Antiphospholipid Antibodies Promote Tissue Factor–Dependent Angiogenic Switch and Tumor Progression
2014; Elsevier BV; Volume: 184; Issue: 12 Linguagem: Inglês
10.1016/j.ajpath.2014.07.027
ISSN1525-2191
AutoresYuanyuan Wu, Andrew Nguyen, Xiao-Xuan Wu, Mingyu Loh, Michelle N. Vu, Yiyu Zou, Qiang Liu, Peng Guo, Yanhua Wang, Leslie L. Montgomery, Amos Orlofsky, Jacob H. Rand, Elaine Y. Lin,
Tópico(s)Monoclonal and Polyclonal Antibodies Research
ResumoProgression to an angiogenic state is a critical event in tumor development, yet few patient characteristics have been identified that can be mechanistically linked to this transition. Antiphospholipid autoantibodies (aPLs) are prevalent in many human cancers and can elicit proangiogenic expression in several cell types, but their role in tumor biology is unknown. Herein, we observed that the elevation of circulating aPLs among breast cancer patients is specifically associated with invasive-stage tumors. By using multiple in vivo models of breast cancer, we demonstrated that aPL-positive IgG from patients with autoimmune disease rapidly accelerates tumor angiogenesis and consequent tumor progression, particularly in slow-growing avascular tumors. The action of aPLs was local to the tumor site and elicited leukocytic infiltration and tumor invasion. Tumor cells treated with aPL-positive IgG expressed multiple proangiogenic genes, including vascular endothelial growth factor, tissue factor (TF), and colony-stimulating factor 1. Knockdown and neutralization studies demonstrated that the effects of aPLs on tumor angiogenesis and growth were dependent on tumor cell–derived TF. Tumor-derived TF was essential for the development of pericyte coverage of tumor microvessels and aPL-induced tumor cell expression of chemokine ligand 2, a mediator of pericyte recruitment. These findings identify antiphospholipid autoantibodies as a potential patient-specific host factor promoting the transition of indolent tumors to an angiogenic malignant state through a TF-mediated pathogenic mechanism. Progression to an angiogenic state is a critical event in tumor development, yet few patient characteristics have been identified that can be mechanistically linked to this transition. Antiphospholipid autoantibodies (aPLs) are prevalent in many human cancers and can elicit proangiogenic expression in several cell types, but their role in tumor biology is unknown. Herein, we observed that the elevation of circulating aPLs among breast cancer patients is specifically associated with invasive-stage tumors. By using multiple in vivo models of breast cancer, we demonstrated that aPL-positive IgG from patients with autoimmune disease rapidly accelerates tumor angiogenesis and consequent tumor progression, particularly in slow-growing avascular tumors. The action of aPLs was local to the tumor site and elicited leukocytic infiltration and tumor invasion. Tumor cells treated with aPL-positive IgG expressed multiple proangiogenic genes, including vascular endothelial growth factor, tissue factor (TF), and colony-stimulating factor 1. Knockdown and neutralization studies demonstrated that the effects of aPLs on tumor angiogenesis and growth were dependent on tumor cell–derived TF. Tumor-derived TF was essential for the development of pericyte coverage of tumor microvessels and aPL-induced tumor cell expression of chemokine ligand 2, a mediator of pericyte recruitment. These findings identify antiphospholipid autoantibodies as a potential patient-specific host factor promoting the transition of indolent tumors to an angiogenic malignant state through a TF-mediated pathogenic mechanism. The identification of mechanisms that trigger the malignant transition of indolent, avascular tumors is an important goal for cancer