CD137 Expression in Tumor Vessel Walls High Correlation with Malignant Tumors

Anatomic Pathology / CD137 EXPRESSION IN TUMOR VESSEL WALLS

CD137 Expression in Tumor Vessel Walls High Correlation With Malignant Tumors

Karin Broll, Georg Richter, MD, Susanne Pauly, Ferdinand Hofstaedter, MD, and Herbert Schwarz, PhD

Key Words: CD137; Blood vessel; Tumor Downloaded from https://academic.oup.com/ajcp/article/115/4/543/1758017 by guest on 28 September 2021

Abstract CD137 (ILA/4-1BB) is a member of the tumor necrosis CD137 (ILA/4-1BB), a member of the tumor factor receptor family.1 It was isolated from activated T necrosis factor receptor family, and its ligand are lymphocytes in mice and humans.2,3 While expression of expressed on activated T lymphocytes and on antigen- CD137 messenger RNA (mRNA) can be detected in all acti- presenting cells, respectively. Via bidirectional signal vated hematopoietic cells, expression of CD137 protein is transduction, this receptor-ligand system regulates the specific for T lymphocytes, implying a stringent posttran- activation, proliferation, and survival of T and B scriptional control for protein expression.4,5 lymphocytes and monocytes. We used Costimulation through CD137 enhances T-lymphocyte immunohistochemical studies on human tissue samples activation, comparable to costimulation through CD28, and to determine in vivo CD137 expression in nonimmune induces rejection of tumors in mice.6-8 The CD137 ligand is tissue samples. Strong CD137 expression was found in expressed by antigen-presenting cells, and bidirectional blood vessel walls, on the endothelial layer, and on the signaling has been demonstrated for CD137.9,10 Cross- vascular smooth muscle cells. But in 32 healthy tissue linking of the membrane-bound CD137 ligand inhibits T- samples examined, none contained CD137-positive lymphocyte proliferation and induces apoptosis.6,11 In mono- vessels. Also, in benign tumors (2/14) and in cytes, cross-linking of the CD137 ligand causes activation inflammatory tissues (2/9) only a minority had CD137- and prolongation of survival and proliferation.12-14 expressing vessels. However, malignant tumors had a Soluble forms of CD137 are generated by differential significantly enhanced frequency of CD137-expressing splicing and are expressed specifically by activated T blood vessels (11/34). lymphocytes.5 Their levels correlate with activation-induced cell death in T lymphocytes.15 Expression of CD137 is not restricted to immune cells. CD137 mRNA has been detected in activated chondrocytes and epithelial and hepatoma cells.4 Expression of CD137 protein has been verified in chondrocytes.16 In all primary cells, expression of CD137 mRNA and protein is strictly dependent on activation. A constitutive expression of CD137 mRNA was found only in several tumor cell lines, implying a potential growth advantage of CD137 expression for tumor cells.4 This hypothesis prompted us to study whether CD137 protein also would be expressed constitutively by tumor cells in vivo. By using immunohistochemical studies, we found that CD137 can be expressed on blood vessel walls by the

