocking solution and incubated with the cells for 60 minutes at room temperature. After two washes with washing buffer, the cells were incubated with fluorescein-labelled anti-mouse IgG (diluted to a 1:4000 ratio in blocking buffer) for 1 hour. The cells were then washed twice in washing buffer, stained with DAPI for 1 minute and rinsed with PBS. The cells were then viewed through a fluorescence microscope.

Cells expressing CK18 were counted in five typical fields of view per well. As an additional counting method, photographs were imported to the Image J program and the cells expressing CK18 were counted. The cell numbers were expressed as a percentage per field of view and the results reported as a mean ± SEM of triplicates.

Data analysis

Results were expressed as mean ± SEM and the data were analysed by one-way analysis of variance (ANOVA), followed by a Bonferroni post hoc test using SPSS v. 16.0 (SPSS Inc., Chicago, IL, USA). However, in the case of the CK18 cleavage product experiment, the data were analysed using Student's t-test. Differences were considered statistically significant at P < 0.05 and each experiment was performed three times.

Results

Hepatic stellate cell identification with vitamin A autofluorescence staining of lipid and immunocytochemistry detection of glial fibrillary acidic protein

In the normal rat liver, the HSCs that had a star-like appearance were identified by vitamin A autofluorescence. They exhibited a striking, rapidly fading blue-green autofluorescence when excited with a light of approximately 330 nm (Figure 1A). Under the light microscope examination, 4Oil red O staining was shown to have multivesicular red bodies of fat droplets distributed in the periphery of the cytoplasm (Figure 1B). GFAP is an intermediate protein found in quiescent HSCs that disappears 2 days after isolation. It is a cell type marker for qHSCs, which may allow a distinction between HSCs and other fibroblastic liver cells (Figure 1C).

FIGURE 1

Quiescent HSCs identification. (A) Vitamin A autofluorescence excited at 330 nm, magnification × 40. (B) Visualization of lipid droplets within the hepatic stellate cells incubated with Oil Red O to stain lipid vesicles red, magnification × 40. (C) Merged images of primary qHSCs (treated with mouse monoclonal GFAP antibody). The blue areas are the DABI-stained nuclei and the green areas are fluorescein isothiocyanate (FITC) stained GFAP, magnification × 20.

6-3-11-fig1.jpg

Sequential staining of Oil Red O and alpha-smooth muscle actin onto plastic culture of quiescent hepatic stellate cells

Hepatic stellate cells in the first 3 days of uncoated plastic culture remain quiescent (Figure 2A and B) and change to myofibroblast-like cells after day 3, as identified by α-SMA and Oil red-O staining. HSCs revealed a loss of lipid droplets after day 3 of culture. The cells progressively spread and flattened after day 3 and had the typical myofibroblast appearance of activated HSCs by day 5 (Figure 2C). Conversely, α-SMA was absent from the early plastic uncoated culture that was represented throughout days 1 and 3 of the sequential staining (Figure 2D and E); however, α-SMA expression was detected on day 5 (Figure 2F).

FIGURE 2

Sequential staining of qHSCs in order to determine the duration of HSCs transdifferentiation. HSCs incubated with Oil red O to stain lipid vesicles red at days 1 (A), 3 (B) and 5 (C). The first and second red pictures, (A) and (B), represent the cell in the quiescent phase with red lipid vesicles. The third picture (C) represents the cell alteration to the active phase, which relies on the external feature and no red lipid vesicles, magnification × 40. Immunocytochemistry expression for actin in cultured HSCs at days 1, 3 and 5 [(D), (E) and (F)] after the isolation. The first and second merged blue pictures, (D) and (E), represent the HSCs in quiescent phase (negative actin expression). The third merged green picture (F) represents the active phase of HSCs (positive actin expression), magnification × 20. The blue areas are the DABI-stained nuclei and the green areas are FITC stained actin.