prevention and treatment. The avascular state may occur as either an early stage in primary tumor development or minimal residual disease after treatment.1Almog N. Molecular mechanisms underlying tumor dormancy.Cancer Lett. 2010; 294: 139-146Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar The transition of avascular tumors to a malignant state depends on the induction of angiogenesis (angiogenic switch),1Almog N. Molecular mechanisms underlying tumor dormancy.Cancer Lett. 2010; 294: 139-146Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar but the mechanisms that drive this transition have not been well defined. It would be especially valuable to elucidate patient-specific host factors that modulate tumor angiogenesis and progression, but few such factors that are assessable in patients have been identified. The antiphospholipid antibodies (aPLs) are a class of autoantibodies that recognize complexes of phospholipid-binding proteins bound to anionic phospholipids on the cell surface.2Rand J.H. Molecular pathogenesis of the antiphospholipid syndrome.Circ Res. 2002; 90: 29-37Crossref PubMed Scopus (126) Google Scholar Anionic phospholipids are absent from the surface of most cells but translocate to the surface on activation of endothelial cells, monocytes, and platelets, as well as during apoptosis and malignant transformation.3Goth S.R. Stephens R.S. Rapid, transient phosphatidylserine externalization induced in host cells by infection with Chlamydia spp.Infect Immun. 2001; 69: 1109-1119Crossref PubMed Scopus (45) Google Scholar, 4Riedl S. Rinner B. Asslaber M. Schaider H. Walzer S. Novak A. Lohner K. Zweytick D. In search of a novel target: phosphatidylserine exposed by non-apoptotic tumor cells and metastases of malignancies with poor treatment efficacy.Biochim Biophys Acta. 2011; 1808: 2638-2645Crossref PubMed Scopus (228) Google Scholar, 5Boyle Jr., E.M. Pohlman T.H. Cornejo C.J. Verrier E.D. Endothelial cell injury in cardiovascular surgery: ischemia-reperfusion.Ann Thorac Surg. 1996; 62: 1868-1875Abstract Full Text Full Text PDF PubMed Scopus (153) Google Scholar The incidence of aPLs in the general population is approximately 1% to 5%.6Rand J.H. The antiphospholipid syndrome.Hematology Am Soc Hematol Educ Program. 2007; : 136-142Crossref PubMed Scopus (24) Google Scholar Chronic aPL elevation can lead to a thrombogenic state known as antiphospholipid syndrome (APS),2Rand J.H. Molecular pathogenesis of the antiphospholipid syndrome.Circ Res. 2002; 90: 29-37Crossref PubMed Scopus (126) Google Scholar, 7de Groot P.G. Urbanus R.T. Derksen R.H. Pathophysiology of thrombotic APS: where do we stand?.Lupus. 2012; 21: 704-707Crossref PubMed Scopus (25) Google Scholar and aPL elevation also can occur transiently in association with viral and several other infections8Uthman I.W. Gharavi A.E. Viral infections and antiphospholipid antibodies.Semin Arthritis Rheum. 2002; 31: 256-263Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar as well as in patients with other autoimmune disorders, such as lupus or rheumatoid arthritis.9Rojas-Villarraga A. Toro C.E. Espinosa G. Rodriguez-Velosa Y. Duarte-Rey C. Mantilla R.D. Iglesias-Gamarra A. Cervera R. Anaya J.M. Factors influencing polyautoimmunity in systemic lupus erythematosus.Autoimmun Rev. 2010; 9: 229-232Crossref PubMed Scopus (54) Google Scholar Numerous studies have shown a significantly increased frequency of circulating aPLs in cancer across multiple tumor types, compared with matched controls, with incidence generally 15% to 25%.10Tincani A. Taraborelli M. Cattaneo R. Antiphospholipid antibodies and malignancies.Autoimmun Rev. 2010; 9: 200-202Crossref PubMed Scopus (58) Google Scholar, 11Font C. Vidal L. Espinosa G. Tassies D. Monteagudo J. Farrus B. Visa L. Cervera R. Gascon P. Reverter J.C. Solid cancer, antiphospholipid antibodies, and venous thromboembolism.Autoimmun Rev. 2011; 10: 222-227Crossref PubMed Scopus (33) Google Scholar, 12Reinstein E. Shoenfeld Y. Antiphospholipid syndrome and cancer.Clin Rev Allergy Immunol. 