© American Society of Clinical Pathologists Am J Clin Pathol 2001;115:543-549 543 Broll et al / CD137 EXPRESSION IN TUMOR VESSEL WALLS endothelial cells and the vascular smooth muscle cells Reverse Transcriptase–Polymerase Chain Reaction (VSMCs). CD137-positive vessels are not present in healthy Total RNA was isolated from peripheral blood mononu- tissues. They occasionally occur in benign tumors and clear cells using RNAzol B (Tel-Test, Friendswood, TX), inflammatory tissues but are found at a significantly higher and up to 5 µg of RNA were reverse-transcribed in a 20 µL rate in malignant tumors. volume, using random hexanucleotide primers (50 µg/mL), a 25-µmol/L concentration of deoxynucleoside triphosphate (dNTP), a 10-mmol/L concentration of dithiothreitol, 200 U Materials and Methods of SuperScript II RNaseH-RT (BRL, Eggenstein, Germany), and 20 U of RNAsin (Roche, Mannheim, Germany) for 60 Reagents minutes at 42°C. Anti-CD137 antibody (clone BBK-2) and its isotype Two microliters of the reverse transcriptase reaction control, MOPC21, were obtained from Bioscource served as template for the subsequent polymerase chain reac- Downloaded from https://academic.oup.com/ajcp/article/115/4/543/1758017 by guest on 28 September 2021 (Ratingen, Germany) and Sigma (Deisenhofen, Germany), tion, which was performed in a 20-µL volume with 1 U of respectively. Taq DNA polymerase (Roche), a 200-µmol/L concentration of dNTPs, a 1.5-mmol/L concentration of magnesium chlo- Tissue Samples ride, a 10-mmol/L concentration of tris(hydroxymethyl)- Grossly normal tissue was taken from a site distant from aminomethane (pH 8.3), a 50-mmol/L concentration of the lesion of fresh tissue after surgical removal. We studied potassium chloride, and a 10-µmol/L concentration of each the following 32 fresh specimens from various organs: lung primer. After a 5-minute denaturation step at 94°C, the reac- and liver, 5 (16%) each; colon and soft tissue, 4 (12%) each; tion proceeded in 30 cycles of 30 seconds at 94°C, 30 thyroid gland, 3 (9%); stomach, parathyroid gland, and seconds at 58°C, and 1 minute at 72°C, followed by 10 spleen, 2 (6%) each; parotid gland, heart, aorta, kidney, and minutes at 72°C. lymph node, 1 (3%) each ❚Table 1❚. The mean age of the The primers used were as follows: patients was 54 years (range, 15-78 years), and the CD137 sense: 5'-ATCATGGGAAACAGCTGTTAC- male/female ratio was 2.3 (21:9). AAC In addition, we studied the following 61 fresh specimens CD137 antisense: 5'-TGGTCCACAGACCACGTC- from diseased tissues: gastrointestinal tract (including liver, CCTCTC parotid gland, gallbladder, and pancreas), 15 (25%); Cyclophilin sense: 5'-GTCCAGCATTTGCCATGGAC lymphatic system, 12 (20%); endocrine system and female Cyclophilin antisense: 5'-GACAAGGTCCCAAA- genital tract, 8 (13%) each; soft tissue, 6 (10%); respiratory GACAGC tract, 4 (7%); and urinary tract, male genital tract, cardiovas- cular system, and skin, 2 (3%) each (Table 1). Statistics There were 14 benign tumors (23%), 34 malignant Significance values were calculated using the chi-square tumors (56%), 9 specimens of inflammatory tissue (15%), test.17 and 4 samples not grouped (7%). In all cases, the diagnosis was based on histologic examination. Results Immunohistochemical Studies We undertook an immunohistochemical screening study Frozen tissue sections were fixed with 2% to determine expression of CD137 in vivo under physiologic paraformaldehyde for 10 minutes. Endogenous peroxidases and pathologic conditions. We studied 93 human tissue were inactivated by 2% hydrogen peroxide in methanol for samples from healthy and diseased organs (Table 1). 15 minutes. Unspecific staining was blocked by 3% dry milk Strong expression of CD137 was found on blood vessel in phosphate-buffered saline for 30 minutes. Two micro- walls in 15 tissue samples. In most cases (10), the endothelial grams per milliliter of anti-CD137 (clone BBK-2) or an cell layer and the VSMCs stained positive for CD137 ❚Image isotype control antibody (MOPC21) in 3% dry milk was 1❚. However, in 2 cases, Crohn disease and cystadenocarci- added overnight. Positive hybridization was detected by noma of the ovary, only the endothelium expressed CD137. using the ABC staining kit (Dako, Hamburg, Germany) at In 3 tumors, leiomyosarcoma, fibrosarcoma, and rhab- 37°C using diaminobenzidine as the substrate. After each domyosarcoma, the endothelial layers were negative, while step, the samples were washed 3 times with phosphate- the VSMCs displayed strong staining for CD137 ❚Image 2❚. buffered saline. Tissue sections were stained with hema- CD137-expressing blood vessels could not be found in toxylin and embedded in Entellan (Merck, Darmstadt, any of the 32 healthy tissue samples. Furthermore, in inflam- Germany). matory tissues and in benign tumors, only 2 (22%) of 9 and 2

544 Am J Clin Pathol 2001;115:543-549 © American Society of Clinical Pathologists Anatomic Pathology / ORIGINAL ARTICLE

❚Table 1❚ ❚Table 1❚ CD137 Expression in Tissues* (cont)

