European Journal of Endocrinology (1998) 138 104–112

Integrins in thyroid tissue: upregulation of a2b1 in anaplastic thyroid carcinoma

The´re`se Dahlman, Lars Grimelius1,Go¨ran Wallin2, Kristofer Rubin3 and Kerstin Westermark Departments of Internal Medicine and 1Pathology, University Hospital, S-751 85 Uppsala, Sweden, 2Department of Surgery, Karolinska Hospital, S-104 01 Stockholm, Sweden and 3Department of Medical and Physiological Chemistry, Biomedical Center, S-751 23 Uppsala, Sweden (Correspondence should be addressed to K Westermark, Department of Internal Medicine, University Hospital, S-751 85 Uppsala, Sweden)

Abstract Objective: To evaluate the integrin pattern in the normal thyroid gland and in different pathological disorders including malignant tumors, because the aggressiveness of several malignant tumors correlates with alterations in the expression of one or more integrins. Design: We examined the expression of integrins and E-cadherin immunohistochemically in a large and well-defined sample of normal and pathological human thyroid tissue. Methods: Cryosections of 58 thyroid tissue specimens from normal tissue, thyrotoxicosis, nodular goiter, oxyphilic adenoma, follicular adenoma, follicular carcinoma, papillary carcinoma and ana- plastic carcinoma, and three lymph node metastases were investigated immunohistochemically using monoclonal antibodies specifically recognizing the integrin b1-, b4-, a1-, a2-, a3-, a5- and a6-subunits, or E-cadherin. Results: All thyroid epithelial cells expressed integrin b1- and a3-subunits. Immunostaining of the b4- subunit and the a6-subunits was found only in tumors. The staining pattern in the three lymph node metastases from papillary carcinomas did not differ from that in their primaries. Anaplastic carcinomas demonstrated neoexpression of the integrin a2-subunit. E-cadherin was detected in all tissues except anaplastic carcinomas. Conclusions: Neoexpression of a6b4 was seen in most malignant tumors, whereas a2 was exclusively found in anaplastic carcinomas. In the latter, a loss of E-cadherin expression was also seen. These changes in cell adhesion molecule expression strongly suggest an association with the acquisition of proliferative and invasive properties.

European Journal of Endocrinology 138 104–112

decreases in the a2- and a3- or the a6- and b4- Introduction subunits, or in all of them, have been consistently noted To date, four major families of adhesion receptors have in certain kinds of tumors (14–29). been identified: the immunoglobulin superfamiliy (1, 2), Cadherins are Ca2þ-dependent homophilic adhe- the cadherins (3, 4), the integrins (5–10) and the sion receptors mediating cell–cell adhesion. The selectins (11–13). Adhesion receptors have vital roles in expression of cadherins, as detected by immunohisto- epithelial morphogenesis, in the maintenance of epithe- chemistry, is reduced in many invasive carcinomas lial integrity and in epithelial cell migration. Changes in (30–32). expression and function of these receptors occur during Thyroid neoplasms account for about 1% of all development and during progression of various cancers. malignant tumors. Papillary carcinomas are the most Integrins constitute a large family of adhesion common, comprising 50–70% of all thyroid malignan- receptors that mediate both cell–cell and cell–matrix cies, followed by follicular carcinoma (10–15%) and adhesion. Structurally, integrins are heterodimeric anaplastic carcinoma (10%); medullary thyroid carci- transmembrane glycoproteins composed of non-cova- nomas, which originate from parafollicular cells, lently associated a- and b-subunits. At least eight b- give rise to only 1–2% of thyroid malignant tumors. subunits and 16 a-subunits have been isolated, these Whereas papillary cancer grows slowly and spreads via associate in various combinations to form the 20 or so the intraglandular lymphatic system, follicular carcino- different integrins that have been described to date. mas metastasize hematogeneously. Anaplastic carcino- Available data suggest that all nucleated cells express mas represent the most malignant tumor type, with one or more integrins. The aggressiveness of several rapid invasion of surrounding organs and tissues. The malignant tumors correlates with alterations in the patient generally succumbs to the disease within a few expression of one or more integrins. In particular, months after diagnosis (33).

