ANTICANCER RESEARCH 25: 4141-4148 (2005)

The Relevance of Adhesion Molecules in the Classification of Squamous Cell of the Head and Neck

ANJA M. LIEDER1, TASH G.T. PRIOR2, KATHRYN J. WOOD3 and JOCHEN A. WERNER1

1Department of Otolaryngology, Head and Neck Surgery, Philipps-University Marburg, Germany; 2The Cardiac Department, The Harley Street Clinic, London; 3Nuffield Department of Surgery, University of Oxford John Radcliffe Hospital, Oxford, United Kingdom

Abstract. Background: Clinical studies have demonstrated family of carbohydrate-binding expressed on increased serum levels of E-, P-selectin and VCAM-1 endothelial cells, that are known to promote the adhesion in patients with squamous cell , which correlate of leukocytes to the and to provide with expression in the primary tumour. For this reason, selectin traffic signals for leukocyte migration (1). E-selectin expression may also support the diagnosis and be of prognostic (CD62E) expression is induced on vascular endothelial cells value in squamous cell carcinoma of the head and neck following exposure to -1 (IL-1), TNF-· and (SCCHN). Materials and Methods: Using immuno- lipopolysaccharide (LPS) and requires de novo mRNA and histochemistry, the expressions of E- and P-selectin and synthesis. In contrast, P-selectin (CD62P) is stored VCAM-1 in SCCHN were characterised and analysed. Results: preformed in Weibel-Palade bodies and is rapidly mobilised The expressions of E-selectin and VCAM-1 were increased. The to the plasma membrane in response to mediators of acute magnitude of expression was unrelated to the location or size inflammation (2, 3). The vascular molecule of the primary tumour. With increasing de-differentiation, the (VCAM-1) is also up-regulated by TNF-·, IL-1 and LPS, its expression of E-selectin increased, whereas the number of cells up-regulation facilitating the adherence of peripheral blood expressing VCAM-1 was highly variable. In contrast, E-selectin (3, 4). are constitutively expressed on and VCAM-1 expressions decreased with progressive metastatic human vascular endothelium in vivo and on human disease. Conclusion: These data demonstrate that the umbilical vein endothelial cells (HUVEC) in vitro. In expression patterns of E-selectin and VCAM-1 may depend on addition to this, their expression is also demonstrable on differentiation and lymphogenic metastatic disease in SCCHN, the vasculature of squamous cell carcinomas (5-10). and suggest that these molecules may have a dual role, Corresponding to these findings, the serum levels of depending on the stage of tumour development. These findings E-selectin (11-13), P-selectin (12) and VCAM-1 (11, 14-16) support the hypothesis that expression of E-selectin and have been found to be elevated in patients with squamous VCAM-1 may be initiated by the release of tumour necrosis cell carcinomas. It has, thus, been postulated that selectins factor-alpha (TNF-·) at an early stage of tumour development. play an important role in the lymphogenous and In contrast, the decrease in selectin expression, observed as the haematogenous metastasis of squamous cell carcinoma of metastatic disease progresses, may serve as a protective the head and neck (SCCHN). In these tumours, the up- mechanism to prevent an influx of effector cells. regulation of selectins may contribute to changes in invasive phenotype by promoting certain endothelial cell Adhesion molecules, selectins in particular, are expressed adhesion properties and , and by acting as in squamous cell carcinomas, and it has been hypothesised chemo- attractants for infiltrating leukocytes (10, 17, 18). that these molecules may facilitate disease progression and In this study, the expressions and distribution of VCAM-1, metastasis. The selectin family of adhesion molecules is a E-and P-selectin were examined in samples of human SCCHN using . The objective of this study was to analyse the expression patterns of these selectins, and to correlate these findings with leukocyte Correspondence to: Anja Lieder, D.Phil, Klinik für Hals-, Nasen-, und infiltration and neovascularisation in human SCCHN, using Ohrenheilkunde, am Klinikum der Philipps-Universität Marburg, tumour-free inflammatory and non-inflammatory tissues as Deutschhausstr. 3, 35037 Marburg, Germany. Tel: +496421-2862850, Fax: +496421-2862842, e-mail: [email protected] controls, in order to determine if this analysis has potential prognostic value with regard to tumour stage and, thus, Key Words: Selectins, SCCHN, adhesion molecules, metastasis. disease progression.

