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In situ expression of integrins in human hepatocellular carcinoma
International
Hepatology colIxlunicatio~ Int Hepatol Commun 2 (1994) 87-93
In situ expression of integrins in human hepatocellular carcinoma Ichinosuke Hyodo”,*, Kenji Jinnoa,b, Masahito Tanimizu” Departments of “Internal Medicine and bClinical Research, National Shikoku Cancer Center Hospital, 13 Horinouchi, Matsuyama 790, Japan
(Received 28 September 1993;accepted 12 November 1993)
Abstract Integrins are plasma membrane integral proteins, composed of a anda subunits and distributed on various normal and neoplastic cells. They serve a function as cellular receptors for extracellular matrix proteins. In order to reveal the distinct distribution of integrins on hepatocellular carcinoma (HCC) cells, we studied for the expression of a2, a3, a4, a5, a6, av, /Il, 83, and /34 integrin subunits in 12 HCC and adjacent inflamed liver, and three normal liver tissues, using monoclonal antibodies against each subunit by indirect immunoperoxidase method. The expression of a2 subunit in cytoplasm, and that of a6, pl, and /?3 subunits mainly on cell membrane was constantly observed in HCC cells. The a6 subunit showed remarkable de novo expression on all HCC cells without any relation to their differentiation grade, but not on hepatocytes in inflamed liver except for severe inflamed and fibrotic lesions. Others showed no difference in expression patterns between HCC cells and hepatocytes. a3, a4, av, and 84 subunits were absent from HCC cells and inflamed hepatocytes, and the a5 subunit was weakly stained on them. In the normal liver tissues, the expression of all integrin subunits examined was almost negative or faint on hepatocytes. We conclude that the strong de novo expression of a6 integrin subunit is the most conspicuous feature of HCC cells, when compared with normal and inflamed hepatocytes, and that it is a possible immunocytochemical marker of HCC cells to distinguish them from non-neoplastic hepatocytes. Key words: Adhesion molecule; Integrin subunit; Extracellular matrix; Henatoma
1. Introduction
It is known that both normal and neoplastic cells recognize many extracellular and cellular proteins using cell surface receptors. Integrins composed of transmembrane subunits 01 and ,L?are examples of these receptors [1,2]. The CI and /? subunits are *Corresponding author. 0928-4346/94/$07.00 0 1994 Elsevier Science B.V. All SSDI 0928-4346(93)E0058-X
rights reserved
88
I. Hyodo et al. lint Hepatol Commun 2 (1994) 87-93
noncovalently bound to each other and the ligand specificity of integrins depends on the a//3 subunit combinations. More than 15 members of this family have been identified and are generally expressed in a tissue-specific manner. During the process of tumor cell growth, invasion and metastasis, tumor cells interact with extracellular matrix proteins using these cell surface receptors. The alterations of integrin expression by tumor cells have been reported to be of potential significance in these processes [3,4]. The purpose of this study is to reveal the distinct distribution of various integrin subunits on HCC cells and hepatocytes, and to know the differences of integrin expression between them. 2. Materials and methods 2.1. Patients HCC and adjacent liver tissues were obtained from 12 patients (of whom 11 and 1 were positive for anti-hepatitis C virus antibody (ClOO-3; Chiron, Emeryville, CA) and hepatitis B surface antigen, respectively) undergoing surgical resection. Histologically normal liver tissues were obtained from three patients with cholelithiasis and hemangioma. Informed consent was obtained from each patient. Half of each tissue specimen was fixed in 10% buffered formalin for routine histological examination, and the other half was fixed in periodate-lysine-2% paraformaldehyde (PLP) fixative [5] for immunohistochemical examination. Two specimens of adjacent liver tissue from the HCC patients showed chronic active hepatitis and the remaining ten showed cirrhosis. The differentiation of HCC was determined according to the EdmondsonSteiner classification. Two tumors were classified as Edmondson’s grade I-II (well to moderately differentiated), eight tumors were grade II, and two tumors were grade III (poorly differentiated). 2.2. Immunohistochemical analysis of integrins Cryostat sections of liver specimens fixed with PLP were incubated for 12 h with each monoclonal antibody (MoAb) as shown in Table 1. Then the sections were reacted for 4 h with horseradish peroxidase-labeled Fab’ fragments of rabbit antimouse (a2,3,4,5,v and/31,3,4) or anti-rat (a6) immunoglobulins, followed by reaction with diaminobenzidine solution containing H202 counterstained with methylgreen, dehydrated and mounted. Control sections were incubated with diluted normal mouse or rat serum, or phosphate-buffered saline (PBS) instead of with the MoAb.
