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Thyroid carcinoma distinctively expresses intracellular fibronectin in vivo
Cancer Letters 121 (1997) 189–193
Thyroid carcinoma distinctively expresses intracellular fibronectin in vivo Shinso Ryu*, Shiro Jimi, Shigeo Takebayashi The Second Department of Pathology, School of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-08, Japan Received 11 July 1997; received in revised form 15 July 1997; accepted 15 July 1997
Abstract Fibronectin is a multifunctional protein that plays a role in tumor invasion. We immunohistochemically examined the in vivo expression of fibronectin in thyroid carcinoma in comparison with other carcinomas, such as hepatocellular carcinoma, transitional cell carcinoma and gastric adenocarcinoma. Intracellular localization of fibronectin was found in almost all cases of thyroid carcinoma. In contrast, hepatocellular carcinoma showed a lower expression rate and the other carcinomas were all negative. These results indicate that the intracellular expression of fibronectin is not a common phenomenon in carcinoma, but rather is distinctive for thyroid carcinoma. 1997 Elsevier Science Ireland Ltd. Keywords: Fibronectin; Thyroid carcinoma; In vivo; Immunohistochemistry
1. Introduction Fibronectin is a ubiquitous glycoprotein found in the extracellular matrix and plasma [5]. It plays various biological roles in cellular adhesion, spreading, migration, oncogenic transformation, invasion and metastasis [1,4,16]. In vitro experiments have shown that suppressed fibronectin synthesis indicates a high likelihood of tumor cell invasion [17] through the liberation of adhesive glycoprotein. Fibronectin also has anchorage and stabilizing functions in cell locomotion [10] and thus acts to counteract malignancy. It has been demonstrated that fibronectin, which may be synthesized by tumor cells [7,11], is primarily loca-
* Corresponding author. Tel.: +81 92 8011011, ext. 3285; fax: +81 92 8638383; e-mail: [email protected]
lized in peripheral areas of malignant tumors including the basement membrane [2,6,9,14]. Therefore, it has been speculated that such tumors are highly likely to exhibit invasion. However, the clinical importance of fibronectin in tumor invasion has not yet been elucidated. To clarify the roles of fibronectin in malignant tumors, we examined the expression of fibronectin in thyroid carcinoma in comparison with other carcinomas by means of immunohistochemical techniques.
2. Materials and methods The specimens used in the present study were obtained from surgical or biopsy samples taken from March 1988 to June 1996 at Fukuoka University Hospital, Fukuoka, Japan. The samples were from 62
0304-3835/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3835 (97 )0 0353-4
190
S. Ryu et al. / Cancer Letters 121 (1997) 189–193
patients (36 male, 26 female) with a mean age of 68 years (range 38–87 years), which included a total of 28 cases of thyroid carcinoma (15 cases of papillary carcinoma (six cases well differentiated, nine cases poorly differentiated), seven cases of follicular carcinoma (three cases well differentiated, four cases poorly differentiated), three cases of medullary carcinoma and three cases of undifferentiated carcinoma), 13 cases of hepatocellular carcinoma (three cases well differentiated, seven cases moderately differentiated, three cases poorly differentiated), 11 cases of transitional cell carcinoma (eight cases grade II, three cases grade III) and 10 cases of gastric adenocarcinoma (five cases well differentiated, one case moderately differentiated, four cases poorly differentiated). All of the cases of thyroid carcinoma were shown to have metastasized into other organs. In all of the patients, histological diagnosis was made according to the classification established by the World Health Organization. All samples were fixed in 10% buffered formalin and embedded in paraffin. Deparaffinized sections (3 mm thick) were washed in PBS (pH 7.4) and treated with protease (DAKO, Denmark). Immunohistochemical staining was performed by the alkaline phosphatase–antialkaline phosphatase method. In brief, the primary antibody against human plasma fibronectin (DAKO) was diluted 1:600 and incubated overnight at 4°C. After washing in PBS, the sections were incubated with alkaline phosphatase-conjugated second antibody against rabbit immunoglobulin (DAKO) for 60 min at room temperature. After washing, the sections were incubated with alkaline phosphataseconjugated third antibody against alkaline phosphatase (DAKO) for 60 min at room temperature. As a