᭧ 1998 Society of the European Journal of Endocrinology

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In this study, we have examined the distribution of from normal glandular tissue adjacent to adenomas E-cadherin and integrin b1, b4, a1–3, a5 and a6 (three) or papillary carcinomas (four). Seven specimens chains in a series of well-defined normal and abnormal showed thyrotoxicosis, seven nodular goiter, four human thyroid glands and lymph node metastases from oxyphilic adenoma, six follicular adenoma, seven papillary carcinoma. By using immunohistochemistry, follicular carinoma, ten papillary carcinoma and four it was possible to distinguish the pattern of expression of anaplastic carcinoma. integrins by the epithelial tumor cells from that of the surrounding vessels. Immunohistochemistry Cryosections 6 mm thick were fixed in ice-cold acetone Materials and methods for 10 min and then stained with hematoxylin–eosin to confirm that they were representative of the histopatho- Antibodies logical diagnosis or immunostained to investigate Monoclonal antibodies (mAbs) against the integrin b1, expression of integrin subunits or E-cadherin. The a1–3, a5 and a6 chains were kindly donated as follows: peroxidase–anti-peroxidase method was used for P4C10, anti-b1 by Dr Kurt Gehlsen (San Diego, CA, immunostaining with 3-amino-9 ethylcarbazole as USA); TS2/7, anti-a1 by Dr Timothy Springer (Boston, chromogen, followed by a short counterstaining in MA, USA); PIH5, anti-a2, PIB5, anti-a3 and PID6, anti- hematoxylin. In addition, immunostaining with anti- a5 by Dr William Carter (Seattle, WA, USA); GOH3, bodies to laminin was used to demonstrate basement anti-a6 by Dr Arnold Sonnenberg (Utrecht, Holland). membranes, and antibodies to PAL-E and Factor VIII All antibodies were of mouse origin except for the were used to get an overview of the distribution of blood rat mAb, GOH3. Antibodies were purchased from vessels. The cryosections were pretreated with 0.3% Telios, San Diego, CA, USA (3E1, anti-b4), Beckton hydrogen peroxide in phosphate buffer for 15 min and Dickinson, San Jose´, CA, USA (PIE6, anti-a2), British then incubated in normal rabbit serum (for mAbs) or Biotechnology Products Ltd, Abingdon, UK (HECD-1, normal swine serum (for polyclonal antibodies, pAbs) anti-E-cadherin), Sigma Immunochemicals, St Louis, for 5 min at room temperature. The primary mouse MO, USA (L-9393, anti-laminin), Sanbio, Amuden, mAbs were applied overnight at þ4 ЊC. After washing in Holland (6001–1, anti-Pal-E) and Dakopatts, Glostrup, PBS, rabbit anti-mouse conjugate (for mAbs) or swine Denmark (61601, anti-vWF). An additional anti-b1 anti-rabbit conjugate (for pAbs) was applied for 30 min antibody (mAb 13) purchased from Beckton Dickinson at room temperature, followed by peroxidase–anti- was used in double immunofluorescence staining peroxidase conjugate (Dakopatts) for 30 min at room together with the anti-a2 antibody, PIE6 (see above), temperature. Every tumor was stained several times with to demonstrate co-localisation of b1 and a2in the same antibodies, and showed the same results. anaplastic carcinoma. All antibodies were mouse Negative controls were obtained by omitting the primary monoclonals except for the rabbit polyclonal L-9393. antibody or replacing it with non-immune sera. Sections Because one of the main findings in this study was the from human skin and colon were used as positive expression of the integrin a2-subunit in anaplastic controls. carcinoma but not in any other thyroid tissue investi- gated, we chose to use two different anti-a2 antibodies to ensure that our data were valid. Immunofluorescence Double immunofluorescence staining against the b1- and a2-subunits was performed with a technique that Human tissue samples enables the sequential use of two mouse mAbs as Tissue specimens from 58 normal and abnormal glands primary antibodies. After being blocked with avidin and and three lymph node metastases from papillary biotin for 15 min each, sections were incubated with carcinomas from a total of 52 patients were included normal horse serum for 5 min. Cryosections were then in the present study. All the specimens were obtained at incubated with the first primary antibody (PIE6, anti- surgery at the Karolinska Hospital, Stockholm, Sweden. a2) for 30 min, rinsed and incubated with biotinylated The tissue specimens were fixed immediately in 10% horse anti-mouse IgG diluted 1 : 200 for 30 min. buffered formalin for 18–24 h or snap frozen in liquid Sections were rinsed and incubated with Texas red nitrogen and stored at ¹70 ЊC until required for avidin diluted 1 : 100 for 30 min, rinsed again and staining. Paraffin sections 4 mm thick and cryosections blocked with normal rabbit serum for 5 min. The 6 mm thick were cut and attached to glass slides. sections were then incubated with the second primary Routine histopathological diagnosis was performed in antibody (mAb 13, anti-b1) for 30 min, rinsed and hematoxylin–eosin stained deparaffinized sections incubated with rabbit anti-mouse IgG fluorescein according to the criteria of the WHO histological isothiocyanate (FITC) conjugate diluted 1 : 20 for classification of thyroid tumors (34); 13 specimens 30 min. The mounting medium was fluoromount. showed normal histology; seven of these were taken The same technique was used for staining against the