3. Results Expression patterns and staining intensity of integrin subunits were summarized in Table 2. 3.1. Integrin a6 subunit a6 subunit was expressed strongly on the cell membrane of all HCC cells without any relation to their differentiation grade, and weakly in cytoplasm of some of those
I. Hyodo et al. lint Hepatol Commun 2 (1994) 87-93
89
cells (Fig. 1). While its expression on hepatocytes was almost negative or weakly positive only for the hepatocyte membrane surrounded with active fibrosis (Fig. 1A). 3.2. Other integrim subunits a2 subunit was diffusely distributed in the cytoplasm of both HCC cells (Fig. 2A) and hepatocytes in inflamed liver tissues. The staining for a2 subunit was very weak on normal hepatocytes. a3 subunit was constantly positive for bile duct epithelial cells (Fig. 2B), sinusoidal endothelial cells, and endothelial cells of blood space of HCC, but was negative or faint for HCC cells and hepatocytes. a4 and av subunits were never seen on HCC cells or hepatocytes in any tissues. The staining for a5 subunit was observed weakly on sinusoidal endothelial cells and endothelial cells of blood space of HCC in all tissues. The staining on both HCC cells and hepatocytes was negative or very weak. Although the various degrees of staining of/?1 and/33 subunits from weak to strong were seen on the cell membrane of HCC cells, no remarkable difference was noticed when compared with hepatocytes in inflamed liver. /31 subunit was mainly distributed on the cell membrane of HCC cells and the staining was intense in seven of HCC tissues (Fig. 2C). Eight HCC tissues showed strong staining of/33 subunit on the cell membrane of HCC cells, and they were accompanied with its cytoplasmic expression (Fig. 2D). 84 subunit was only located along the basement membrane of vessels and bile ducts in the portal area. The positive staining of /?4 subunit was neither seen on HCC cells nor on hepatocytes. No specific reaction was observed in control sections incubated with normal mouse or rat serum, or PBS.
Table 1 Profiles of integrin subunits directed against by used monoclonal antibodies and their counter-receptors Integrin Adhesion subunit molecule a2
VLA”-2(a2/?1)
a3
VLA-3(c13/?1)
a4 a5 a6 av
VLA-4(a4/?1) VLA-S(a5pl) VLA-6(a6Bl) av/31,3,5
;:
al-641 avpl.3
84
VLA-6 alt (a6B4)
Counter-receptor/ ligand
Monoclonal antibody source (purchased from)
laminin, collagen fibronectin, laminin, collagen fibronectin, VCAMb-1 fibronectin laminin fibronectin, vitronectin, fibrinogen, von Willebrand factor
Oncogene science, Inc., Telios pharmaceuticals, Inc. Oncogene science, Inc., Telios pharmaceuticals, Inc.
vitronectin, fibrinogen, fibronectin, von Willebrand factor laminin
Telios pharmaceuticals, Chemicon international, Chemicon international, Chemicon international,
Inc. Inc. Inc. Inc.
Takara biochemicals (Kyoto, Japan) Takara biochemicals (Kyoto, Japan)
Chemicon international, Inc.
“VLA, very late antigen; ?‘CAM, vascular cell adhesion molecule.