negative control, vehicle alone or an identical concentration of non-immune animal IgG was used. To check the specificity of the first antibody, an excess amount of antigen, fibronectin (Sigma, St. Louis, MO), was mixed with the first-antibody solution and incubated for 1 h at 37°C. This solution was used as a first-antibody solution for immunohistochemistry. Thereafter, the sections were colored by 0.01% new fucshin (Merck, Germany), 0.01% NaNO2, 10 mg naphtol AS-BI phosphate (Sigma), 0.1 ml n,Ndimethyl formamide (Wako, Osaka) and 10 mg levamisol (Sigma) in 40 ml of 0.2 M Tris–HCl buffer (pH 8.2). Specimens were observed under a microscope. As a result, all of the stained materials in specimens treated with the antibody were fibronectin. Immunohistological evaluation was performed with the following criteria to classify tissue as positive: in tumor cells/normal cells adjacent to the tumor, when one or more tumor cells/normal cells was intracellularly positive; in stroma in tumor/normal tissue adjacent to the tumor, when extracellular matrix including the basement membrane in the tissue was positive; in blood vessels, when the luminal space was positive.
3. Results We examined fibronectin expression in four different types of malignant tumor, including thyroid carcinoma, hepatocellular carcinoma, transitional cell carcinoma and gastric adenocarcinoma. Since most of the samples contained a tumor lesion and adjacent normal tissue, both types of tissue were subjected to immunohistochemical analysis. Table 1 shows the
Table 1 Fibronectin in cells and stroma in different types of carcinoma Carcinoma
Thyroid carcinoma Hepatocellular carcinoma Transitional cell carcinoma Gastric adenocarcinoma
Total cases
28 13 11 10
Values represent the number of positive cases. a Samples did not contain normal tissue.
Tumor
Normal
Cell (%)
Stroma (%)
Cell (%)
Stroma (%)
27 4 0 0
28 7 1 5
0 (0) 3 (23.1) –a 0 (0)
3 (10.7) 5 (38.5) –a 1 (10.0)
(96.4) (30.7) (0) (0)
(100) (54.0) (9.0) (50.0)
S. Ryu et al. / Cancer Letters 121 (1997) 189–193
results of our immunohistochemical analysis. In thyroid carcinoma, although they showed different types of thyroid carcinoma, almost all of the cases (27/28 cases; 96.4%) showed positive staining for fibronec-
191
tin, whereas no fibronectin was found in any of the normal epithelial cells adjacent to the carcinoma (Fig. 1A). The cases of hepatocellular carcinoma showed a positive reaction in 4/13 cases (30.7%) of varied dif-
Fig. 1. Fibronectin localization in cases of (A) papillary thyroid carcinoma (54 years, female), (B) hepatocellular carcinoma (44 years, male), (C) transitional cell carcinoma (65 years, female) and (D) gastric adenocarcinoma (84 years, male). (A) The invading carcinoma is positive for fibronectin in the cells and in the stroma in some places. On the other hand, normal thyroid glands adjacent to the carcinoma possess no fibronectin. (B) The invading carcinoma is positive for fibronectin in the cells, while the tumor stroma possesses almost no fibronectin. (C) The carcinoma cells in the upper part are entirely negative for fibronectin, while the edematous extracellular matrix, including some vessels, is positive for fibronectin (lower part). (D) The adenocarcinoma cells and stroma in the upper part are entirely negative for fibronectin, while the vessel lumen is slightly positive for fibronectin (lower part). Bar, 40 mm.
