TECHNICAL NOTE |
Correspondence to: Helen Liapis, Lauren V. Ackerman Lab. of Surgical Pathology at Washington U., Barnes Hospital, One Barnes Hospital Plaza, Suite 300B, Peters Bldg., St Louis, MO 63110.
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Summary |
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Integrins are heterodimeric transmembrane receptors, which are expressed in many cells. In vitro experiments have demonstrated that integrins may be important in tumor progression and organ development. The functions of integrins were previously studied in cell cultures and their tissue expression was detected by immunofluorescence or immunoperoxidase in frozen sections. The purpose of this study was to determine the optimal conditions for detection of integrins in formalin-fixed, paraffin-embedded tissues. We utilized microwave heating and enzyme digestion in routinely processed, surgically removed tissues. Our results demonstrate that integrins can be reliably detected in archival material. This approach will facilitate further investigation of the role played by integrins in human malignancies and in developmental processes. (J Histochem Cytochem 45:737-741, 1997)
Key Words: integrins, immunohistochemistry, formalin fixation
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Introduction |
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Integrins are heterodiameric transmembrane glycoproteins found at specialized cell-cell or cell-extracellular matrix (ECM) sites of contact and/or within hemidesmosomes ( and one ß, which variously combine to form functional receptors with distinct ligand binding specificity (
- and at least nine ß-subunits, and the heterodimers they form can be grouped into subfamilies on the basis of the particular ß-subunit present. The ß-subunit of the integrins interacts with the actin cytoskeleton through several intermediary molecules, including
-actinin, vinculin, and talin (
-subunit. For example the
2ß1 integrin is a collagen, laminin, and fibronectin receptor,
5ß1 is a fibronectin receptor, and
6ß1 is a laminin receptor. Some integrins function as receptors for multiple ligands, i.e., the
vß3 complex binds to vitronectin, fibronectin, thrombospondin, osteopontin, laminin, and von Willebrand factor. There is now evidence that integrins expressed in diverse cell types can display different ligand specificities. In addition, during dynamic and complex processes, such as organ development and tumor progression and metastases, the cellular distribution and/or intensity of integrin expression may change (
3,
6ß4, and
2ß1 participate in normal mammary development (
vß3, are markedly increased during breast cancer growth and invasion (
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Materials and Methods |
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Antibodies
We have used monoclonal or polyclonal antibodies (Abs) to integrins to include Abs to common ß1-chain, several -integrins, ß5, and the
vß3 complex. Abs identifying integrins that bind primarily basement membrane components (
2, ß1,
3,
6) or primarily extracellular matrix proteins (
vß3, ß5) were assayed. These Abs were chosen because in vitro experiments have suggested that integrins with basement membrane or matrix binding specifies are involved in cancer cell invasion and metastases. The integrin specificity and sources of Abs are shown in Table 1.
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Tissues
Surgically removed specimens accessioned in our pathology laboratory were used. Non-neoplastic breast (n = 4), normal kidney (n = 4), bone marrow with prominent osteoclasts (n = 3), and ovarian adenocarcinoma of serous papillary type (of various grades) (n = 4) were selected on the basis of literature-documented integrin expression in these tissues and our own experience, as shown in Table 1. Fresh samples were divided so that one half was frozen and the remaining half was fixed overnight in 10% buffered formalin. The maximal dimensions of tissue submitted for paraffin blocks were approximately 1 x 1.5 cm. No attempt was made to compare various fixation times. Frozen tissue sections were fixed in cold acetone for 10-20 min. Bone biopsy specimens were decalcified in EDTA or hydrochloric acid for 4-6 hr before overnight fixation formalin. Sections were cut at 4 µm, mounted on Microprobe slides (Fisher #15-188-52; Pittsburgh, PA), or lysine-coated slides dried overnight at 37C.