I. Hyodo et al. lint Hepatol Commun 2 (1994) 87-93
90
Table 2 Expression patterns and staining intensity of integrins on hepatocellular carcinoma (HCC) cells and on hepatocytes of adjacent inflamed and normal liver Integrin subunit
HCC cell
Hepatocyte
Other positive staining site
in inflamed liver in normal liver a2 a3 a4 a5 a6
c(+)-(++’
av
c(+‘-(++’ (-‘-W+’ (-’ (-‘-MC+’ MC++) cc+’ (-’
;:
MC+‘-(++) MC+‘-(++)
84
cc+) t-1
M(+)-(++I M(+H++’ cc+)
W) (-’ (-’
bile duct epithelium endothelial cells, bile duct epithelium
t-‘-W+’
(-’
C-‘-M(+) cc-)-(+’
(-’
endothelial cells (weak) endothelial cells, bile duct epithelium
(-)
(-’ W-H+-) M(-‘-(+’
bile duct epithelium endothelial cells, bile duct epithelium endothelial cells, bile duct epithelium
IT”“’
(-’
CC-’
(-’
basement membrane of vessels and bile duct
C, cytoplasmic expression; M, membranous expression. The staining intensity was evaluated semiquantitatively as follows: (-) = negative staining; (+) = faint staining; (+) = weak staining; (++) = strong staining.
4. Discussion From this immunohistochemical study, we demonstrated that HCC cells mainly express integrin a2 subunit in their cytoplasm and integrin 016,j?l , and j33 subunits on their cell membrane. Among these integrins, the increased expression of a6 subunit was the most striking feature of HCC cells when compared with normal and inflamed hepatocytes. The distribution of integrins is of interest in the pathology of inflammatory liver diseases. It has been reported that a5 subunit is expressed weakly on normal hepatocytes and the expression is up-regulated in inflamed liver [6,7]. Furthermore, a2 and a6 subunits are also induced on some hepatocyes in inflammatory liver. This upregulated or induced expression of integrins is considered to be relevant with cytokine stimulation in inflammation, fibrogenic process, and liver cell regeneration. On the other hand, a3 and a4 subunits are not distributed on hepatocytea. a3 subunit is mainly expressed on sinusoidal and vascular endothelial cells, and bile duct epitheliurn. a4 subunit is located on lymphocytes and Kupffer cells. Our results for the distribution of integrin subunits in inflammatory liver tissues were almost compatible with previous reports [6,7]. The distribution of integrins in human HCC tissues is still not well known. Volpes et al. have recently reported immunohistochemical distribution of integrins in HCC tissues [S]. Their observations were that well-differentiated HCC cells expressed al and j?l integrin subunits but, in addition, poorly differentiated HCC cells de novo expressed a2, a3, and a6 subunits. These findings are different from ours in the point that we recognized a2 and a6 integrin subunits clearly in all HCC cells without any
I. Hyodo et al. lht Hepatol Commun 2 (1994) 87-93
91
Fig. 1. Immunohistochemical staining of integrin a6 subunit. a6 subunit is clearly recognized on the cell membrane of well-differentiated HCC cells and partly in their cytoplasm (right side), but it is not expressed on normal hepatocytes in the cirrhotic liver (left side) (A). Arrows show the invasive front of HCC cells. Similar distribution of a6 subunit is observed on moderately (B) and poorly (C) differentiated HCC cells. Counterstained with methylgreen. x300.
relation to their differentiation grade. This discrepancy is probably due to the following causes: fixation of the specimens and/or difference of binding activity of usedMoAbs reacting with their epitopes. It seems that our positive staining of a2 and a6 subunits is also reliable because our specimens were immediately fixed in PLP fixative to avoid antigen dissolution and movement from original sites and, moreover, their
92
I. Hyodo et al. IInt Hepatol Commun 2 (1994) 87-93
Fig. 2. Immunohistochemical staining of integrin ~2, ~3, /31, and /33 subunits. a2 subunit is expressed diffusely in the cytoplasm of HCC cells (A). a3 subunit is distributed on the vascular endothelial cells (arrows) and bile duct epithelial cells (arrow heads) (B). /Tl (C) andp3 (D) subunits are expressed diffusely on the cell membrane of HCC cells and partly in their cytoplasm. Counterstained with methylgreen. x300.