6-3-11-fig2.jpg

Hepatic stellate cells co-cultured with colon cancer cell lines of HT-29 and Colo-741

Hepatic stellate cell co-cultures with a tumour metastatic cell line, HT-29, and non-tumour metastatic cell line, Colo-741, exerted different influences on the qHSCs, as judged by the expression of α-SMA. For example, on day 1 of the HSC HT-29 co-culture, α-SMA was revealed and increased continuously throughout the duration of the HSCs co-culture (Figure 3 and Table 1). In contrast, the HSC Colo-741 co-cultures expressed α-SMA only on days 7 and 10, as identified by staining with primary antibody (Abcam, Cambridge, UK), diluted to a 1:100 ratio in blocking solution (Figure 4 and Table 1), and the α-SMA count was low when compared with control HSCs, maintained in single culture (Figure 5 and Table 1). α-SMA expression in HSCs control was expressed after day 3, which is concurrent with the result of the qHSCs in Figure 2F and the expression of α-SMA continued until day 10 (Figure 5).

FIGURE 3

Quiescent HSCs control. Immunocytochemistry expressions for actin in HSCs cultured throughout 10 days. The primary HSCs seeded on uncoated plastic culture. (A), (B) and (C) represent the first 3 days of qHSCs culture (negative actin expression) and (D), (E) and (F) represent the days 5, 7 and 10 of activated HSCs phase (positive actin expression). The blue areas are the DABI-stained nuclei and the green areas are FITC stained actin. Magnification × 20.

6-3-11-fig3.jpg
TABLE 1

The table shows the data of actin expression represented as percentages (mean ± SEM) in HSCs control, HSCs co-cultured with HT-29 and HSCs co-cultured with Colo-741 (n = 5)

Days Mean control ± SEM Mean HT-29 ± SEM Mean Colo-741 ± SEM
Day 1 0 ± (0.00) 29.92 ± (0.970) 0 ± (0.00)
Day 2 0 ± (0.00) 50.61 ± (1.430) 0 ± (0.00)
Day 3 3.016 ± (0.218) 63.16 ± (0.366) 0.707 ± (0.395)
Day 5 10.74 ± (0.542) 88.87 ± (0.863) 1.23 ± (0.397)
Day 7 31.186 ± (0.600) 95.356 ± (0.380) 19.914 ± (0.763)
Day 10 41.199 ± (0.307) 97.263 ± (0.464) 34.286 ± (0.734)
FIGURE 4

Quiescent HSCs co-cultured with Colo-741. Immunocytochemistry expressions for actin in HSCs co-cultured with Colo-741 over a period of 10 days. (A), (B), (C) and (D) represent the negative actin expression days of HSCs Colo-741 cell line co-culture (negative actin expression), and (E) and (F) represent the days 7 and 10 of the activated HSCs phase (positive actin expression). The blue areas are the DABI-stained nuclei and the green areas are FITC-stained actin. The red area is Mito-Tracker red stain of tumour cell line. Table 1 shows the actin expression represented as percentage (mean ± SEM) and differences were considered statistically significant at P < 0.05 using one-way ANOVA followed by a Bonferroni post hoc test. Magnification × 20.

6-3-11-fig4.jpg
FIGURE 5

Quiescent HSCs co-cultured with HT-29. Immunocytochemistry expressions for actin in HSCs co-cultured with HT-29 over a period of 10 days. All pictures represent the positive actin expression days of HSCs HT-29 cell line co-culture (positive actin expression). The blue areas are the DABI-stained nuclei and the green areas are FITC-stained actin. The red area is Mito-Tracker red stain of tumour cell line. Table 1 shows the actin expression represented as percentage (mean ± SEM) and differences were considered statistically significant at P < 0.05 using one-way ANOVA followed by a Bonferroni post-hoc test. Magnification × 20.

6-3-11-fig5.jpg

Proliferation assessment of hepatic stellate cells co-cultured with colon cancer cell lines conditioned medium using Ki67

The HT-29 conditioned medium enhanced qHSC proliferation on day 1 of co-culture rather than express actin as in the cell–cell contact. However, qHSCs co-cultured with Colo-741 conditioned medium did not develop any changes (Figure 6).