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S. Ryu et al. / Cancer Letters 121 (1997) 189–193
ferentiation and fibronectin-positive normal epithelial cells adjacent to the tumor were found in 3/13 cases (23.1%) (Fig. 1B). The cases of transitional cell carcinoma obtained by biopsy did not contain normal tissue and all of these cases showed negative staining for fibronectin (Fig. 1C). All of the cases of gastric adenocarcinoma showed a negative reaction in both tumor cells and adjacent normal epithelial cells (Fig. 1D). In contrast, all of the cases in the luminal space in blood vessels showed a positive reaction (Fig. 1C,D).
4. Discussion The present immunohistochemical study demonstrated that thyroid carcinoma is distinctive regarding the intracellular expression of fibronectin compared to the other carcinomas examined, i.e. hepatocellular carcinoma, transitional cell carcinoma and gastric adenocarcinoma, in which the expression rates of fibronectin were quite low. While fibronectin expression is not a common phenomenon in cells that have transformed into carcinoma, it is common for thyroid carcinoma regardless of the subtype. The cases of thyroid carcinoma were subclassified as papillary carcinoma, follicular carcinoma, medullary carcinoma and undifferentiated carcinoma. Their prognosis tended to vary depending upon the type of thyroid carcinoma (data not shown). However, no difference was found in any of the carcinomas in that almost all of the cases possessed fibronectin in tumor cells. A quantitative analysis for fibronectin in tumor cells of thyroid carcinoma is necessary. Fibronectin can be produced by various kinds of cells including epithelial cells [13], fibroblasts and endothelial cells and exists primarily in extracellular matrix and plasma [5], in which it is referred to as matrix protein and cold insoluble-globulin, respectively. Our results also indicated that fibronectin was found more or less in the extracellular matrix in tumor and adjacent normal tissue and in the luminal space of blood vessels. Such fibronectin may be a component of matrix protein associated with collagens [8] and plasma protein. Although the liver is the primary source of fibronectin [3], only 23.1% of the cases with normal hepatocytes were positive for fibronectin expression based
on an immunohistochemical analysis. As in other studies [15], we found fibronectin in the intercellular spaces of hepatocellular carcinoma. The positive rate for cell-associated fibronectin in this carcinoma was slightly increased (30.7%) relative to those among the other carcinomas, although it was almost the same as that in normal hepatocytes. In hepatocellular carcinoma, there was no relationship between the expression of intracellular fibronectin and the type of differentiation. Moreover, although the cases of transitional cell carcinoma and gastric adenocarcinoma showed various levels of differentiation, fibronectin was not found in the cells of either of these carcinomas. It is likely that fibronectin secretion is not a uniform characteristic of carcinoma. On the other hand, fibronectin was found in some normal gastric glands, in which severe inflammation occurred (data not shown). It has been shown that fibronectin occurs at sites of inflammation [12]. Tumor stroma not only contribute to tumor structure but also play a role in creating a foothold for tumor cells. In some carcinomas in vivo, stromal fibronectin has been identified as a characteristic of carcinoma [2,6,14]. However, stromal fibronectin in normal tissue also possesses fibronectin and cases of stromal fibronectin in tumor lesions showed slightly higher rates of fibronectin expression. However, this difference was not significant except among the cases of thyroid carcinoma. Therefore, the presence of stromal fibronectin may not be a characteristic of carcinoma in general. We found that in thyroid carcinoma, in comparison with other carcinomas, a large amount of fibronectin was accumulated in the stroma. Moreover, some of the cases of thyroid carcinoma had a