Downloaded from Bioscientifica.com at 09/30/2021 08:02:55PM via free access 106 T Dahlman and others EUROPEAN JOURNAL OF ENDOCRINOLOGY (1998) 138 b4- and a6-subunits. The antibody against the a6- After washing in PBS, the secondary rabbit anti-mouse subunit (GOH3) was used as the first primary antibody FITC-conjugated antibody was applied for 30 min and and the antibody 3E1 (anti-b4) was used as the second the sections washed quickly in PBS. The procedure was primary antibody. In this case the sections were then then repeated, but using normal swine serum, another investigated with an image analyzer in order to establish primary anibody (L-9393, anti-laminin) and a swine whether b4 and a6 were co-localized. anti-rabbit rhodamine-conjugated secondary antibody. Double immunofluorescence used one mouse mAb Sections were examined by Zeiss 410 confocal laser and one rabbit pAb as primary antibodies. The cryosec- scanning microscopy and microphotographs were taken tions (6 mm) were incubated in normal rabbit serum in RGB-mode, simultaneously showing laminin in the for 10 min at room temperature. The first primary red channel, b4 in the green channel and co-localized antibody (3E1, anti-b4) was then applied for 60 min. signals as white.

Table 1 Expression of b1, b4, a2, a3, a6 and E-cadherin (Ec) in human thyroid epithelium. No expression of a1 or a5 was seen in any of the patients.