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Table I. Origin, differentiation and TNM classification of tumour samples. Statistical analysis. The Student’s unpaired two-sided t-test with unequal variance was utilized for the determination of statistical Number Localisation Grade TNM classification significance between groups. Values of p<0.05 were considered statistically significant. The values are presented as mean±standard Size Nodes Metastasis deviation.

1 Hypopharynx G1 T2 N0 M0 2 Hypopharynx G2 T2 N1 M0 Results 3 Hypopharynx G2 T4 N2b M0 4 Hypopharynx G3 T2 N1 M0 5 Larynx G1 T1 N0 M0 The expression of the endothelial marker CD31 and the 6 Larynx G2 T2 N2b M0 selectins VCAM-1, E- and P-selectin were delineated using 7 Larynx G2 T2 N1 M0 immunohistochemistry. In addition, cells staining positively 8 Larynx G3 T2 N0 Mx for CD45, CD4 and MHC class II were identified. Human 9 Oropharynx G1 T3 N2 M0 internal mammary arteries showed a continuous 10 Oropharynx G1 T4 N2c M0 11 Oropharynx G2 T1 N2 M0 endothelial lining. This was confirmed by the uniform 12 Oropharynx G2 T1 N0 M0 expression of CD31. The endothelium also stained 13 Oropharynx G2 T2 N1 M0 positively for HLA-DP,-DQ and -DR. No infiltrating 14 Oropharynx G2 T3 N2 M0 leukocytes or VCAM-1, E- or P-selectin were detectable 15 Oropharynx G2 T3 N1 M0 in the side branches of the human internal mammary 16 Oropharynx G2 T4 N2 M0 17 Oropharynx G3 T2 N2b M0 artery (data not shown). The expression of human CD45 18 Oropharynx G3 T3 N2 M0 was high in the chronic tonsillitis controls. Most of these CD45+ cells also expressed HLA-DP, -DQ, -DR and CD4 (Figure 1A and C). infiltration was significantly sparser in tissue samples of SCCHN compared with the controls, and it was not related to the level of de-differentiation (G1-G3), or lymph node metastasis (Figure 1 A and C). Similarly, the Materials and Methods location of the tumour and tumour size (T1-T4) were not related to lymphocytic infiltration (data not shown). The SCCHN samples. Samples from squamous cell carcinoma of the hypopharynx, larynx and oropharynx (Table I) were procured from expression of CD31, indicating the density of microvessels, patients undergoing surgery at the Department of Otolaryngology was similar in the controls and in SCCHN (Figure 1B and and Head and Neck Surgery at the Philipps-University of Marburg, D). The expressions of E- and P-selectin were very low in Germany. Samples of tissue to act as non-inflammatory and the controls, whereas moderate VCAM-1 expression was inflammatory controls for the study were obtained with informed observed (Figure 2A,C and E). consent from patients undergoing coronary artery bypass grafting E-selectin expression was low in well-differentiated and tonsillectomy, respectively. All samples were immediately SCCHN (G1; n=4), but this was dramatically increased in embedded in OCT compound on extraction, snap-frozen in liquid nitrogen and stored at 4ÆC. de-differentiated SCCHN (G3; n=4) (Figure 1B). In contrast, E-selectin expression was high in SCCHN without . The primary antibodies (mouse anti-human CD4, lymph node metastases (N0; n=4), whereas the expression CD31, CD45, CD62E, CD62P, CD106 and HLA-DP/DQ/DR) level was found to decrease with progressive metastatic were purchased from PharMingen (BD Biosciences), and the disease (N1; n=5 and N2; n=9; Figure 1D). In some cases, secondary (rabbit anti-mouse) immunoglobulin from Dako. the expression of E-selectin was confined to the vascular endothelium, while in others the tumour cells also expressed Immunohistochemistry. Immunohistochemistry was performed by adding appropriate dilutions of the primary antibodies to 5-Ìm E-selectin (Figure 2B). However, no correlation between frozen sections fixed in acetone and incubating for 1 hour. E-selectin distribution patterns and tumour localization or Following three washes in phosphate-buffered saline with added disease progression was observed. bovine serum albumin and secondary incubation, the The expression of VCAM-1 was low in well- and slides were washed again and colour developed with moderately-differentiated tumours (G1-G2; n=14), and was 3’-diaminobenzidine tetrahydrochloride (DAB) solution (Sigma). found to increase in de-differentiated tumours (G3; n=4), Coverslips were mounted using DPX compound. The degree of where it reached levels comparable to those found in the cellular infiltration in the histological sections was determined by point counting the positively-stained cells. The sections were control tissues (Figure 1B). Although the expression of viewed under a microscope at x400 magnification. Within a VCAM-1 was highly variable in metastatic disease, there single cross-section, the positively-stained cells at the intersections appeared to be a trend towards reduced expression of of an overlay graticule containing 121 intersections were counted. VCAM-1 with progressive lymph node metastasis (Figure