2007; 32: 184-187Crossref PubMed Scopus (24) Google Scholar Because the linkage of aPLs to coagulation is well-known, studies of the pathological implications of aPLs in cancer have focused on thromboembolic disorders, a common complication in many malignancies.11Font C. Vidal L. Espinosa G. Tassies D. Monteagudo J. Farrus B. Visa L. Cervera R. Gascon P. Reverter J.C. Solid cancer, antiphospholipid antibodies, and venous thromboembolism.Autoimmun Rev. 2011; 10: 222-227Crossref PubMed Scopus (33) Google Scholar Although comparatively few studies have examined aPLs as a potential risk factor for cancer development or progression, studies with relatively short-time follow-up found an association between aPLs and non–coagulation-related cancer mortality,13Endler G. Marsik C. Jilma B. Schickbauer T. Vormittag R. Wagner O. Mannhalter C. Rumpold H. Pabinger I. Anti-cardiolipin antibodies and overall survival in a large cohort: preliminary report.Clin Chem. 2006; 52: 1040-1044Crossref PubMed Scopus (11) Google Scholar and aPL-positive patients experienced a higher than expected frequency of non-Hodgkin lymphoma.14Finazzi G. The Italian Registry of Antiphospholipid Antibodies.Haematologica. 1997; 82: 101-105PubMed Google Scholar The potential for aPL as an angiogenic factor has long been evident. aPLs can induce the expression of vascular endothelial growth factor (VEGF) and tissue factor (TF) in monocytes and endothelial cells.15Satta N. Kruithof E.K. Fickentscher C. Dunoyer-Geindre S. Boehlen F. Reber G. Burger D. de Moerloose P. Toll-like receptor 2 mediates the activation of human monocytes and endothelial cells by antiphospholipid antibodies.Blood. 2011; 117: 5523-5531Crossref PubMed Scopus (86) Google Scholar, 16Cuadrado M.J. Buendia P. Velasco F. Aguirre M.A. Barbarroja N. Torres L.A. Khamashta M. Lopez-Pedrera C. Vascular endothelial growth factor expression in monocytes from patients with primary antiphospholipid syndrome.J Thromb Haemost. 2006; 4: 2461-2469Crossref PubMed Scopus (79) Google Scholar, 17Williams F.M. Parmar K. Hughes G.R. Hunt B.J. Systemic endothelial cell markers in primary antiphospholipid syndrome.Thromb Haemost. 2000; 84: 742-746PubMed Google Scholar, 18Motoki Y. Nojima J. Yanagihara M. Tsuneoka H. Matsui T. Yamamoto M. Ichihara K. Anti-phospholipid antibodies contribute to arteriosclerosis in patients with systemic lupus erythematosus through induction of tissue factor expression and cytokine production from peripheral blood mononuclear cells.Thromb Res. 2012; 130: 667-673Abstract Full Text Full Text PDF PubMed Scopus (19) Google Scholar TF, a central factor in coagulation cascade, can stimulate angiogenesis via either procoagulant function and platelet activation or, alternatively, the direct activation of protease-activated receptor-2 (PAR2; alias coagulation factor II receptor-like 1) and proangiogenic signaling in endothelial cells.19Mackman N. Davis G.E. Blood coagulation and blood vessel development: is tissue factor the missing link?.Arterioscler Thromb Vasc Biol. 2011; 31: 2364-2366Crossref PubMed Scopus (13) Google Scholar, 20Kasthuri R.S. Taubman M.B. Mackman N. Role of tissue factor in cancer.J Clin Oncol. 2009; 27: 4834-4838Crossref PubMed Scopus (307) Google Scholar Many tumor cells produce TF, and tumor cell–derived TF has been shown to promote tumor progression.20Kasthuri R.S. Taubman M.B. Mackman N. Role of tissue factor in cancer.J Clin Oncol. 