FIGURE 6

The Ki-67 proliferation assay illustrates the influence of the HT-29 and Colo-741 conditioned media on the qHSCs. (A) The negative control represents the qHSCs. (B) The positive control represents the HT-29 colon cell line. (C) The qHSCs co-cultured with Colo-741 conditioned medium (the qHSCs have not proliferated). (D) The qHSCs co-cultured with HT-29 condition medium (the qHSCs have proliferated). Magnification × 20.

6-3-11-fig6.jpg

Cytokeratin-18 cleavage staining in hepatic stellate cells co-cultured with colon cancer cell lines

The HSC apoptosis in culture was quantified in vitro by M30 CytoDEATH™ mouse monoclonal antibody staining. Apoptotic cells were detected and counted under fluorescence as demonstrated in (Figure 7 and Table 2). The Colo-741 non-metastatic cancer cell line showed a significant apoptotic effect (P < 0.05) on the qHSCs in co-culture. Compared with this, the metastatic cancer cell line, HT-29, showed a low apoptotic effect on the qHSCs in co-culture.

FIGURE 7

CK18 cleavage product expression (mean ± SEM). Differences were considered statistically significant at P < 0.05 using an independent-sample t-test (n = 5).

6-3-11-fig7.jpg
TABLE 2

The data of CK18 cleavage product represented as percentages (mean ± SEM) in HSCs co-cultured with HT-29 or Colo-741

Cells Mean (± SEM)
HT-29 10.55 (± 0.548)
Colo-741 31.22 (± 0.582)

Discussion

In this study, HSCs were isolated and identified using three different markers, vitamin A autofluorescence, Oil red O staining of lipid droplets and GFAP staining. These markers are significant in characterizing the fresh qHSCs. For example, GFAP expression is present in qHSCs and expression is high after isolation but reduces with the age of the culture.2425 Vitamin A autofluorescence and lipid droplets are lost in the active phase of HSCs and synthesize extracellular matrix instead.7,26,27 However, according to previous studies,12,2829 induction of α-SMA is a marker of stellate cell activation.

The loss of lipid droplets is considered by some authors a sign of HSC transdifferentiation into myofibroblast-like cells14,30 as well as α-SMA expression.7 Therefore, in order to determine the duration of qHSC transdifferention, the present in vitro study using serial immunocytochemistry staining showed that the qHSCs expressed α-SMA at day 5, whereas, sequential staining of lipid or no fat droplets was noted on day 3 and completely disappeared by day 5, suggesting that activation had started by day 3. This alteration of HSCs to the active phase occurs gradually as in α-SMA sequential staining, the expression on the first days (1 and 3) were absent, which has also been reported also by other studies.12,28,29

In the present study, α-SMA expression was compared in HSCs co-cultured with (1) a metastatic colorectal adenocarcinoma cell line tumour, HT-29 and (2) a non-metastatic colon carcinoma cell line tumour, Colo-741. The results have shown that following the co-culture of HSCs with HT-29, α-SMA expression occurred after 24 hours, which marked rapid activation and transdifferentiation to myofibroblast-like cells. However, this is not the case in the HSCs sequential staining of α-SMA and co-cultured experiment control. Furthermore, the percentage myofibroblast-like cells that expressed α-SMA increased progressively throughout the experiment with HT-29 (P < 0.05).