4142 Lieder et al: Selectin Expression in SCCHN

Figure 1. Quantification by point counting of cells staining positively for CD45, CD4 and HLA-DP,-DQ and-DR (A, C) and for VCAM-1, E- and P- selectin (B, D) in SCCHN compared to control tissue (chronic tonsillitis) detected in relation to tumour differentiation (A, B) and lymph node metastatic disease (C, D). Counts were obtained per 121 fields of a grid and established from five independent counts per section. Mean values including standard deviation are reported (*p<0.05)

1D). Cells staining positive for VCAM-1 tended to be Discussion widespread and scattered throughout the tumour tissues (Figure 2F). The localization and size of the primary The expressions of VCAM-1, E- and P-selectin and their tumour did not have a significant impact on the expressions association with lymphocytic cell infiltration in samples of of VCAM-1 and E-selectin (data not shown). The P-selectin SCCHN were studied and compared to controls (chronic expression did not vary greatly and no statistically significant inflammation and healthy arteries). In addition, the differences were found with regards to either relationship between selectin expression and the location of differentiation, presence of metastases (Figure 1B, D and the tumour, tumour size, evidence of lymph node metastasis Figure 2D), or location or size of the primary tumour (data and differentiation was analysed. Clinical studies have not shown). demonstrated elevated serum levels of E-selectin (11, 12,

4143 ANTICANCER RESEARCH 25: 4141-4148 (2005)

Figure 2. Representative samples for the expressions of E-selectin CD62E (A-B), P-selectin CD62P (C-D) and VCAM-1 (E-F) in controls of chronic tonsillitis (A, C, E) and in SCCHN (B, D, F), as detected by immunohistochemistry (magnification x 400).