large volume of stromal fibronectin. One case of papillary thyroid carcinoma (poor differentiation) failed to express intracellular fibronectin, although a large amount of fibronectin was found in its stroma. Together with the intracellular expression of fibronectin, the stroma may act as a reservoir after the secretion of fibronectin from tumor cells. Further studies are necessary to determine the relationship between stromal fibronectin and intracellular fibronectin in thyroid carcinoma cells. In conclusion, we immunohistochemically examined fibronectin expression in different carcinomas in vivo using a conventional method and found that
S. Ryu et al. / Cancer Letters 121 (1997) 189–193
this intracellular expression was distinct for thyroid carcinoma regardless of the subtype. References [1] I.U. Ali, V.M. Mautner, R.P. Lanza, R.O. Hynes, Restoration of normal morphology, adhesion and cytoskeleton in transformed cells by addition of a transformation-sensitive surface protein, Cell 11 (1977) 115–126. [2] S.K. Chintala, R. Sawaya, Z.L. Gokaslan, G. Fuller, J.S. Rao, Immunohistochemical localization of extracellular matrix proteins in human glioma, both in vivo and in vitro, Cancer Lett. 101 (1996) 107–114. [3] B.S. Christiansen, J. Ingerslev, L. Heickendorff, C.M. Petersen, Human hepatocytes in culture synthesize and secrete fibronectin, Scand. J. Clin. Lab. Invest. 48 (1988) 685–690. [4] F. Grinnell, M. Feld, D. Minter, Fibroblast adhesion to fibrinogen and fibrin substrata. Requirement for cold-insoluble globulin (plasma fibronectin), Cell 19 (1980) 517–525. [5] R.O. Hynes, K.M. Yamada, Fibronectin: multifunctional modular glycoproteins, J. Cell Biol. 95 (1982) 369–377. [6] S. Kannan, P. Balaram, G.J. Chandran, M.R. Pillai, B. Mathew, K.R. Nalinakumari, M.K. Nair, Alterations in expression of basement membrane proteins during tumor progression in oral mucosa, Histopathology 24 (1994) 531–537. [7] E. Kawahara, Y. Okada, I. Nakanishi, K. Iwata, S. Kojima, E. Yamamoto, The expression of invasive behavior of differentiated squamous carcinoma cell line evaluated by an in vitro invasion model, Jpn. J. Cancer Res. 84 (1993) 409–418. [8] H.K. Kleiman, R.J. Klebe, G.R. Martin, Role of collagenous matrices in the adhesion and growth of cell, J. Cell Biol. 88 (1981) 473–485.
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[9] J. Klominek, K.H. Robert, K.G. Sundqvist, Chemotaxis and haptotaxis of human malignant mesothelioma cells: effects of fibronectin, laminin, type IV collagen and autocrine motility factor-like substance, Cancer Res. 53 (1993) 4376–4382. [10] H. Mohri, Fibronectin and integrin interactions, J. Invest. Med. 44 (1996) 429–441. [11] G. Olt, A. Berchuck, A.P. Soisson, C.M. Boyer, R.C. Bast, Fibronectin is an immunosuppressive substance associated with epithelial ovarian cancer, Cancer 70 (1992) 2137–2142. [12] T.M. Saba, E. Jaffe, Plasma fibronectin (opsonic glycoprotein): its synthesis by endothelial cells and role in cardiopulmonary integrity after trauma as related to reticuloendothelial function, Am. J. Med. 68 (1980) 577–594. [13] H.S. Smith, J.L. Riggs, M.W. Mosesson, Production of fibronectin by human epithelial cells in culture, Cancer Res. 39 (1979) 4138–4144. [14] H. Takei, Y. Iino, J. Horiguchi, T. Yokoe, Immunohistochemical fibronectin staining pattern and prognosis in invasive breast carcinoma, Oncology 52 (1995) 106–111. [15] T. Torimura, T. Ueno, S. Inuzuka, M. Kin, H. Ohira, Y. Kimura, Y. Majima, M. Sato, H. Abe, K. Tanikawa, The extracellular matrix in hepatocellular carcinoma shows different localization patterns depending on the differentiation and the histological pattern of tumors: immunohistochemical analysis, J. Hepatol. 21 (1994) 37–46. [16] K.M. Yamada, K. Olden, Fibronectins – adhesive glycoproteins of cell surface and blood, Nature 275 (1978) 179–184. [17] K.M. Yamada, S.S. Yamada, I. Pastan, Cell surface protein partially restores morphology, adhesiveness and contact inhibition of movement to transformed fibroblasts, Proc. Natl. Acad. Sci. USA 73 (1976) 1217–1221.