In the Colo-741 co-culture, α-SMA expression was detected at days 7 and 10 only. The non-metastatic cell line, Colo-741, had a significant apoptotic effect on the fresh isolated HSCs (P < 0.05). This result may explain why the α-SMA expression consistency increased in the HT-29 co-culture, whereas the Colo-741 co-culture expressed α-SMA at a low percentage on days 7 and 10 compared with the control. In previous studies, activation shown by α-SMA-positive HSCs was observed in liver parenchyma adjacent to metastases.10,15 The uPA mRNA-positive cells were identified by combined immunohistochemistry and in situ hybridization to be primarily α-SMA-positive myofibroblasts.31 In colon cancer liver metastases, uPA mRNA was also expressed by stromal α-SMA-positive myofibroblasts, at the periphery of the desmoplastic growth pattern metastases, which may indicate that they have a role in growth and invasion of the liver by tumour cells.16 Additionally, metastasized tumour cells alter the supporting mesenchymal tissue in which they grow. This phenomenon, known as the stromal reaction, includes the activation of fibroblasts or myofibroblast-like cells.32 The stromal reaction plays a role in colorectal carcinoma either at the primary or metastatic site. For example, stromal fibroblasts express activation markers such as α-SMA33 and fibroblast-activating protein-α;34 these markers are associated with tumour progression and, in addition, tumour-associated myofibroblasts stimulate tumour invasiveness.35 Moreover, instead of cell-to-cell contact co-culture, qHSCs co-cultured with HT-29 conditioned medium show proliferation by Ki-67 rather than α-SMA expression, whereas the qHSCs co-cultured with Colo-741 did not reveal any change.

The CK18 cleavage product liberates a neoepitope that is specifically recognized by the M30 CytoDEATH™ monoclonal antibody. Specific proteolytic cleavage of CK18 is an event that takes place before disruption of membrane asymmetry and induction of DNA strand breaks. Numerous studies confirm that the M30 CytoDEATH™ antibody detects only apoptotic but not viable or necrotic cells.3638 The CK18 intermediate filament of epithelial cells are cleaved in vitro by caspase-6, 3 and 7.39,40 The cleavage of CK18 by caspase marks an early event in the apoptotic process.41 Colo-741 and HT-29 tumour cell lines differ in terms of the occurrence of apoptosis as detected by CK18 cleavage products. Colo-741 had a significant apoptotic effect on the fresh isolated HSCs (P < 0.05), which may explain the finding of the HT-29 co-culture that had increased the α-SMA expression consistently, whereas Colo-741 expressed α-SMA in low percentage at days 7 and 10 compared with the control. A malignancy may be required for successful establishment because the sequential accumulation of mutations may be necessary to block apoptosis. For example, dysregulation of apoptosis may be necessary to promote growth, prevent elimination by cytotoxic lymphocytes and allow survival.42 A novel mechanism to escape immune recognition by neoplastic cells is loss of Fas-receptor expression and the development of Fas ligand expression by the cancer cells. Expression of the Fas ligand results in apoptosis of Fas-receptor-expressing cytotoxic lymphocytes as they attempt to attack the neoplastic cell; loss of Fas receptors by the neoplastic cell ensures its survival despite recognition by the cytotoxic lymphocytes.43,44 These observations may have a relation with the HT-29 co-culture finding, as the desmoplastic growth pattern did not induce apoptosis despite the dense infiltration of lymphocytes at the tumour periphery.45 The mechanism underlying the proapoptotic Colo-741 co-culture may relate to Fas and Fas ligand as detected in the fully activated HSCs (on day 7), whereas HSCs in resting and transitional phase show a higher resistance to CD95-mediated apoptosis.46 However, in the current experiment, the HSC apoptosis was detected on day 4 and this observation may relate to Fas ligand-expressing tumour cells.47

In conclusion, the metastatic colon cancer cell line, HT-29, stimulates HSCs to transform into myofibroblast-like cells. Low α-SMA expression with the non-metastatic colon cancer cell line, Colo-741, co-culture may reflect the level of HSC apoptosis. This result may be linked with the non-metastatic behaviour of Colo-741. These data provide a critical link between metastatic and non-metastatic colon cancer cell lines and HSC activation, which ultimately needs further investigation to clarify the molecular mechanism by which the HSCs or tumour cells promote tumorigenesis.

References

1. 

Jemal A, Tiwari RC, Murray T, et al. Cancer statistics, 2004. CA: a cancer journal for clinicians 2004; 54:8–29. http://dx.doi.org/10.3322/canjclin.54.1.8

2. 