4144 Lieder et al: Selectin Expression in SCCHN

19) and P-selectin (12) in patients with squamous cell with a strong endothelial expression and binding of carcinoma or adenocarcinoma (for example, renal, breast, intercellular adhesion molecule-1 (ICAM-1) and vascular urothelial and squamous cell lung carcinoma). Similar adhesion protein-1 (VAP-1) to T cells, and with increased results have also been obtained for VCAM-1 (11, 19, 20). In utilization of the ICAM-1/LFA-1 and VAP-1/VAP-receptor some studies, the level of adhesion molecule expression pathways (26). In SCCHN in particular, the serum levels of positively correlated with advanced disease and invasiveness soluble VCAM-1 and E-selectin have been found to be of the tumour (11, 12), whereas others suggested a limited increased in carcinoma of the oropharynx or larynx, but, in role for selectins as prognostic markers (19). Reports of some of these studies, there was no relationship between the enhanced selectin expression by tumour tissues correspond serum levels and disease stage (7, 15, 27). In other studies, with these data. Increased expressions of VCAM-1, E- and VCAM-1 expression was correlated with increased influx of P-selectin have all been reported in various types of T lymphocytes (22), whereas E- and P-selectin expressions squamous cell carcinomas (5, 8, 10, 21) and were correlated with the amount of infiltrating adenocarcinomas (13, 14). In our study, VCAM-1, E- and (10). In this study, an association between tumour de- P-selectin were expressed on samples of human SCCHN of differentiation and increased expression of E-selectin and, the oropharynx, hypopharynx and larynx, whereas these to a lesser extent, P-selectin were observed (Figure 1B). selectin molecules were absent in the control tissue (Figure This could indicate increased recruitment of circulating 1). With the exception of VCAM-1, selectin expression was leukocytes with more progressive disease. However, the confined to the vascular endothelial cells of the tumour. human lymphocyte infiltrate remained consistent This is confirmed by other reports, where E-selectin and throughout all the stages of differentiation (Figure 1A). VCAM-1 were found to be expressed on endothelial cells in Studies have revealed that E-selectin expression is the tumour microvasculature of squamous cell carcinomas accompanied by increased adhesion of human colon (5, 8, 10) and adenocarcinomas (13, 22, 23). As seen in adenocarcinoma cells to HUVECs (28). Furthermore, pre- other studies, the expression of CD31 on the tumour treatment with TNF-· greatly enhanced the expressions of microvessels appears to be unrelated to the extent of E-selectin and VCAM-1 on HUVECs, but this effect was selectin expression (23). negated by tumour-conditioned medium from breast This staining pattern, observed on most endothelial cells, adenocarcinoma. These findings indicate that, under certain supports the idea of an active status of the tumour circumstances, tumour-secreted factors can have inhibitory microvasculature, similar to that proposed in vasculitis (8). potential on selectin-mediated leukocyte adherence (29). It is widely understood that -mediated mechanisms Similarly, breast carcinoma cell lines have been found to play a crucial role in the defence against solid organ bind and activate eosinophilic granulocytes. This was tumours. The reason why some tumours attract more of a associated with the expressions of ICAM-1 and VCAM-1, leukocyte infiltrate than others is not entirely clear, but is which increased after pre-treatment with TNF-· (30). In a likely to depend on adhesive interactions between rodent model of lung carcinoma, treatment with TNF-‚ circulating lymphocytes and the tumour vascular caused enhanced E- and P-selectin-dependent leukocyte endothelium. Therefore, as a matter of host defence, it is rolling and adhesion (31). Selectin-mediated leukocyte thought that the adhesion of inflammatory cells to the extravasation has also been shown to generate fewer vascular endothelium is a prerequisite for the recruitment metastases (32). In contrast, VCAM-1, E- and P-selectin of inflammatory effector cells into the tumour to combat expressions are suppressed in squamous cell lung carcinoma disease. VCAM-1 expression in gastric carcinoma has been and adenocarcinoma in humans (9), and also in a rodent found to correlate with more invasive and advanced disease model of adenocarcinoma (33). Metastatic cells are rarely (10, 14). The presence of selectins may, on the other hand, cleared by blood-borne leukocytes, so it is possible that promote tumour growth and metastasis, since P-selectin- carcinomatous lesions might escape the host’s defences by deficient mice showed significantly slower growth of interfering with the mechanism of effector cell implanted human adenocarcinoma and generated fewer extravasation. In our study, evidence of metastatic disease metastases. This is thought to occur because selectin- was associated with a steep decrease in E-selectin deficient fail to adhere to the tumour cell surface expression, whereas the P-selectin and VCAM-1 expressions and, hence, there is a reduction of tumour- remained unchanged. These findings are compatible with a microembolization (24). In a rodent model of human lung tumour survival mechanism that facilitates the avoidance of carcinoma, up-regulation of P-selectin was found to an inflammatory infiltrate with tumour-combating correlate with increased metastatic disease (25), while in a properties (Figure 1D). study of human hepatocellular carcinomas, the primary In conclusion, the increased expression of E-selectin seen tumour was heavily infiltrated by T-lymphocytes in relation in de-differentiated SCCHN and the decreased expression to the extent of metastatic disease. This, in turn, correlated of this molecule seen in metastatic disease may be

4145 ANTICANCER RESEARCH 25: 4141-4148 (2005) manifestations of a bi-directional role of selectins in tumour 10 Hemmerlein B, Scherbening J, Kugler A and Radzun HJ: biology. The expression of selectins may be initially up- Expression of VCAM-1, ICAM-1, E- and P-selectin and regulated by TNF-·, with the aim of facilitating an effective tumour-associated macrophages in renal cell carcinoma. Histopathology 37: 78, 2000. immune response against the tumour. However, a 11 O'Hanlon DM, Fitzsimons H, Lynch J, Tormey S, Malone C subsequent decrease in selectin expression following and Given HF: Soluble adhesion molecules (E-selectin, ICAM- immune modulation may also serve to protect the tumour 1 and VCAM-1) in breast carcinoma. Eur J Cancer 38: 2252, from effector cell infiltration. Therefore, selectins are 2002. unlikely to serve as straightforward markers of prognostic 12 Roselli M, Mineo TC, Martini F, Mariotti S, Ambrogi V, Spila significance. 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