Thyroid carcinoma distinctively expresses intracellular fibronectin in vivo Shinso Ryu*, Shiro Jimi, Shigeo Takebayashi The Second Department of Pathology, School of Medicine, Fukuoka University, 7-45-1 Nanakuma, Jonan-ku, Fukuoka 814-08, Japan Received 11 July 1997; received in revised form 15 July 1997; accepted 15 July 1997
Abstract Fibronectin is a multifunctional protein that plays a role in tumor invasion. We immunohistochemically examined the in vivo expression of fibronectin in thyroid carcinoma in comparison with other carcinomas, such as hepatocellular carcinoma, transitional cell carcinoma and gastric adenocarcinoma. Intracellular localization of fibronectin was found in almost all cases of thyroid carcinoma. In contrast, hepatocellular carcinoma showed a lower expression rate and the other carcinomas were all negative. These results indicate that the intracellular expression of fibronectin is not a common phenomenon in carcinoma, but rather is distinctive for thyroid carcinoma. 1997 Elsevier Science Ireland Ltd. Keywords: Fibronectin; Thyroid carcinoma; In vivo; Immunohistochemistry
1. Introduction Fibronectin is a ubiquitous glycoprotein found in the extracellular matrix and plasma [5]. It plays various biological roles in cellular adhesion, spreading, migration, oncogenic transformation, invasion and metastasis [1,4,16]. In vitro experiments have shown that suppressed fibronectin synthesis indicates a high likelihood of tumor cell invasion [17] through the liberation of adhesive glycoprotein. Fibronectin also has anchorage and stabilizing functions in cell locomotion [10] and thus acts to counteract malignancy. It has been demonstrated that fibronectin, which may be synthesized by tumor cells [7,11], is primarily loca-
* Corresponding author. Tel.: +81 92 8011011, ext. 3285; fax: +81 92 8638383; e-mail: [email protected]
lized in peripheral areas of malignant tumors including the basement membrane [2,6,9,14]. Therefore, it has been speculated that such tumors are highly likely to exhibit invasion. However, the clinical importance of fibronectin in tumor invasion has not yet been elucidated. To clarify the roles of fibronectin in malignant tumors, we examined the expression of fibronectin in thyroid carcinoma in comparison with other carcinomas by means of immunohistochemical techniques.
2. Materials and methods The specimens used in the present study were obtained from surgical or biopsy samples taken from March 1988 to June 1996 at Fukuoka University Hospital, Fukuoka, Japan. The samples were from 62
0304-3835/97/$17.00 1997 Elsevier Science Ireland Ltd. All rights reserved PII S0304-3835 (97 )0 0353-4
190
S. Ryu et al. / Cancer Letters 121 (1997) 189–193
patients (36 male, 26 female) with a mean age of 68 years (range 38–87 years), which included a total of 28 cases of thyroid carcinoma (15 cases of papillary carcinoma (six cases well differentiated, nine cases poorly differentiated), seven cases of follicular carcinoma (three cases well differentiated, four cases poorly differentiated), three cases of medullary carcinoma and three cases of undifferentiated carcinoma), 13 cases of hepatocellular carcinoma (three cases well differentiated, seven cases moderately differentiated, three cases poorly differentiated), 11 cases of transitional cell carcinoma (eight cases grade II, three cases grade III) and 10 cases of gastric adenocarcinoma (five cases well differentiated, one case moderately differentiated, four cases poorly differentiated). All of the cases of thyroid carcinoma were shown to have metastasized into other organs. In all of the patients, histological diagnosis was made according to the classification established by the World Health Organization. All samples were fixed in 10% buffered formalin and embedded in paraffin. Deparaffinized sections (3 mm thick) were washed in PBS (pH 7.4) and treated with protease (DAKO, Denmark). Immunohistochemical staining was performed by the alkaline phosphatase–antialkaline phosphatase method. In brief, the primary antibody against human plasma fibronectin (DAKO) was diluted 1:600 and incubated overnight at 4°C. After washing in PBS, the sections were incubated with alkaline phosphatase-conjugated second antibody against rabbit immunoglobulin (DAKO) for 60 min at room temperature. After washing, the sections were incubated with alkaline phosphataseconjugated third antibody against alkaline phosphatase (DAKO) for 60 min at room temperature. As a