Diagnosis Patient b1 b4 a2† a3 a6 Ec

N 1–12 þþ ––þþþ – þþþ 13 þþþ ––þþþ – þþþ TT 14 þþþ ––þþþ – þþþ 15–17 þ ––þþþ – þþþ 18–20 þþ ––þþþ – þþþ NG 21 þþ ––þþ – þþþ 22–23 þþ ––þþþ – þþþ 24–25 þþþ ––þþþ – þþþ 26–27 þþ=þ ––þþþ – þþþ OA 28 þþþ=þ ––þþ – þþþ=þ 29 þþþ=þ ––þþ – þþþ=¹ 30 þþþ=þ ––þ –þþ=¹ 31 þ ––þþ – þþ=þ FA 32, 36, 37 þþ ––þþ – þþþ 33 þþþ ––þ –þþþ 34 þþþ ––þþ – þþ 35 þþ ––þþþ – þþþ=þþ FC 38 þ=¹ ––þþþ – þþþ 39 þ ––þþ – þþ=þ 40 – þþ=¹ – þþþ –* þþþ 41 – – – þþþ – þþþ 42 þþþþ=¹–þþþ –* þþþ=þþ 43 þ=¹þþþ=¹– þþþ –* þþþ 44 þþþ=¹–þþ –* þþþ PC 45, 48–50 þ=¹þþ=¹– þþþ –* þþþ 46 0 – – þþ – þþþ 47 þ=¹ ––þþ – þþþ 51 þþþ=¹–þþþ –* þþþ 52 þþ=þþþþ=¹– þþþ –* þþþ 52 met þþ=þþþþ=¹– þþþ –* þþþ 53 þ=¹þþ=¹– þþþ –* þþþ 53 met þ=¹þþ=¹– þþþ –* þþþ 54 þ=¹þþ=¹– þþþ –* þþþ 54 met þþ=þþþ=¹– þþþ –* þþþ AC 55 0 þþ(þ)ðþÞ=¹ – 56 þþþ þþ=þ þþ þþþ þ=ðþÞ ðþÞ=¹ 57 þ ðþÞ þ – þ=¹ – 58 þ ðþÞ=¹þþ=þþþ=¹¹ þþ=¹ –

† The anti-a2-antibody PIH5 was routinely used and the results are shown here. * a6 immmunostaining co-localized with the b4-subunit could be detected with double immunofluorescence staining and examination by image analyzer. Immunostaining scored as follows: ðþÞ very weak, þ weak, þþ moderate, þþþstrong. þþþ=þ, staining varied within the section from þþþto þ; —, no immunostaining; 0, section not representative. N, normal histology; TT, thyrotoxicosis; NG, nodular goiter; OA, oxyphilic adenoma; FA, follicular adenoma; FC, follicular carcinoma; PC, papillary carcinoma; AC, anaplastic carcinoma. met, metastasis.

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Results follicular arrangement, but was less strong in more solid areas. In the differentiated carcinomas, immuno- Expression of integrins in human thyroid staining appeared focally. Tumor cells from anaplastic follicular cells carcinomas showed immunoreactive cytoplasm. Positive b4 immunostaining was present only in The epithelial cells in all thyroid tissue specimens tumors and was localized basally in the neoplastic cells, examined, with a few exceptions, displayed a basally for example against the fibrovascular stroma (Fig. 1b–d). localized b1 immunostaining (Table 1). In normal thyroid This immunoreactivity, however, did not show a linear glands and in benign lesions, the immunostaining for the distribution and was often interrupted. In the anaplastic b1 integrin was, in most cases, stronger in columnar carcinomas, the immunostaining was seen along the than in cuboidal cells. In adenomas, the immunostaining border of the tumor cells and also in the cytoplasm of was predominantly seen in areas with preserved some neoplastic cells (Fig. 1d). In normal, hyperfunctioning and goitrous glands, a basal a3 immunostaining of the epithelial cells was seen. In columnar epithelium from hyperplastic and goitrous glands, additional immunostaining between the cells was detected. Adenomas and differentiated tumors also displayed immunoreactivity at their lateral (intercellular) cell border. In anaplastic carcinomas, the immunostaining appeared around the tumor cells. Immunostaining of the a2-subunit was seen exclu- sively in anaplastic carcinoma (Fig. 1g) and was detected with two different anti-a2 antibodies (PIH5 and PIE6). The anti-a2 antibody, PIH5, was routinely used (Table 1), whereas PIE6 was used on two representative sections from each diagnostic group. Both antibodies gave the same result: positive immu- nostaining could be detected only in tumor cells from anaplastic carcinoma. Immunostaining with PIE6 gave a somewhat stronger immunostaining than PIH5. The a2 immunostaining was seen between the cells, whereas the a6 immunostaining appeared along the tumor cell surfaces facing the matrix; however, some tumor cells also showed cytoplasmatic staining. The integrin a1- and a5-subunits were not detected immunohistochemically in epithelia of any of the thyroid tissues investigated. E-cadherin displayed a strong staining of the lateral cell borders in all tissues examined, except in anaplastic carcinomas, in which the staining was weak and in some specimens absent. In tissues from oxyphilic