Immunohistochemistry on Formalin-fixed Tissues
All Abs were first tested and titrated in frozen sections. Subsequently, the dilution with the best signal-to-noise ratio was selected and adapted for the formalin-fixed, paraffin-embedded sections from the same tissue. For each experimental slide and for each Ab, a negative control was included in which the primary Ab was omitted during the assay. Immunohistochemistry on frozen sections was performed as previously described (
Slides deparaffinized in xylene and hydrated through 100% and 95% alcohol were placed in a solution of methanol containing 30% hydrogen peroxide in equal volumes (50:50) for 20 min at room temperature (RT) to block endogenous peroxidase. Slides were then thoroughly rinsed in distilled water and then digested with ficin [1:50 dilution of the 2 x suspension preparation from Sigma Chemical (St Louis, MO)] for 20 min at 37C, or slides were placed in Coplin jars filled with citrate buffer (0.01 M, pH 6). Coplin jars were capped with a vented plastic lid and microwaved (Kenmore microwave) for antigen retrieval on full power twice for 3.5-5 min, followed by 20-min incubation each time at RT (
All experiments were preferably performed on the Microprobe. Positive results were consistent with MW heating when the automated system was used, whereas manual assays at RT were less often successful.
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Results |
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More than half of the Abs we used (8/14) successfully detected integrins in formalin-fixed, paraffin-embedded tissue (Table 1). With the exception of three Abs (Ab33, GoH3, and DH12), integrin detection was achieved only through MW heating before incubation with the primary antibody. GoH3, Ab 33, and DH12 Abs gave equally good detection signal with ficin compared to MW heating. Combination of both ficin and MW treatment did not improve signal detection with any of the Abs used in this study. Experiments without MW or enzyme digestion carried out at RT for 2 hr or overnight at 4C were not successful. Doubling the MW cycles (total of four 3.5-5 min heating cycles) did not prove helpful.
Examples of positive reactions are shown in Figure 1A through F. GoH3 ad (Figure 1A) to laminin receptor (6) strongly stained myoepithelial cells in mammary ducts. Immunoreactivity in side-by-side frozen breast tissue preparations was of equal intensity, as shown in Figure 1B. Stromal vessels demonstrated staining within their thin walls in both the frozen and formalin-fixed tissue (arrows in Figure 1A and Figure 1B). Our results are as previously reported for
6 integrin localization in mammary glands (
vß3 complex showed a plasma membrane type of immunoreactivity within osteoclasts (Figure 1C).
vß3 integrin was previously detected in vitro by immunofluorescence on osteoclasts at the sealing zone of the osteoclast attachment apparatus to bone, and a role for the
vß3 integrin in bone resorption has been suggested (
vß3 reactivity observed in EDTA decalcified sections compared to acid decalcification. However, the difference, in our opinion, was insignificant. We recently demonstrated abundant ß3 mRNA by in situ hybridization in T545-positive osteoclasts of breast cancer metastases to bone (
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Ab33 Ab to fibronectin receptor showed strong immunoreactivity within cells of papillary serous ovarian adenocarcinoma (Figure 1E). The fibronectin receptor, when genetically overexpressed in Chinese Hamster ovary (CHO) cells, inhibits tumor growth in nude mice (
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Discussion |
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Formalin fixation has long been known to potentially induce "masking" of antigenic epitopes in tissues. Enzyme treatments to digest aldehyde bonds induced by formalin fixation or, more recently, MW heating is used for detection of a wide range of antigens (v, ß3, and
5ß1. As a result, studies of integrin expression in human tissues are limited to frozen material. Even though the exact mechanisms through which microwave heating works are still under investigation (
The practical aspects of tissue availability, as well as the image superiority of immunohistochemistry or formalin-fixed tissue vs immunofluorescence on frozen sections, cannot be overemphasized. The described modifications provide the potential to study integrin receptors in many common as well as rare neoplasms that might be available only as formalin-fixed, paraffin-embedded blocks.
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Acknowledgments |
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We wish to thank all of the investigators who provided the Abs to integrins listed in Table 1.
Received for publication August 21, 1996; accepted December 23, 1996.