negative control, vehicle alone or an identical concentration of non-immune animal IgG was used. To check the specificity of the first antibody, an excess amount of antigen, fibronectin (Sigma, St. Louis, MO), was mixed with the first-antibody solution and incubated for 1 h at 37°C. This solution was used as a first-antibody solution for immunohistochemistry. Thereafter, the sections were colored by 0.01% new fucshin (Merck, Germany), 0.01% NaNO2, 10 mg naphtol AS-BI phosphate (Sigma), 0.1 ml n,Ndimethyl formamide (Wako, Osaka) and 10 mg levamisol (Sigma) in 40 ml of 0.2 M Tris–HCl buffer (pH 8.2). Specimens were observed under a microscope. As a result, all of the stained materials in specimens treated with the antibody were fibronectin. Immunohistological evaluation was performed with the following criteria to classify tissue as positive: in tumor cells/normal cells adjacent to the tumor, when one or more tumor cells/normal cells was intracellularly positive; in stroma in tumor/normal tissue adjacent to the tumor, when extracellular matrix including the basement membrane in the tissue was positive; in blood vessels, when the luminal space was positive.
3. Results We examined fibronectin expression in four different types of malignant tumor, including thyroid carcinoma, hepatocellular carcinoma, transitional cell carcinoma and gastric adenocarcinoma. Since most of the samples contained a tumor lesion and adjacent normal tissue, both types of tissue were subjected to immunohistochemical analysis. Table 1 shows the
Table 1 Fibronectin in cells and stroma in different types of carcinoma Carcinoma
Thyroid carcinoma Hepatocellular carcinoma Transitional cell carcinoma Gastric adenocarcinoma
Total cases
28 13 11 10
Values represent the number of positive cases. a Samples did not contain normal tissue.
Tumor
Normal
Cell (%)
Stroma (%)
Cell (%)
Stroma (%)
27 4 0 0
28 7 1 5
0 (0) 3 (23.1) –a 0 (0)
3 (10.7) 5 (38.5) –a 1 (10.0)
(96.4) (30.7) (0) (0)
(100) (54.0) (9.0) (50.0)
S. Ryu et al. / Cancer Letters 121 (1997) 189–193
results of our immunohistochemical analysis. In thyroid carcinoma, although they showed different types of thyroid carcinoma, almost all of the cases (27/28 cases; 96.4%) showed positive staining for fibronec-
191
tin, whereas no fibronectin was found in any of the normal epithelial cells adjacent to the carcinoma (Fig. 1A). The cases of hepatocellular carcinoma showed a positive reaction in 4/13 cases (30.7%) of varied dif-
Fig. 1. Fibronectin localization in cases of (A) papillary thyroid carcinoma (54 years, female), (B) hepatocellular carcinoma (44 years, male), (C) transitional cell carcinoma (65 years, female) and (D) gastric adenocarcinoma (84 years, male). (A) The invading carcinoma is positive for fibronectin in the cells and in the stroma in some places. On the other hand, normal thyroid glands adjacent to the carcinoma possess no fibronectin. (B) The invading carcinoma is positive for fibronectin in the cells, while the tumor stroma possesses almost no fibronectin. (C) The carcinoma cells in the upper part are entirely negative for fibronectin, while the edematous extracellular matrix, including some vessels, is positive for fibronectin (lower part). (D) The adenocarcinoma cells and stroma in the upper part are entirely negative for fibronectin, while the vessel lumen is slightly positive for fibronectin (lower part). Bar, 40 mm.