Hardingham JE, Kotasek D, Sage RE, et al. Detection of circulating tumor cells in colorectal cancer by immunobead-PCR is a sensitive prognostic marker for relapse of disease. Mol Med 1995; 1:789–94.

3. 

Khatri VP, Petrelli NJ, Belghiti J. Extending the frontiers of surgical therapy for hepatic colorectal metastases: is there a limit? J Clin Oncol 2005; 23:8490–9. http://dx.doi.org/10.1200/JCO.2004.00.6155

4. 

Mueller L, Goumas FA, Affeldt M, et al. Stromal fibroblasts in colorectal liver metastases originate from resident fibroblasts and generate an inflammatory microenvironment. Am J Pathol 2007; 171:1608–18. http://dx.doi.org/10.2353/ajpath.2007.060661

5. 

Friedman SL. Molecular regulation of hepatic fibrosis, an integrated cellular response to tissue injury. J Biol Chem 2000; 275:2247–50. http://dx.doi.org/10.1074/jbc.275.4.2247

6. 

Gressner AM, Weiskirchen R. Modern pathogenetic concepts of liver fibrosis suggest stellate cells and TGF-beta as major players and therapeutic targets. J Cell Mol Med 2006; 10:76–99. http://dx.doi.org/10.1111/j.1582-4934.2006.tb00292.x

7. 

Friedman SL. Hepatic stellate cells: protean, multifunctional, and enigmatic cells of the liver. Physiol Rev 2008; 88:125–72. http://dx.doi.org/10.1152/physrev.00013.2007

8. 

Rockey DC, Boyles JK, Gabbiani G, et al. Rat hepatic lipocytes express smooth muscle actin upon activation in vivo and in culture. J Submicroscop Cytol Pathol 1992; 24:193–203.

9. 

Uyama N, Zhao L, Van Rossen E, et al. Hepatic stellate cells express synemin, a protein bridging intermediate filaments to focal adhesions. Gut 2006; 55:1276–89. http://dx.doi.org/10.1136/gut.2005.078865

10. 

Lunevicius R, Nakanishi H, Ito S, et al. Clinicopathological significance of fibrotic capsule formation around liver metastasis from colorectal cancer. J Cancer Res Clin Oncol 2001; 127:193–9. http://dx.doi.org/10.1007/s004320000199

11. 

Weinstein DE, Shelanski ML, Liem RK. Suppression by antisense mRNA demonstrates a requirement for the glial fibrillary acidic protein in the formation of stable astrocytic processes in response to neurons. J Cell Biol 1991; 112:1205–13. http://dx.doi.org/10.1083/jcb.112.6.1205

12. 

Jiroutova A, Majdiakova L, Cermakova M, et al. Expression of cytoskeletal proteins in hepatic stellate cells isolated from normal and cirrhotic rat liver. Acta Medica (Hradec Kralove) 2005; 48:137–44.

13. 

Weiskirchen R, Gressner AM. Isolation and culture of hepatic stellate cells. Method Mol Med 2005; 117:99–113.

14. 

Pinzani M. Novel insights into the biology and physiology of the Ito cell. Pharmacol Ther 1995; 66:387–412. http://dx.doi.org/10.1016/0163-7258(94)00072-B

15. 

Shimizu S, Yamada N, Sawada T, et al. In vivo and in vitro interactions between human colon carcinoma cells and hepatic stellate cells. Jpn J Cancer Res 2000; 91:1285–95. http://dx.doi.org/10.1111/j.1349-7006.2000.tb00916.x

16. 

Illemann M, Bird N, Majeed A, et al. Two distinct expression patterns of urokinase, urokinase receptor and plasminogen activator inhibitor-1 in colon cancer liver metastases. Int J Cancer 2009; 124:1860–70. http://dx.doi.org/10.1002/ijc.24166

17. 