staining was very strong in many tissues. Therefore, the latter might be a main cause of the discrepancy. Our result for a3 and /I1 expression was almost the same as their report. av/?3 integrin serves to function as a vitronectin receptor. /33 subunit was expressed strongly on HCC cells, but av subunit was absent from WCC cells. It is not known whether or not this /33 subunit on HCC cells is expressed with any other partner of a subunit. a6B4 and as/31 subunit combination is known to be a laminin receptor. 84 subunit was expressed on bile duct epithelium and vascular endothelium in the portal area, but not on HCC cells and hepatocytes. From our results, the bile duct epithelium and vascular endothelium in the portal area have both sets of a6jIl and a6B4 immunocytochemically, while HCC cells have only a set of a6Bl. a6 subunit was not seen on hepatocytes of normal and inflamed liver except for severe inflammation and fibrotic lesions, but it was strongly and diffusely expressed on all HCC cells. Based on these findings, we suggest that it is possible to discriminate HCC cells from
I. Hvodo et al. IInt Hepatol
Commun 2 (1994) 87-93
93
hepatocytes immunocytochemically by using this commercially available antibody (Chemicon international Inc.), even if HCC cells are well differentiated and greatly resemble to normal hepatocytes. So-called ‘atypical’ adenomatous hyperplasia is occasionally observed with HCC. However, our examined tissues unfortunately did not include any foci of the adenomatous hyperplasia. Further investigation is necessary to determine the expression of integrin subunits, especially 0~6,in the adenomatous hyperplasia cells. Laminin receptor has been reported to play an important role in tumor growth, invasion and metastasis in various tumors [3,9]. Taken together with these reports and our present findings, it is likely that as/?1 integrin strongly expressed on HCC cells acts as a laminin receptor and relates profoundly to HCC progression.
Acknowledgements The authors thank Dr. Shousuke Moriwaki, Dr. Koichi Mandai, Dr. Minoru Tanada, and Dr. Masanori Akiyama for their cooperation. This study was supported in part by Grant-in-Aid for Cancer Research from the Ministry of Health and Welfare of Japan.
References [I] Hynes RO. Integrins: a family of cell surface receptors. Cell 1987; 48:549-554. [2] Ruoslahti E, Pierschbacher MD. New perspectives in cell adhesion: RGD and integrins. Science 1987;238:491497. [3] Liotta LA. Tumor invasion and metastasis - Role of the extracellular matrix. Cancer Res 1986;46:1-7. [4] Ruoslahti E, Giancotti FG. Integrins and tumor cell dissemination. Cancer Cells 1989;1:119-126. [5] McLean SD, Nakane PK. Periodate-lysine-paraformaldehyde fixative: a new fixative for immunoelectron microscopy. J Histochem Cytochem 1974;22:1077-1083. [6] Volpes R, van den Oord JJ, Desmet VJ. Distribution of the VLA family of integrins in normal and pathological human liver tissue. Gastroenterology 1991;101:200-206. [7] Staeinhoff G, Behrend M, Schrader B, Pichalmayr R. Intercellular immune adhesion molecules in human liver transplants: Overview on expression patterns of leukocyte receptor and ligand molecules. Hepatology 1993;18:44&453. [8] Volpes R, van den Oord JJ, Desmet VJ. Integrins as differential cell lineage markers of primary liver tumors. Am J Pathol. 1993;142:1483-1492. [9] Grigioni WF, Garbisa S, D’Errico A, et al. Evaluation of hepatocellular carcinoma aggressiveness by a panel of extracellular matrix antigens. Am J Path01 1991;138:647-654,
Hepatology colIxlunicatio~ Int Hepatol Commun 2 (1994) 87-93
In situ expression of integrins in human hepatocellular carcinoma Ichinosuke Hyodo”,*, Kenji Jinnoa,b, Masahito Tanimizu” Departments of “Internal Medicine and bClinical Research, National Shikoku Cancer Center Hospital, 13 Horinouchi, Matsuyama 790, Japan
(Received 28 September 1993;accepted 12 November 1993)