192
S. Ryu et al. / Cancer Letters 121 (1997) 189–193
ferentiation and fibronectin-positive normal epithelial cells adjacent to the tumor were found in 3/13 cases (23.1%) (Fig. 1B). The cases of transitional cell carcinoma obtained by biopsy did not contain normal tissue and all of these cases showed negative staining for fibronectin (Fig. 1C). All of the cases of gastric adenocarcinoma showed a negative reaction in both tumor cells and adjacent normal epithelial cells (Fig. 1D). In contrast, all of the cases in the luminal space in blood vessels showed a positive reaction (Fig. 1C,D).
4. Discussion The present immunohistochemical study demonstrated that thyroid carcinoma is distinctive regarding the intracellular expression of fibronectin compared to the other carcinomas examined, i.e. hepatocellular carcinoma, transitional cell carcinoma and gastric adenocarcinoma, in which the expression rates of fibronectin were quite low. While fibronectin expression is not a common phenomenon in cells that have transformed into carcinoma, it is common for thyroid carcinoma regardless of the subtype. The cases of thyroid carcinoma were subclassified as papillary carcinoma, follicular carcinoma, medullary carcinoma and undifferentiated carcinoma. Their prognosis tended to vary depending upon the type of thyroid carcinoma (data not shown). However, no difference was found in any of the carcinomas in that almost all of the cases possessed fibronectin in tumor cells. A quantitative analysis for fibronectin in tumor cells of thyroid carcinoma is necessary. Fibronectin can be produced by various kinds of cells including epithelial cells [13], fibroblasts and endothelial cells and exists primarily in extracellular matrix and plasma [5], in which it is referred to as matrix protein and cold insoluble-globulin, respectively. Our results also indicated that fibronectin was found more or less in the extracellular matrix in tumor and adjacent normal tissue and in the luminal space of blood vessels. Such fibronectin may be a component of matrix protein associated with collagens [8] and plasma protein. Although the liver is the primary source of fibronectin [3], only 23.1% of the cases with normal hepatocytes were positive for fibronectin expression based
on an immunohistochemical analysis. As in other studies [15], we found fibronectin in the intercellular spaces of hepatocellular carcinoma. The positive rate for cell-associated fibronectin in this carcinoma was slightly increased (30.7%) relative to those among the other carcinomas, although it was almost the same as that in normal hepatocytes. In hepatocellular carcinoma, there was no relationship between the expression of intracellular fibronectin and the type of differentiation. Moreover, although the cases of transitional cell carcinoma and gastric adenocarcinoma showed various levels of differentiation, fibronectin was not found in the cells of either of these carcinomas. It is likely that fibronectin secretion is not a uniform characteristic of carcinoma. On the other hand, fibronectin was found in some normal gastric glands, in which severe inflammation occurred (data not shown). It has been shown that fibronectin occurs at sites of inflammation [12]. Tumor stroma not only contribute to tumor structure but also play a role in creating a foothold for tumor cells. In some carcinomas in vivo, stromal fibronectin has been identified as a characteristic of carcinoma [2,6,14]. However, stromal fibronectin in normal tissue also possesses fibronectin and cases of stromal fibronectin in tumor lesions showed slightly higher rates of fibronectin expression. However, this difference was not significant except among the cases of thyroid carcinoma. Therefore, the presence of stromal fibronectin may not be a characteristic of carcinoma in general. We found that in thyroid carcinoma, in comparison with other carcinomas, a large amount of fibronectin was accumulated in the stroma. Moreover, some of the cases of thyroid carcinoma had a