Mazzocca A, Coppari R, De Franco R, et al. A secreted form of ADAM9 promotes carcinoma invasion through tumor–stromal interactions. Cancer Res 2005; 65:4728–38. http://dx.doi.org/10.1158/0008-5472.CAN-04-4449

18. 

Enzan H, Himeno H, Iwamura S, et al. Alpha-smooth muscle actin-positive perisinusoidal stromal cells in human hepatocellular carcinoma. Hepatology 1994; 19:895–903.

19. 

Schmitt-Graeff A, Jing R, Nitschke R, et al. Synemin expression is widespread in liver fibrosis and is induced in proliferating and malignant biliary epithelial cells. Hum Pathol 2006; 37:1200–10. http://dx.doi.org/10.1016/j.humpath.2006.04.017

20. 

Olaso E, Salado C, Egilegor E, et al. Proangiogenic role of tumor-activated hepatic stellate cells in experimental melanoma metastasis. Hepatology 2003; 37:674–85. http://dx.doi.org/10.1053/jhep.2003.50068

21. 

Olaso E, Santisteban A, Bidaurrazaga J, et al. Tumor-dependent activation of rodent hepatic stellate cells during experimental melanoma metastasis. Hepatology 1997; 26:634–42. http://dx.doi.org/10.1002/hep.510260315

22. 

Faouzi S, Lepreux S, Bedin C, et al. Activation of cultured rat hepatic stellate cells by tumoral hepatocytes. Lab Invest 1999; 79:485–93.

23. 

Vidal-Vanaclocha F (eds.). The Tumour Microenvironment at Different Stages of Hepatic Metastases. Houten, Netherlands: Springer Netherlands; 2011.

24. 

Neubauer K, Knittel T, Aurisch S, et al. Glial fibrillary acidic protein – a cell type specific marker for Ito cells in vivo and in vitro. J Hepatol 1996; 24:719–30. http://dx.doi.org/10.1016/S0168-8278(96)80269-8

25. 

Knittel T, Kobold D, Saile B, et al. Rat liver myofibroblasts and hepatic stellate cells: different cell populations of the fibroblast lineage with fibrogenic potential. Gastroenterology 1999; 117:1205–21. http://dx.doi.org/10.1016/S0016-5085(99)70407-5

26. 

Senoo H, Kojima N, Sato M. Vitamin A-storing cells (stellate cells). Vitam Horm 2007; 75:131–59. http://dx.doi.org/10.1016/S0083-6729(06)75006-3

27. 

Geerts A. History, heterogeneity, developmental biology, and functions of quiescent hepatic stellate cells. Semin Liver Dis 2001; 21:311–35. http://dx.doi.org/10.1055/s-2001-17550

28. 

Friedman SL, Roll FJ, Boyles J, et al. Maintenance of differentiated phenotype of cultured rat hepatic lipocytes by basement membrane matrix. J Biol Chem 1989; 264:10756–62.

29. 

Gressner AM. The cell biology of liver fibrogenesis – an imbalance of proliferation, growth arrest and apoptosis of myofibroblasts. Cell Tissue Res 1998; 292:447–52. http://dx.doi.org/10.1007/s004410051073

30. 

Friedman SL. Seminars in medicine of the Beth Israel Hospital, Boston. The cellular basis of hepatic fibrosis. Mechanisms and treatment strategies. N Engl J Med 1993; 328:1828–35. http://dx.doi.org/10.1056/NEJM199306243282508

31. 

Illemann M, Bird N, Majeed A, et al. Possible evidence of a protease-independent invasion mechanism in colon cancer liver metastases with ‘pushing’ growth pattern. Clin Exp Metastasis 2011; 28:179–80.

32. 

Tsukamoto H, Mishima Y, Hayashibe K, et al. Alpha-smooth muscle actin expression in tumor and stromal cells of benign and malignant human pigment cell tumors. J Invest Dermatol 1992; 98:116–20. http://dx.doi.org/10.1111/1523-1747.ep12496020

33. 