CD137 Expression CD137 Expression

Vascular Smooth Vascular Smooth Endothelium Muscle Cells Endothelium Muscle Cells

Healthy tissue Carcinomatous peritonitis – – Stomach (2) – – Renal cell carcinoma – – Colon (4) – – Carcinoma of the portio uteri + + Parotid gland – – Thyroid cancer Liver (5) – – Follicular – – Kidney – – Papillary – – Heart – – Anaplastic – – Aorta – – Medullary – – Downloaded from https://academic.oup.com/ajcp/article/115/4/543/1758017 by guest on 28 September 2021 Spleen (2) – – Melanoma – – Lymph node – – Basal cell carcinoma + + Parathyroid gland (2) – – Others Thyroid gland (3) – – Endometrial cyst – – Soft tissue (4) – – Mastopathy – – Lung (5) – – Arteriosclerosis – – Inflammatory tissues Fatty liver – – Chronic polypous rhinitis – – Chronic fibrosing alveolitis – – +, positive; –, negative. Crohn disease + – * When more than one specimen of a tissue type was studied, the number is indicated Diverticulitis – – in parentheses. Cholecystitis – – Mastitis – – Thyroiditis – – Graves disease + + (14%) of 14 tissue samples, respectively, contained CD137- Osteomyelitis – – positive vessels. However, 11 (32%) of 34 malignant tumors Benign tumors Polyp (2) – – contained CD137-positive vessels, a significantly higher rate Adenoma of the parotid gland – – (P < .01). Hyperplasia of the thyroid – – Adenoma of the liver – – Several methods were used to verify that the monoclonal Fibroadenoma of the breast – – antibody used (clone BBK-2) recognizes CD137 rather than Leydig cell tumor + + Myxoma – – merely cross-reacting with an unrelated antigen. First, BBK-2 Thymoma – – was incubated with CD137-transfected COS or mock-trans- Morbus Castleman – – fected COS cells for 1 hour at room temperature before being Pheochromocytoma – – Adrenal adenoma – – used in immunohistochemical studies. This preabsorption on Chondroma – – CD137-expressing cells abolished staining on a parallel Ameloblastoma + + Malignant tumors section of the Leydig cell tumor sample that was used in Squamous cell carcinoma Image 1. Second, the immunohistochemical staining was Lung – – Tongue – – repeated on selected tissue samples with a polyclonal chicken Vulva + + anti-CD137 serum that yielded identical results (not shown). Tonsil – – Adenocarcinoma Third, a mixed culture of primary endothelial cells and Lung – – VSMCs expressed CD137 mRNA on activation ❚Image 3❚. Stomach (2) – – No significant difference in CD137 expression could be Colon – – Pancreas (2) – – detected between veins (10/15 positive cases) and arteries Breast – – (6/15). In some tumors, all vessels stained positive for Cystadenocarcinoma of the ovary + – Lymphangiosarcoma – – CD137, whereas in others, for example, the Leydig cell Rhabdomyosarcoma – + tumor, CD137-positive vessels were found interspersed with Leiomyosarcoma – + Fibrosarcoma – + CD137-negative vessels (Image 1). Only frozen tissue Neuroblastoma – – samples were used since no CD137 staining could be Nephroblastoma + + Hodgkin disease – – obtained with the anti-CD137 antibody (clone BBK-2) on Immunocytoma paraffin-embedded tissues. Spleen + + Lymph node – – Non-Hodgkin lymphoma Stomach – – Discussion Lymph node (2) + + True histiocytic lymphoma – – We studied in vivo CD137 expression in human tissue (lymph node) samples. No expression of CD137 could be detected in

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❚Image 1❚ Expression of CD137 in the vessel walls of a Leydig C cell tumor. A, H&E staining. B, Isotype control: MOPC21 (Sigma, Deisenhofen, Germany). C, Anti-CD137. (×200)