Abstract Integrins are plasma membrane integral proteins, composed of a anda subunits and distributed on various normal and neoplastic cells. They serve a function as cellular receptors for extracellular matrix proteins. In order to reveal the distinct distribution of integrins on hepatocellular carcinoma (HCC) cells, we studied for the expression of a2, a3, a4, a5, a6, av, /Il, 83, and /34 integrin subunits in 12 HCC and adjacent inflamed liver, and three normal liver tissues, using monoclonal antibodies against each subunit by indirect immunoperoxidase method. The expression of a2 subunit in cytoplasm, and that of a6, pl, and /?3 subunits mainly on cell membrane was constantly observed in HCC cells. The a6 subunit showed remarkable de novo expression on all HCC cells without any relation to their differentiation grade, but not on hepatocytes in inflamed liver except for severe inflamed and fibrotic lesions. Others showed no difference in expression patterns between HCC cells and hepatocytes. a3, a4, av, and 84 subunits were absent from HCC cells and inflamed hepatocytes, and the a5 subunit was weakly stained on them. In the normal liver tissues, the expression of all integrin subunits examined was almost negative or faint on hepatocytes. We conclude that the strong de novo expression of a6 integrin subunit is the most conspicuous feature of HCC cells, when compared with normal and inflamed hepatocytes, and that it is a possible immunocytochemical marker of HCC cells to distinguish them from non-neoplastic hepatocytes. Key words: Adhesion molecule; Integrin subunit; Extracellular matrix; Henatoma
1. Introduction
It is known that both normal and neoplastic cells recognize many extracellular and cellular proteins using cell surface receptors. Integrins composed of transmembrane subunits 01 and ,L?are examples of these receptors [1,2]. The CI and /? subunits are *Corresponding author. 0928-4346/94/$07.00 0 1994 Elsevier Science B.V. All SSDI 0928-4346(93)E0058-X
rights reserved
88
I. Hyodo et al. lint Hepatol Commun 2 (1994) 87-93
noncovalently bound to each other and the ligand specificity of integrins depends on the a//3 subunit combinations. More than 15 members of this family have been identified and are generally expressed in a tissue-specific manner. During the process of tumor cell growth, invasion and metastasis, tumor cells interact with extracellular matrix proteins using these cell surface receptors. The alterations of integrin expression by tumor cells have been reported to be of potential significance in these processes [3,4]. The purpose of this study is to reveal the distinct distribution of various integrin subunits on HCC cells and hepatocytes, and to know the differences of integrin expression between them. 2. Materials and methods 2.1. Patients HCC and adjacent liver tissues were obtained from 12 patients (of whom 11 and 1 were positive for anti-hepatitis C virus antibody (ClOO-3; Chiron, Emeryville, CA) and hepatitis B surface antigen, respectively) undergoing surgical resection. Histologically normal liver tissues were obtained from three patients with cholelithiasis and hemangioma. Informed consent was obtained from each patient. Half of each tissue specimen was fixed in 10% buffered formalin for routine histological examination, and the other half was fixed in periodate-lysine-2% paraformaldehyde (PLP) fixative [5] for immunohistochemical examination. Two specimens of adjacent liver tissue from the HCC patients showed chronic active hepatitis and the remaining ten showed cirrhosis. The differentiation of HCC was determined according to the EdmondsonSteiner classification. Two tumors were classified as Edmondson’s grade I-II (well to moderately differentiated), eight tumors were grade II, and two tumors were grade III (poorly differentiated). 2.2. Immunohistochemical analysis of integrins Cryostat sections of liver specimens fixed with PLP were incubated for 12 h with each monoclonal antibody (MoAb) as shown in Table 1. Then the sections were reacted for 4 h with horseradish peroxidase-labeled Fab’ fragments of rabbit antimouse (a2,3,4,5,v and/31,3,4) or anti-rat (a6) immunoglobulins, followed by reaction with diaminobenzidine solution containing H202 counterstained with methylgreen, dehydrated and mounted. Control sections were incubated with diluted normal mouse or rat serum, or phosphate-buffered saline (PBS) instead of with the MoAb.