Lieubeau B, Garrigue L, Barbieux I, et al. The role of transforming growth factor beta 1 in the fibroblastic reaction associated with rat colorectal tumor development. Cancer Res 1994; 54:6526–32.

34. 

Welt S, Divgi CR, Scott AM, et al. Antibody targeting in metastatic colon cancer: a phase I study of monoclonal antibody F19 against a cell-surface protein of reactive tumor stromal fibroblasts. J Clin Oncol 1994; 12:1193–203.

35. 

Dimanche-Boitrel MT, Vakaet L Jr, et al. In vivo and in vitro invasiveness of a rat colon-cancer cell line maintaining E-cadherin expression: an enhancing role of tumor-associated myofibroblasts. Int J Cancer 1994; 56:512–21. http://dx.doi.org/10.1002/ijc.2910560410

36. 

Walker JA, Quirke P. Viewing apoptosis through a ‘TUNEL’. J Pathol 2001; 195:275–6. http://dx.doi.org/10.1002/path.979

37. 

Krol J, Mengele K, Ottl-Mantchenko I, et al. Ex vivo detection of apoptotic trophoblast cells applying flow cytofluorometry and immunocytochemistry using M30 antibody directed to the cytokeratin 18 neo-epitope. Int J Mol Med 2005; 16:415–20.

38. 

Bantel H, Ruck P, Gregor M, Schulze-Osthoff K. Detection of elevated caspase activation and early apoptosis in liver diseases. Eur J Cell Biol 2001; 80:230–9. http://dx.doi.org/10.1078/0171-9335-00154

39. 

Caulin C, Salvesen GS, Oshima RG. Caspase cleavage of keratin 18 and reorganization of intermediate filaments during epithelial cell apoptosis. J Cell Biol 1997; 138:1379–94. http://dx.doi.org/10.1083/jcb.138.6.1379

40. 

Leers MP, Kolgen W, Bjorklund V, et al. Immunocytochemical detection and mapping of a cytokeratin 18 neo-epitope exposed during early apoptosis. J Pathol 1999; 187:567–72. http://dx.doi.org/10.1002/(SICI)1096-9896(199904)187:5<567::AID-PATH288>3.0.CO;2-J

41. 

Hetz H, Hoetzenecker K, Hacker S, et al. Caspase-cleaved cytokeratin 18 and 20 S proteasome in liver degeneration. J Clin Lab Anal 2007; 21:277–81. http://dx.doi.org/10.1002/jcla.20180

42. 

Bedi A, Pasricha PJ, Akhtar AJ, et al. Inhibition of apoptosis during development of colorectal cancer. Cancer Res 1995; 55:1811–16.

43. 

Hahne M, Rimoldi D, Schroter M, et al. Melanoma cell expression of Fas(Apo-1/CD95) ligand: implications for tumor immune escape. Science 1996; 274:1363–6. http://dx.doi.org/10.1126/science.274.5291.1363

44. 

Moller P, Koretz K, Leithauser F, et al. Expression of APO-1 (CD95), a member of the NGF/TNF receptor superfamily, in normal and neoplastic colon epithelium. Int J Cancer 1994; 57:371–7. http://dx.doi.org/10.1002/ijc.2910570314

45. 

Vermeulen PB, Colpaert C, Salgado R, et al. Liver metastases from colorectal adenocarcinomas grow in three patterns with different angiogenesis and desmoplasia. J Pathol 2001; 195:336–42. http://dx.doi.org/10.1002/path.966

46. 

Saile B, Knittel T, Matthes N, et al. CD95/CD95L-mediated apoptosis of the hepatic stellate cell. A mechanism terminating uncontrolled hepatic stellate cell proliferation during hepatic tissue repair. Am J Pathol 1997; 151:1265–72.

47. 

O'Connell J, O'Sullivan GC, Collins JK, et al. The Fas counterattack: Fas-mediated T cell killing by colon cancer cells expressing Fas ligand. J Exp Med 1996; 184:1075–82. http://dx.doi.org/10.1084/jem.184.3.1075





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