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Literature Cited |
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Albelda SM (1993) Role of integrins and other cell adhesions molecules in tumor progression and metastasis. Lab Invest 68:4-17[Medline]
Alsbeh R, Battifora H (1995) Microwave antigen unmasking: which buffer solution should we use? Lab Invest 72:948A
Berdichevsky F, Alford D, D'Souza B, Taylor-Papadimitriou J (1994) Branching morphogenesis of human mammary epithelial cells in collagen gels. J Cell Sci 10:3557-3568
Brooks PC, Stromblad S, Klemke R, Visscher D, Sarkar FH, Cheresh DA (1995) Antiintegrin vß3 blocks human breast cancer growth and angiogenesis in human skin. J Clin Invest 96:1815-1822[Medline]
Cattoretti G, Pileri S, Parravicini C, Becker MHG, Poggi S, Bifulco C, Key G, D'Amato L, Sabattini E, Feudale E, Reynolds F, Gerdes J, Rilke F (1993) Antigen unmasking on formalin-fixed, paraffin-embedded tissue sections. J Pathol 171:83-98[Medline]
Damsky CH (1995) Starve a tumor: therapeutic advice for breast cancer? J Clin Invest 96:1996-1997
Giancotti FG, Mainiero F (1994) Integrin-mediated adhesion and signaling in tumorigenesis. Biochim Biophys Acta 1198:47-64[Medline]
Giancotti F, Ruoslahti E (1990) Elevated levels of 5ß1 fibronectin receptor suppress the transformed phenotype in CHO cells. Cell 60:849-859[Medline]
Gladson CL, Hancock S, Arnold MM, Faye-Petersen OM, Castleberry RP, Kelly DR (1996) Stage-specific expression of integrin vß3 in neuroblastic tumors. J Pathol 148:1423-1434
Grossfeld GD, Shi S-R, Ginsberg DA, Rich KA, Skinner DG, Taylor CR, Cote RJ (1996) Immunohistochemical detection of thrombospondin-1 in formalin-fixed, paraffin-embedded tissue. J Histochem Cytochem 44:761-766
Horton MA, Davies J (1989) Perspective: adhesion receptors in bone. J Bone Miner Res 4:803-808[Medline]
Hynes RO (1992) Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69:11-25[Medline]
Korhonen M, Ylanne J, Laitinen L, Virtanen (1990) Distribution of ß1 and ß3 integrins in human fetal and adult kidney. Lab Invest 62:616-625[Medline]
Leong ASY, Milios J (1993) An assessment of the efficacy of the microwave antigen-retrieval procedure on a range of tissue antigens. Appl Immunohistochem 1:266-274
Liapis H, Adler LM, Wick MR, Rader JS Expression of vß3 integrin in ovarian epithelial tumors of low malignant potential is less frequent in contrast to ovarian carcinomas. Hum Pathol, in press
Liapis H, Flath A, Kitazawa S (1996) Integrin vß3 expression by bone-residing breast cancer metastases. Diagn Mol Pathol 5:127-135[Medline]
Maemura M, Akiyama SK, Woods VL, Jr, Dickson RB (1995) Expression and ligand binding of 2ß1 integrin on breast carcinoma cells. Clin Exp Metastasis 13:223-235[Medline]
Muller AM, Olert J, Cronen C, Kirkpatrick CJ (1996) Detection of adhesion molecules on endothelial cells in paraffin sections. Pathol Res Pract 192:81A
Pignatelli M, Cardillo MR, Hanby A, Stamp GWH (1992) Integrins and their accessory adhesion molecules in mammary carcinomas: loss of polarization in poorly differentiated tumors. Hum Pathol 23:1159-1166[Medline]
Shi S-R, Gu J, Kalra KL, Chen T, Cote RJ, Taylor CR (1995) Antigen retrieval technique: a novel approach to immunohistochemistry on routinely processed tissue sections. Cell Vis 2:6-8, 10-22
Shi S-R, Key ME, Kalra KL (1991) Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections. J Histochem Cytochem 39:741-748[Abstract]
Tani Y, Phillips T, Key ME (1995) Comparison of water bath, microwave, and autoclave methods of thermal-induced target retrieval for immunohistochemistry and in situ hybridization. Lab Invest 72:988A
Taylor CR, Shi S-R, Chaiwun B, Young L, Imam SA, Cote RJ (1994) Strategies for improving the immunohistochemical staining of various intranuclear prognostic markers in formalin-paraffin sections: androgen receptor, estrogen receptor, progesterone receptor, p53 protein, proliferating cell nuclear antigen, and Ki-67 antigen revealed by antigen retrieval techniques. Hum Pathol 25:263-270[Medline]
Zutter MM, Mazoujian G, Santoro SA (1990) Decreased expression of integrin adhesive protein receptors in adenocarcinoma of the breast. Am J Pathol 137:863-870[Abstract]