tumor cells. This was an unexpected result, since CD137 In most cases of CD137-positive blood vessels, the mRNA expression was expressed constitutively in several endothelial cells and the VSMCs expressed CD137. Two tumor cell lines, while its expression in primary cells is cases in which only the endothelial cells were stained and 3 strictly dependent on activation.4,11 Therefore, our original cases in which only the VSMCs were stained were found. hypothesis that tumor cells may express CD137 because of a These cases may represent intermediary stages of vessels growth or selection advantage could not be confirmed. starting to express CD137. It is noteworthy that the 3 cases However, strong expression of CD137 was found in with solely VSMC staining all were sarcomas. blood vessel walls. Expression of CD137 in blood vessels In the present study, we found tumors in which all blood does not seem to be dependent on the type of organ the vessels expressed CD137 and others in which CD137-posi- vessels are supplying but rather on the nature of the tissue tive and CD137-negative vessels were found side by side. alteration. Even in the malignant tumors, and more so in the The potential significance of this observation is the focus of benign tumors and in inflammatory tissues, only a minority ongoing work in our laboratory. of samples contained CD137-positive vessels. Furthermore, 3 The complete absence of CD137-positive vessels in of 4 sarcomas but only 4 of 15 carcinomas contained CD137- healthy tissues may allow the use of CD137 as a target for an positive vessels. This indicates that distinct physiologic antiangiogenic tumor therapy. CD137 also is expressed on conditions may be required for blood vessels to express activated T lymphocytes, but levels of expression on blood CD137. The nature of these conditions is unknown at present. vessels far exceed those on T lymphocytes. Even in inflamed

546 Am J Clin Pathol 2001;115:543-549 © American Society of Clinical Pathologists Anatomic Pathology / ORIGINAL ARTICLE

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❚Image 2❚ Expression of CD137 by the vascular smooth C muscle cells of a rhabdomyosarcoma. A, H&E staining. B, Isotype control: MOPC21 (Sigma, Deisenhofen, Germany). C, Anti-CD137. (×400)

tissue samples in which strong CD137 staining was obtained CD137 in capillaries and vessels of a normal lung. However, on blood vessels, CD137 could hardly be detected on infil- these authors used grossly normal-looking lung tissues trating T lymphocytes. Therefore, the potential side effects of distant from the site of the bronchial carcinomas. We have a therapy directed against CD137-expressing cells, ie, encountered similar cases in which we found CD137-posi- immunosuppression, may be tolerable. tive vessels in seemingly healthy tissue sections of cancer- Anti-CD137 antibodies have been proven to be potent infested organs. Detection of CD137-positive blood vessels antitumor agents. Injection of anti-CD137 antibodies in therefore may provide a valuable and novel parameter for the mastocytoma and sarcoma-bearing mice led to a complete differentiation of benign from malignant tumors. remission of the tumors and lasting antitumor immunity.7 An CD137 delivers a potent costimulatory signal to T enhancement of the activity of CD137-expressing helper and lymphocytes. Whether CD137 also can deliver a signal cytotoxic T lymphocytes by the anti-CD137 antibodies could to blood vessel cells and the nature of this potential signal be identified as an underlying mechanism.7 Theoretically, are unknown. The CD137 ligand is expressed on anti- antibody-dependent cell cytotoxicity or complement lysis of gen-presenting cells.5,9,10,12-14 Bidirectional signal transduc- CD137-expressing endothelial cells could have been a tion has been shown for the CD137 receptor–ligand system. contributing second mechanism. The signal through the CD137 ligand induces activation While we did not find CD137 in vessels of healthy of and adherence by blood monocytes.12-14 Therefore, tissue samples, Boussaud et al18 described the expression of CD137 on the endothelium could cause monocyte activation

nitric oxide, and arachidonic acid metabolites.21 On the other hand, activated monocytes and macrophages also release extracellular matrix–degrading enzymes such as matrix metal- loproteinases, plasminogen, and plasminogen activator, which 22

No template PMA / Ca-I control Positive may support tumor growth and metastasis. It may be this A latter activity that provides tumors with a growth advantage if they succeed in facilitating monocyte recruitment by inducing CD137 expression on blood vessel walls.

From the Department of Pathology, University of Regensburg, Regensburg, Germany. CD137 Downloaded from https://academic.oup.com/ajcp/article/115/4/543/1758017 by guest on 28 September 2021 Supported by the Deutsche Krebshilfe, Bonn, Germany. Address reprint requests to Dr Schwarz: Cantab Pharmaceuticals, 310 Cambridge Science Park, Cambridge CB4 OWG, England. Acknowledgment: We thank Susanne Fertig and Brigitte Krause for excellent technical work. B

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