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High Expression Levels of Egfl7 Correlate with Low Endothelial Cell Activation in Peritumoral Vessels of Human Breast Cancer

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High Expression Levels of Egfl7 Correlate with Low Endothelial Cell Activation in Peritumoral Vessels of Human Breast Cancer 1422 ONCOLOGY LETTERS 12: 1422-1428, 2016 High expression levels of egfl7 correlate with low endothelial cell activation in peritumoral vessels of human breast cancer DIANE PANNIER1,2*, GÉRALDINE PHILIPPIN‑LAURIDANT1,2*, MARIE‑CHRISTINE BARANZELLI1, DELPHINE BERTIN1, EMILIE BOGART1, VICTOR DELPRAT2, GAËLLE VILLAIN2, VIRGINIE MATTOT2, JACQUES BONNETERRE1 and FABRICE SONCIN2 1Senology Department, Oscar Lambret Center, Université de Lille, 59020 Lille; 2Institut Pasteur de Lille, Centre National de la Recherche Scientifique UMR8161, Université de Lille, 59000 Lille, France Received October 5, 2015; Accepted May 24, 2016 DOI: 10.3892/ol.2016.4791 Abstract. Tumor blood vessels participate in the immune Altogether, these results provide further results that egfl7 response against cancer cells and we previously used pre-clin- serves a regulatory role in endothelial cell activation in rela- ical models to demonstrate that egfl7 (VE‑statin) promotes tion to immune infiltration and that it is a potentialtherapeutic tumor cell evasion from the immune system by repressing target to consider for improving anticancer immunotherapies. endothelial cell activation, preventing immune cells from entering the tumor mass. In the present study, the expression Introduction levels of egfl7 and that of ICAM‑1 as a marker of endothelium activation, were evaluated in peritumoral vessels of human The main therapeutic achievements in treating cancer have breast cancer samples. Breast cancer samples (174 invasive been historically obtained by limiting cancer cell proliferation and 30 in situ) from 204 patients treated in 2005 were immu- or division using non‑specific, thereafter, targeted therapies nostained for CD31, ICAM‑1 and stained for egfl7 usingin situ against these cells. More recently, an increasing interest hybridization. The expression levels of ICAM‑1 and egfl7 were has emerged on the role of the tumor microenvironment in assessed in peritumoral areas using semi-quantitative scales. the development of cancer lesions (1). Within this microen- There was a strong and significant inverse correlation between vironment, immune cells which can mediate the destruction the expression of ICAM‑1 and that of egfl7 in CD31+ blood of cancer cells and the immune system as a whole offer a vessels. When the ICAM‑1 score increased, the egfl7 score number of novel potential targets for additional therapies (2), reduced significantly (P=0.004), and vice‑versa Cuzick's( test a proportion of which have provided practical applications for trend across ordered groups). In order to determine which for treating patients. The first efficient treatments using gene influenced the other gene between egfl7 andICAM‑1, the immunotherapies targeting PD‑1/PD‑L1 were beneficial for expression levels of either gene were modulated in endothe- patients with melanoma (3) and such treatments were recently lial cells. Egfl7 regulated ICAM‑1 expression while ICAM‑1 approved for treating lung cancer as well (4). Blood vessels, had no effects on egfl7 expression in the same conditions. which are part of the tumor microenvironment and already the targets of anti-angiogenic drugs in clinical practice for treating colon or lung cancers, also serve a crucial role as a natural physical barrier which regulates the immune response. Correspondence to: Dr Fabrice Soncin, Institut Pasteur de Lille, Indeed, endothelial cells which line the inner side of blood Centre National de la Recherche Scientifique UMR8161, Université vessels, regulate the extravasation of circulating immune de Lille, 1 Rue du Pr. Calmette, 59000 Lille Cedex, France cells into tissues. Under non-activated, normal conditions, E-mail: [email protected] endothelial cells form a non-adhesive surface which does not allow for strong interactions with circulating immune cells (5). Abbreviations: B2M, β2‑microglobulin; DCIS, ductal carcinoma Upon stimulation by pro‑inflammatory cytokines, endothelial in situ; egfl7, EGF‑like domain‑containing protein 7; ICAM‑1, cells become activated and express high levels of cell surface intercellular adhesion molecule‑1; IDC, invasive ductal carcinoma; leukocyte adhesion molecules such as E‑ and P‑selectin, ILC, invasive lobular carcinoma; siRNA, small interfering RNA; TBS, TRIS buffer saline; VCAM‑1, vascular cell adhesion intercellular adhesion molecule‑1 (ICAM‑1) and vascular cell molecule-1 adhesion molecule‑1 (VCAM‑1). These molecules participate in the capture of circulating immune cells through the rolling, *Contributed equally arrest, firm adhesion, and extravasation of leukocytes from the circulation toward the tissues (5). This process is particularly Key words: breast cancer, blood vessels, endothelium activation, important in the context of cancer where activation of endothe- immunity lial cells directly controls the penetration of cytotoxic immune cells in the tumor foci (6,7). The gene coding for EGF‑like domain‑containing protein 7 (egfl7) is mainly expressed PANNIER et al: EGFL7 REGULATES ICAM‑1 IN HUMAN BREAST CANCER 1423 in blood vessel endothelial cells (8‑10) and its expression is retrieval, solution citrate (Vector Laboratories, Peterborough, deregulated in human cancer. High expression levels of egfl7 UK) for 20 min at 90˚C, then for 20 min at room temperature, were correlated with a more advanced stage of human colon and rinsed twice in TBS. Slides were incubated in TRIS‑HCl cancer (11), with poor survival in human hepatocarcinoma (12), 0.1 M, pH 7.5, NaCl 0.15 M, 20% goat serum, 0.5% blocking and with a higher tumor grade in human gliomas (13). Egfl7 buffer from TSA Biosystem (Perkin Elmer, Beaconsfield, UK) expression was also associated with poor overall survival for 2 h and incubated in rabbit anti‑human ICAM‑1 (1:200) in human pancreatic cancer (14). Furthermore, a previous overnight at 4˚C in a humidified atmosphere. Slides were then study demonstrated in breast cancer tissue samples that a washed in TBS for 5 min twice and incubated in goat bioti- high egfl7 protein score in cancer cells corresponded with nylated anti‑rabbit IgG (1:250) for 45 min. Slides were then low VCAM‑1, ICAM‑1, and interferon‑γ in the tumors (15). washed three times in TBS for 5 min, incubated in Strepta- Using experimental approaches, it has been demonstrated vidin‑A‑HRP (TSA Biosystem kit, 1:100) in blocking buffer that, when expressed by tumor cells, egfl7 promotes tumor for 30 min, washed 3 times in TRIS‑HCl 0.1 M, pH 7.5, NaCl escape from immunity by downregulating the activation 0.15 M, Tween‑20 0.25% (TNT) for 5 min, then incubated of tumor blood vessels (15). Egfl7 expressed constitutively in biotinylated tyramine (1:50) in amplification buffer (TSA by blood vessel endothelial cells continuously inhibits the Biosystem, Perkin Elmer), washed 3 times in TNT for 5 min, expression of leukocyte‑adhesion cell surface receptors and incubated in streptavidin‑A‑HRP (TSA Biosystem kit, Perkin thus limits endothelium activation. In the context of cancer, Elmer, 1:100) in blocking buffer for 30 min, washed twice in egfl7 expressed by tumor blood vessels similarly prevents the TNT for 5 min and the immune complexes revealed using cytotoxic immune cells from entering the tumor mass, thus DAB kit (Vector Laboratories) under microscope observation. protecting tumor from immune destruction (15). Staining was stopped with tap water and slides counterstained To the best of our knowledge, no previous study has with hemalun, dehydrated and mounted. addressed the role and expression of egfl7 in blood vessels CD31 and CD34 immunostainings were performed using a of the tumor microenvironment. In the present study, a large BenchMark ULTRA automat and dedicated reagents (Ventana cohort of human breast cancer lesions were included and the Medical Systems, Roche Diagnostics, Basel, Switzerland). For expression levels of egfl7 and that of ICAM‑1 as a marker of CD31 staining, slides were deparaffinized, incubated at 95˚C endothelium activation, were evaluated in peritumoral vessels for 36 min in cell conditioning buffer 1, washed in Reaction of human breast cancer samples. Buffer. Endogenous peroxydases were inactivated using UV INHIBITOR for 4 min and slides further washed in Reac- Materials and methods tion Buffer. Slides were then incubated in anti‑CD31 (Dako, Agilent Technologies, Inc., Santa Clara, CA, USA, #M0823, Human samples. The patient population is described in details 1:20) for 28 min, rinsed in Reaction Buffer and incubated in in the main section. Human tissue samples were processed and HRP UNIV MULT for 8 min, washed in Reaction Buffer, stored at the pathology laboratory of the Centre Oscar Lambret developed using UV DAB and counterstained in Hematoxylin cancer clinic (Lille, France). Following surgery, tissues were for 4 min, then in Bluing Reagent, washed in Reaction Buffer fixed in 4% paraformaldehyde and embedded in paraffin. before dehydration and mounted. For CD34 staining, slides For each tumor, analyses were performed on sections from were processed similarly except that the cell conditioning the paraffin block which had been used for strogene receptor, buffer 1 treatment was performed for 8 min and the slides were progesterone and HER2 expression analyses and for diagnosis, incubated with mouse anti‑CD34 (DAKO, clone QBend‑10, considered by the pathologists to be the most representative of 1:50) for 20 min. the lesion. Stained slides were analyzed by two independent oncolo- gists, including a breast cancer pathologist, using an Axioplan 2 Antibodies. For staining human tissues,
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