3. Results Expression patterns and staining intensity of integrin subunits were summarized in Table 2. 3.1. Integrin a6 subunit a6 subunit was expressed strongly on the cell membrane of all HCC cells without any relation to their differentiation grade, and weakly in cytoplasm of some of those
I. Hyodo et al. lint Hepatol Commun 2 (1994) 87-93
89
cells (Fig. 1). While its expression on hepatocytes was almost negative or weakly positive only for the hepatocyte membrane surrounded with active fibrosis (Fig. 1A). 3.2. Other integrim subunits a2 subunit was diffusely distributed in the cytoplasm of both HCC cells (Fig. 2A) and hepatocytes in inflamed liver tissues. The staining for a2 subunit was very weak on normal hepatocytes. a3 subunit was constantly positive for bile duct epithelial cells (Fig. 2B), sinusoidal endothelial cells, and endothelial cells of blood space of HCC, but was negative or faint for HCC cells and hepatocytes. a4 and av subunits were never seen on HCC cells or hepatocytes in any tissues. The staining for a5 subunit was observed weakly on sinusoidal endothelial cells and endothelial cells of blood space of HCC in all tissues. The staining on both HCC cells and hepatocytes was negative or very weak. Although the various degrees of staining of/?1 and/33 subunits from weak to strong were seen on the cell membrane of HCC cells, no remarkable difference was noticed when compared with hepatocytes in inflamed liver. /31 subunit was mainly distributed on the cell membrane of HCC cells and the staining was intense in seven of HCC tissues (Fig. 2C). Eight HCC tissues showed strong staining of/33 subunit on the cell membrane of HCC cells, and they were accompanied with its cytoplasmic expression (Fig. 2D). 84 subunit was only located along the basement membrane of vessels and bile ducts in the portal area. The positive staining of /?4 subunit was neither seen on HCC cells nor on hepatocytes. No specific reaction was observed in control sections incubated with normal mouse or rat serum, or PBS.
Table 1 Profiles of integrin subunits directed against by used monoclonal antibodies and their counter-receptors Integrin Adhesion subunit molecule a2
VLA”-2(a2/?1)
a3
VLA-3(c13/?1)
a4 a5 a6 av
VLA-4(a4/?1) VLA-S(a5pl) VLA-6(a6Bl) av/31,3,5
;:
al-641 avpl.3
84
VLA-6 alt (a6B4)
Counter-receptor/ ligand
Monoclonal antibody source (purchased from)
laminin, collagen fibronectin, laminin, collagen fibronectin, VCAMb-1 fibronectin laminin fibronectin, vitronectin, fibrinogen, von Willebrand factor
Oncogene science, Inc., Telios pharmaceuticals, Inc. Oncogene science, Inc., Telios pharmaceuticals, Inc.
vitronectin, fibrinogen, fibronectin, von Willebrand factor laminin
Telios pharmaceuticals, Chemicon international, Chemicon international, Chemicon international,
Inc. Inc. Inc. Inc.
Takara biochemicals (Kyoto, Japan) Takara biochemicals (Kyoto, Japan)
Chemicon international, Inc.
“VLA, very late antigen; ?‘CAM, vascular cell adhesion molecule.