Figure 1 Immunostaining with antibody against the integrin b4- subunit (a–d), against the integrin a2-subunit (e–g) and double immunofluorescence staining of laminin and the integrin b4-subunit (h). Strong b4-immunostaining is seen in capillaries of the normal thyroid gland (a); bar represents 80 mm. Papillary carcinoma (b) and lymph node metastases (c) from the same patient (No. 54) shows a strong positive immunostaining basally and in capillaries. In anaplastic carcinoma (d), positive staining is seen mainly at the border of tumor cells facing the matrix; bar represents 40 mm. Some tumor cells also show cytoplasmatic staining. Immunostaining with antibody against the integrin a2-subunit (PIE6), showing strong immunostaining in capillaries of normal thyroid gland (e) and papillary carcinoma (f). In anaplastic carcinoma (g), there is an additional strong cytoplasmic staining of tumor cells. Confocal laser microscopy of anaplastic carcinoma (h) double stained with the indirect immunofluorescence technique using antibodies against the integrin b4-subunit (pseudocolored green) and laminin (pseudocolored red). The immunostaining of both antibodies is Figure 2 Double immunofluorescence staining against the a2- colocalized (white) at the border of the tumor cells facing the matrix. subunit (a) and the b1-subunit (b) in anaplastic carcinoma, showing Bar represents 20 mm. strong co-localisation. Bar represents 15 mm.

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Table 2 Integrin expression in capillaries of interlobular (a) and interfollicular (b) types.

a2

Diagnosis b1 b4 a1 PIH5 PIE6 a3 a5 a6

N a þþþ þþþ þþþ=þþ – þþ þþþ –– b þþþ=þþþþ=þþ þþþ=þþ – þþ ¹¹ – þþ=ðþÞ TT a þþþ þþþ þþþ=þþ – þþ þþþ –– b þþ=¹þþþ=¹þþþ=þþ þ=¹þþ––þþ NG a þþþ þþþ=þþ þþþ=þþ=¹ þþ þþþ=þþ –– b þþþ=þþþ=þþþþ=þ ðþÞ=¹þþ––þþ=ðþÞ OA þþþ=¹þþþ=þþþþ=þþ=¹ þþþ þþþ=¹ – þþþ=¹ FA þþþ=þþþþ=¹þþþ=þ ðþÞ=¹ þþþ þþþ=¹ – þþþ=¹ FC þþþ=þþþþ=¹þþþ=ðþÞ þ=¹ þþþ þþþ=¹ – þþþ=¹ PC Tumor þþ=¹þþþ=¹þþþ=ðþÞ þ þ þ=¹ þþþ þþþ=¹þ=¹ þþþ=¹ Metastasis þþ þþþ þþþ þþ=¹ þþþ þþþ þ=¹ þþþ=þ AC þþ þ þ þ=¹þþþ=þþþ=¹ þþþ þþ=¹ – þ=¹