large volume of stromal fibronectin. One case of papillary thyroid carcinoma (poor differentiation) failed to express intracellular fibronectin, although a large amount of fibronectin was found in its stroma. Together with the intracellular expression of fibronectin, the stroma may act as a reservoir after the secretion of fibronectin from tumor cells. Further studies are necessary to determine the relationship between stromal fibronectin and intracellular fibronectin in thyroid carcinoma cells. In conclusion, we immunohistochemically examined fibronectin expression in different carcinomas in vivo using a conventional method and found that
S. Ryu et al. / Cancer Letters 121 (1997) 189–193
this intracellular expression was distinct for thyroid carcinoma regardless of the subtype. References [1] I.U. Ali, V.M. Mautner, R.P. Lanza, R.O. Hynes, Restoration of normal morphology, adhesion and cytoskeleton in transformed cells by addition of a transformation-sensitive surface protein, Cell 11 (1977) 115–126. [2] S.K. Chintala, R. Sawaya, Z.L. Gokaslan, G. Fuller, J.S. Rao, Immunohistochemical localization of extracellular matrix proteins in human glioma, both in vivo and in vitro, Cancer Lett. 101 (1996) 107–114. [3] B.S. Christiansen, J. Ingerslev, L. Heickendorff, C.M. Petersen, Human hepatocytes in culture synthesize and secrete fibronectin, Scand. J. Clin. Lab. Invest. 48 (1988) 685–690. [4] F. Grinnell, M. Feld, D. Minter, Fibroblast adhesion to fibrinogen and fibrin substrata. Requirement for cold-insoluble globulin (plasma fibronectin), Cell 19 (1980) 517–525. [5] R.O. Hynes, K.M. Yamada, Fibronectin: multifunctional modular glycoproteins, J. Cell Biol. 95 (1982) 369–377. [6] S. Kannan, P. Balaram, G.J. Chandran, M.R. Pillai, B. Mathew, K.R. Nalinakumari, M.K. Nair, Alterations in expression of basement membrane proteins during tumor progression in oral mucosa, Histopathology 24 (1994) 531–537. [7] E. Kawahara, Y. Okada, I. Nakanishi, K. Iwata, S. Kojima, E. Yamamoto, The expression of invasive behavior of differentiated squamous carcinoma cell line evaluated by an in vitro invasion model, Jpn. J. Cancer Res. 84 (1993) 409–418. [8] H.K. Kleiman, R.J. Klebe, G.R. Martin, Role of collagenous matrices in the adhesion and growth of cell, J. Cell Biol. 88 (1981) 473–485.
193
[9] J. Klominek, K.H. Robert, K.G. Sundqvist, Chemotaxis and haptotaxis of human malignant mesothelioma cells: effects of fibronectin, laminin, type IV collagen and autocrine motility factor-like substance, Cancer Res. 53 (1993) 4376–4382. [10] H. Mohri, Fibronectin and integrin interactions, J. Invest. Med. 44 (1996) 429–441. [11] G. Olt, A. Berchuck, A.P. Soisson, C.M. Boyer, R.C. Bast, Fibronectin is an immunosuppressive substance associated with epithelial ovarian cancer, Cancer 70 (1992) 2137–2142. [12] T.M. Saba, E. Jaffe, Plasma fibronectin (opsonic glycoprotein): its synthesis by endothelial cells and role in cardiopulmonary integrity after trauma as related to reticuloendothelial function, Am. J. Med. 68 (1980) 577–594. [13] H.S. Smith, J.L. Riggs, M.W. Mosesson, Production of fibronectin by human epithelial cells in culture, Cancer Res. 39 (1979) 4138–4144. [14] H. Takei, Y. Iino, J. Horiguchi, T. Yokoe, Immunohistochemical fibronectin staining pattern and prognosis in invasive breast carcinoma, Oncology 52 (1995) 106–111. [15] T. Torimura, T. Ueno, S. Inuzuka, M. Kin, H. Ohira, Y. Kimura, Y. Majima, M. Sato, H. Abe, K. Tanikawa, The extracellular matrix in hepatocellular carcinoma shows different localization patterns depending on the differentiation and the histological pattern of tumors: immunohistochemical analysis, J. Hepatol. 21 (1994) 37–46. [16] K.M. Yamada, K. Olden, Fibronectins – adhesive glycoproteins of cell surface and blood, Nature 275 (1978) 179–184. [17] K.M. Yamada, S.S. Yamada, I. Pastan, Cell surface protein partially restores morphology, adhesiveness and contact inhibition of movement to transformed fibroblasts, Proc. Natl. Acad. Sci. USA 73 (1976) 1217–1221.