I. Hyodo et al. lint Hepatol Commun 2 (1994) 87-93
90
Table 2 Expression patterns and staining intensity of integrins on hepatocellular carcinoma (HCC) cells and on hepatocytes of adjacent inflamed and normal liver Integrin subunit
HCC cell
Hepatocyte
Other positive staining site
in inflamed liver in normal liver a2 a3 a4 a5 a6
c(+)-(++’
av
c(+‘-(++’ (-‘-W+’ (-’ (-‘-MC+’ MC++) cc+’ (-’
;:
MC+‘-(++) MC+‘-(++)
84
cc+) t-1
M(+)-(++I M(+H++’ cc+)
W) (-’ (-’
bile duct epithelium endothelial cells, bile duct epithelium
t-‘-W+’
(-’
C-‘-M(+) cc-)-(+’
(-’
endothelial cells (weak) endothelial cells, bile duct epithelium
(-)
(-’ W-H+-) M(-‘-(+’
bile duct epithelium endothelial cells, bile duct epithelium endothelial cells, bile duct epithelium
IT”“’
(-’
CC-’
(-’
basement membrane of vessels and bile duct
C, cytoplasmic expression; M, membranous expression. The staining intensity was evaluated semiquantitatively as follows: (-) = negative staining; (+) = faint staining; (+) = weak staining; (++) = strong staining.
4. Discussion From this immunohistochemical study, we demonstrated that HCC cells mainly express integrin a2 subunit in their cytoplasm and integrin 016,j?l , and j33 subunits on their cell membrane. Among these integrins, the increased expression of a6 subunit was the most striking feature of HCC cells when compared with normal and inflamed hepatocytes. The distribution of integrins is of interest in the pathology of inflammatory liver diseases. It has been reported that a5 subunit is expressed weakly on normal hepatocytes and the expression is up-regulated in inflamed liver [6,7]. Furthermore, a2 and a6 subunits are also induced on some hepatocyes in inflammatory liver. This upregulated or induced expression of integrins is considered to be relevant with cytokine stimulation in inflammation, fibrogenic process, and liver cell regeneration. On the other hand, a3 and a4 subunits are not distributed on hepatocytea. a3 subunit is mainly expressed on sinusoidal and vascular endothelial cells, and bile duct epitheliurn. a4 subunit is located on lymphocytes and Kupffer cells. Our results for the distribution of integrin subunits in inflammatory liver tissues were almost compatible with previous reports [6,7]. The distribution of integrins in human HCC tissues is still not well known. Volpes et al. have recently reported immunohistochemical distribution of integrins in HCC tissues [S]. Their observations were that well-differentiated HCC cells expressed al and j?l integrin subunits but, in addition, poorly differentiated HCC cells de novo expressed a2, a3, and a6 subunits. These findings are different from ours in the point that we recognized a2 and a6 integrin subunits clearly in all HCC cells without any
I. Hyodo et al. lht Hepatol Commun 2 (1994) 87-93
91
Fig. 1. Immunohistochemical staining of integrin a6 subunit. a6 subunit is clearly recognized on the cell membrane of well-differentiated HCC cells and partly in their cytoplasm (right side), but it is not expressed on normal hepatocytes in the cirrhotic liver (left side) (A). Arrows show the invasive front of HCC cells. Similar distribution of a6 subunit is observed on moderately (B) and poorly (C) differentiated HCC cells. Counterstained with methylgreen. x300.
relation to their differentiation grade. This discrepancy is probably due to the following causes: fixation of the specimens and/or difference of binding activity of usedMoAbs reacting with their epitopes. It seems that our positive staining of a2 and a6 subunits is also reliable because our specimens were immediately fixed in PLP fixative to avoid antigen dissolution and movement from original sites and, moreover, their
92
I. Hyodo et al. IInt Hepatol Commun 2 (1994) 87-93
Fig. 2. Immunohistochemical staining of integrin ~2, ~3, /31, and /33 subunits. a2 subunit is expressed diffusely in the cytoplasm of HCC cells (A). a3 subunit is distributed on the vascular endothelial cells (arrows) and bile duct epithelial cells (arrow heads) (B). /Tl (C) andp3 (D) subunits are expressed diffusely on the cell membrane of HCC cells and partly in their cytoplasm. Counterstained with methylgreen. x300.