2009; 27: 4834-4838Crossref PubMed Scopus (307) Google Scholar, 21Schaffner F. Yokota N. Ruf W. Tissue factor proangiogenic signaling in cancer progression.Thromb Res. 2012; 129: S127-S131Abstract Full Text PDF PubMed Scopus (29) Google Scholar Despite these studies, the effects of aPLs from patients on tumor cells and angiogenesis have not been previously examined. In this study, we present evidence that aPL activates proangiogenic gene expression in breast cancer cells, induces TF-dependent vascularization in small, slow-growing avascular tumors, and promotes tumor progression in multiple mouse models of breast cancer. Collectively, these findings indicate a potential protumor role for these autoantibodies. All procedures were approved by the Institutional Animal Care and Use Committees of Albert Einstein College of Medicine (Einstein; New York, NY) and by the Animal Care and Use Review Office (US Department of Defense). All mice were housed in the pathogen-free barrier facility at Einstein. Nude mice in NCr background were obtained from Taconic Farms (Albany, New York) and bred at Einstein. Polyoma virus middle T antigen (PyMT) mice were kindly provided by Dr. Jeffrey W. Pollard (Albert Einstein College of Medicine, Bronx, NY) and backcrossed to mice with C57BL/6 background for >10 generations. MDA-MB-436 breast cancer cells were obtained from the National Cancer Institute Tumor Repository (Frederick, MD). The breast cancer lines MDA-MB-468 and MDA-MB-231, as well as the prostate cancer cell line PC3, were obtained from ATCC (Manassas, VA). Cells were cultured according to the supplier's recommendations. Four TF-knockdown shRNA constructs were selected from the shRNA library of The RNAi Consortium (vector backbone: pGIPZ). The target regions for three of the shRNA sequences, TFKD321 (5′-CTTCTATGGTTGACATTGT-3′), TFKD322 (5′-CTGTTATTACCATTAGCAT-3′), and TFKD323 (5′-TGGAGCTACTGCAAATGCT-3′), were in the 3′ untranslated region of the TF (F3) gene; a fourth, 5′TFKD324 (5′-AAGTCTACACTGTTCAAAT-3′), was within the coding sequence of exon 2. The lentiviral expression vector carried turbo-green fluorescent protein (GFP). Virus-carrying turbo-GFP alone was used as a positive control for transfection. Selection and lentiviral packaging of the constructs, along with an empty vector control, were performed by the shRNA Core Facility at Einstein. Lentivirus carrying the above constructs was transfected into human breast cancer cells following the GIPZ lentiviral shRNA technical manual from Thermo Scientific (Waltham, MA). Lentivirally transduced cells were selected in 6 μg/mL of puromycin for 7 days, after which >90% of cells were GFP positive. Procedures using human samples were approved by the Institutional Review Board of Montefiore Medical Center (MMC)/Einstein (09-06-190X). Residual sera, collected for preoperational tests from patients with abnormal breast mass at MMC, were examined for aPL titer (described below). The diagnosis and staging of breast lesions was performed by the surgical pathologists (Y.W. and other on-service pathologists) at MMC. Serum samples were assayed for aPL by the Immunodiagnostic Laboratory at MMC using a commercial ELISA kit from BIO-RAD (Hercules, CA). The assays included the following: anti-cardiolipin (IgM, IgG), anti–β-2 glycoprotein I (IgM, IgG, IgA), and anti-phosphatidylserine (aPS; IgM, IgG). Samples with medium/high titer, according to criteria established by international consensus,22Miyakis S. Lockshin M.D. Atsumi T. Branch D.W. Brey R.L. Cervera R. Derksen R.H. DE Groot P.G. Koike T. Meroni P.L. Reber G. Shoenfeld Y. Tincani A. Vlachoyiannopoulos P.G. Krilis S.A. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS).J Thromb Haemost. 