For scoring and explanation of abbreviations, see Table 1. adenomas, the immunostaining showed a similar staining pattern for the neoexpressed integrin a2- pattern, but varied from very strong to negative. subunit in anaplastic tumour cells closely resembled The three lymph node metastases investigated that of the integrin b1-subunit, suggesting that these displayed the same pattern of staining as the primary cells express a2b1. tumor of the papillary carcinomas (Fig. 1b,c). With the use of double immunofluorescence stain- ing, together with image analysis, it was evident that the basal staining of 4 found in some of the dif- Human thyroid tissue capillaries b ferentiated carcinomas was co-localized with a6 (data The results are summarized in Table 2. not shown). Immunostaining with b1-, b4- and a1-subunits was Confocal laser scanning microscopy was used to seen both in small capillaries localized between the analyze anaplastic carcinoma double stained with follicles and in large capillaries in the interlobular septa antibodies against the integrin b4-subunit and laminin, (Fig. 1a–c). In addition, positive staining for a3- and using the indirect immunofluorescence technique. a6-subunits was seen in capillaries of the interlobular Immunostaining with the anti-b4 antibody (pseudoco- and interfollicular types respectively. lored green) was partly co-localized with staining from A weak, but focal staining of the a2-subunit (PIH5) the anti-laminin antibody (pseudocolored red) at the was seen in some abnormal glands of all diagnostic border between tumor cells and the surrounding categories. In all normal, and in the majority of the matrix; this co-localization gave a white signal (Fig. 1h). abnormal glands, a2 immunostaining with the anti-a2 antibody, PIH5, was lacking. The other anti-a2 anti- body, PIE6, however, gave moderate to strong staining Discussion in capillaries throughout (Fig. 1e,f). The availability of specific monoclonal antibodies The immunostaining pattern of vessels in three against individual integrin subunits has allowed inves- lymph node metastases from papillary carcinomas did tigation of the localization of integrins in tissues and not differ from that of the corresponding primary tumor, changes in their expression during development and in except for a greater variation in the staining intensity in specific disease conditions (14–21, 34–46). We have the primary tumors (Fig. 1b,c). In vessels of the now presented data on integrin expression in a large metastases, positive staining for a2 seemed to be more and well-defined sample of normal and diseased human frequent compared with that in the primary tumor. thyroid glands. In agreement with what has been reported for most epithelial cells, we found that thyroid follicle cells expressed the integrin 1- and 3-subunits. Immunofluorescence b a Among thyroid follicle cells studied previously using Using double immunofluorescence staining, we were flow cytometry, subpopulations of cells were found also able to demonstrate co-localisation of the integrin b1- to express a1-, a5- and a6-subunits (47). By using and a2-subunits in anaplastic carcinoma (Fig. 2). The immunohistochemistry, we could clearly distinguish