staining was very strong in many tissues. Therefore, the latter might be a main cause of the discrepancy. Our result for a3 and /I1 expression was almost the same as their report. av/?3 integrin serves to function as a vitronectin receptor. /33 subunit was expressed strongly on HCC cells, but av subunit was absent from WCC cells. It is not known whether or not this /33 subunit on HCC cells is expressed with any other partner of a subunit. a6B4 and as/31 subunit combination is known to be a laminin receptor. 84 subunit was expressed on bile duct epithelium and vascular endothelium in the portal area, but not on HCC cells and hepatocytes. From our results, the bile duct epithelium and vascular endothelium in the portal area have both sets of a6jIl and a6B4 immunocytochemically, while HCC cells have only a set of a6Bl. a6 subunit was not seen on hepatocytes of normal and inflamed liver except for severe inflammation and fibrotic lesions, but it was strongly and diffusely expressed on all HCC cells. Based on these findings, we suggest that it is possible to discriminate HCC cells from
I. Hvodo et al. IInt Hepatol
Commun 2 (1994) 87-93
93
hepatocytes immunocytochemically by using this commercially available antibody (Chemicon international Inc.), even if HCC cells are well differentiated and greatly resemble to normal hepatocytes. So-called ‘atypical’ adenomatous hyperplasia is occasionally observed with HCC. However, our examined tissues unfortunately did not include any foci of the adenomatous hyperplasia. Further investigation is necessary to determine the expression of integrin subunits, especially 0~6,in the adenomatous hyperplasia cells. Laminin receptor has been reported to play an important role in tumor growth, invasion and metastasis in various tumors [3,9]. Taken together with these reports and our present findings, it is likely that as/?1 integrin strongly expressed on HCC cells acts as a laminin receptor and relates profoundly to HCC progression.
Acknowledgements The authors thank Dr. Shousuke Moriwaki, Dr. Koichi Mandai, Dr. Minoru Tanada, and Dr. Masanori Akiyama for their cooperation. This study was supported in part by Grant-in-Aid for Cancer Research from the Ministry of Health and Welfare of Japan.
References [I] Hynes RO. Integrins: a family of cell surface receptors. Cell 1987; 48:549-554. [2] Ruoslahti E, Pierschbacher MD. New perspectives in cell adhesion: RGD and integrins. Science 1987;238:491497. [3] Liotta LA. Tumor invasion and metastasis - Role of the extracellular matrix. Cancer Res 1986;46:1-7. [4] Ruoslahti E, Giancotti FG. Integrins and tumor cell dissemination. Cancer Cells 1989;1:119-126. [5] McLean SD, Nakane PK. Periodate-lysine-paraformaldehyde fixative: a new fixative for immunoelectron microscopy. J Histochem Cytochem 1974;22:1077-1083. [6] Volpes R, van den Oord JJ, Desmet VJ. Distribution of the VLA family of integrins in normal and pathological human liver tissue. Gastroenterology 1991;101:200-206. [7] Staeinhoff G, Behrend M, Schrader B, Pichalmayr R. Intercellular immune adhesion molecules in human liver transplants: Overview on expression patterns of leukocyte receptor and ligand molecules. Hepatology 1993;18:44&453. [8] Volpes R, van den Oord JJ, Desmet VJ. Integrins as differential cell lineage markers of primary liver tumors. Am J Pathol. 1993;142:1483-1492. [9] Grigioni WF, Garbisa S, D’Errico A, et al. Evaluation of hepatocellular carcinoma aggressiveness by a panel of extracellular matrix antigens. Am J Path01 1991;138:647-654,