Downloaded from Bioscientifica.com at 09/30/2021 08:02:55PM via free access EUROPEAN JOURNAL OF ENDOCRINOLOGY (1998) 138 Integrins and E-cadherin in the thyroid gland 109 those integrins expressed by normal and tumor cells and less invasive type of cell (56). Expression of a2b1is from those seen in the surrounding vessels, and found reduced or lost in most invasive (Duke’s stage C/D) that the a1- and a6-subunits were expressed by the human colon carcinomas (14, 17, 18, 25). Furthermore, capillaries. One major finding of our study was that the in colon carcinoma, the loss of continuity of a2b1and integrin a2-subunit was detected only in tumor cells a3b1 expression at the basolateral surface of tumor in anaplastic carcinoma and not in follicle or tumor glands correlates with tumor malignancy (42). In the cells of any of the other thyroid tissues investigated. present study, we have demostrated that expression of However, a2 was expressed in capillaries in most tissues a2b1 in thyroid carcinoma correlated positively with investigated. We also detected an upregulation of the malignancy. Papillary and follicular carcinomas grow integrin a2-subunit in capillaries of adenomas and unencapsulated in papillary structures of columnar carcinomas. The fact that, in contrast to other types of epithelial cells or encapsulated in follicle arrangements carcinomas, tumor cells in anaplastic thyroid carcino- respectively. Anaplastic thyroid cancers, however, display mas display an upregulation of the integrin a2b1, a totally dedifferentiated growth pattern, extending suggests that this integrin has diverse functions that are widely into the gland and its surrounding tissues and of importance for the biology of a highly malignant forming distant metastases at an early stage of the tumor. In contrast to the present findings, it has disease. The expression of a2b1 by tumor cells in all previously been reported (48) that the integrin a2- four investigated cases of anaplastic carcinomas is thus subunit was expressed by tumor cells in both adenomas in closer agreement with those experimental studies and carcinomas of the thyroid gland. It could be that showed that this integrin would favour tumor speculated that part of the a2 signal recorded in invasiveness (51–54). the previous studies (47, 48) reflected the presence of The a6b4 integrin is selectively present in hemi- a2-positive vascular cells, and not glandular epithelial desmosomes of several epithelia, where it anchors the cells. In the present study we used an immunohisto- intermediate filament system to the basement mem- chemical staining technique with two different integrin brane (57–60). To our knowledge, there are no reports anti-a2 antibodies. Both of these detected a2in on hemidesmosomes in epithelia of the normal thyroid epidermis of normal skin and in colonic epithelium gland, and the present data support this, as a6b4 (data not shown). Serini et al. (49) reported a lack of a2 integrin was not detected in acinar epithelia. However, expression in thyroid tissue, but they used an a2 a6b4 was detected in capillaries of the interfollicular antibody distinct from those we used and did not report type (Table 2) and in epidermis and in colonic the use of positive controls. Neoexpression of the integrin epithelium (data not shown), using the same reagents a2-subunit in normal thyrocytes cultured as mono- and staining methods. Apart from being expressed in layers has also been reported (50) and, furthermore, blood vessels, the a6- and b4-subunits were also the increase in a2 preceded cell proliferation and its detected in most of the well-differentiated tumors, and expression was downregulated when cell cultures in all the anaplastic carcinomas. The neoexpression of reached confluence, that is, when cell–cell contacts a6b4 in most of the carcinomas investigated is had re-established. Thus it seems reasonable to suggest consistent with previously published data on integrin that the integrin a2b1 is upregulated on thyrocytes expression in thyroid tumors (49). The biological when these cells proliferate and/or migrate. Such a significance of the observed expression of a6- and b4- hypothesis seems likely in the light of the upregulation of subunits remains unknown. In many carcinomas, such a2 in anaplastic thyroid carcinoma that we have as oral squamous carcinoma (29, 61) and basal cell reported here. carcinoma of the skin (62–64), and in prostate Several studies have indicated that tumor cell variants carcinoma (65, 66) expression of a6b4 is reduced or expressing the collagen–laminin receptor a2b1 are more lost, whereas it is enhanced in the neoplastic progres- tumorigenic, invasive or metastatic than cell variants sion of cervical epithelia (67) and squamous cell that do not express this integrin (51–54). This is carcinoma of the skin (62–64). In some types of exemplified by melanoma cells, in which the upregula- tumor, a decrease in expression and polarization of tion of a2b1 coincides with more aggressive behavior a6b4 coincides with a decrease in basement membrane and the ability to reorganize collagen matrices (55). In components, as has been observed in carcinoma of the contrast, decreases in the a2- and a3- or a6- and b4- breast (22) and bladder (27), and oral squamous cell subunits, or all of them, have been consistently noted in carcinoma (29). The distribution of both integrin a6- many types of carcinomas. This pattern is exemplified in and b4-subunits at the margins of tumor cell masses in carcinoma of the breast (15, 19, 22–24), lung (16), anaplastic carcinomas demonstrated in the present colon (14, 17, 18, 25), kidney (20, 21), pancreas (26), study suggests the presence of basement membrane bladder (27), basal cell carcinoma of the skin (28) and components at these sites. Using double immunofluo- oral squamous cell carcinoma (29). Re-expression of the rescence staining, we were able to demonstrate at these a2b1 integrin in a poorly differentiated mammary sites a presence of laminin that coincided mainly with carcinoma resulted in an alteration in phenotype from the integrin b4-subunit in anaplastic carcinomas. It is a fibroblastoid, invasive cell to an epithelioid, less motile noteworthy that anaplastic carcinoma, being the most

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