2006; 4: 295-306Crossref PubMed Scopus (5155) Google Scholar were classified as aPL positive. Plasma samples, obtained from two patients with APS, were designated as aPL#1 and aPL#3. These samples, as well those from five healthy donors (referred to as control IgG herein), were prepared from discard plasma samples obtained during diagnostic or other unrelated clinical procedures (Institutional Review Board number 09-06-190X). Serum was prepared from plasma by incubation with 1 U/mL human thrombin (Sigma-Aldrich, St. Louis, MO) at 37°C for 30 minutes, followed by clot removal. Total IgG was isolated, as described previously,23Rand J.H. Wu X.X. Quinn A.S. Ashton A.W. Chen P.P. Hathcock J.J. Andree H.A. Taatjes D.J. Hydroxychloroquine protects the annexin A5 anticoagulant shield from disruption by antiphospholipid antibodies: evidence for a novel effect for an old antimalarial drug.Blood. 2010; 115: 2292-2299Crossref PubMed Scopus (194) Google Scholar using protein G Sepharose 4, according to the manufacturer's recommended procedure (Amersham Pharmacia Biotech, Piscataway, NJ). Purified IgGs were dialyzed against phosphate-buffered saline (PBS), and the protein concentration was determined by absorbance at 280 nm. A third sample of patient IgG with elevated aPL was obtained from LifeSpan BioSciences, Inc. (Seattle, WA) and designated aPL#2. aPL titers of all samples were determined as described above. IgGs from the three patients are referred to collectively as aPL+ IgGs. Affinity-purified aPS/anti-cardiolipin IgG was prepared by modification of a published method.24Donohoe S. Kingdom J.C. Mackie I.J. Affinity purified human antiphospholipid antibodies bind normal term placenta.Lupus. 1999; 8: 525-531Crossref PubMed Scopus (16) Google Scholar Briefly, phosphatidylserine and cardiolipin (Sigma-Aldrich) in 100% ethanol were mixed at a 2:3 molar ratio in a 50-mL conical tube, dried, and suspended in Tris-HCl buffer (pH 7.4). The tube was rotated at 37°C for 1 hour for liposome formation. aPL#1 serum was added, and the tube was rotated for 2 hours. Liposomes were then pelleted, washed with the same buffer, and resuspended in 20 mmol/L phosphate buffer containing 0.1% Tween 20 (pH 7.4). The affinity-purified IgGs were then isolated with protein G Sepharose 4 (Amersham Pharmacia Biotech). Affinity-purified IgG was approximately 14-fold enriched in aPL titer relative to unpurified IgG, as assessed by aPS activity. IgGs from patients or normal controls were treated with Detoxi-Gel Endotoxin Removing Gel (Thermo Scientific, Rockford, IL) following the manufacturer's recommended protocol. No significant differences were observed in the effect of human IgGs on tumor cells' proangiogenic gene expression nor tumor growth and leukocytic infiltration in xenografted breast model between Detoxi-Gel endotoxin-treated and untreated human IgGs. MDA-MB-436 cancer cells (1 × 105 per well) were plated in 96-well plates in RPMI 1640 medium for overnight growth. Cells were then treated with or without control or aPL+ IgG at the indicated concentrations for 24 hours and then assayed for viability or proliferation. To assay viability, cells were incubated for 4 hours with 0.5 μg/mL of MTT assay (Sigma-Aldrich). The reduction of MTT was measured using a standard method. To assay proliferation, cells were incubated with 1 μmol/L bromodeoxyuridine (BrdU; Sigma-Aldrich), and BrdU incorporation was determined by immunoassay according to the manufacturer's instructions (Millipore-Calbiochem, Billerica, MA). Cells (5 × 105 per well) were placed in 6-well plates in RPMI 1640 medium containing 1% fetal bovine serum for overnight growth. Cells were then treated for the indicated times with 0.2 to 0.4 mg/mL aPL+ or control IgGs. RNA was extracted using TRIzol (Invitrogen/Life Technologies, Grand Island, NY) and reverse transcribed with the Superscript II system (Invitrogen/Life Technologies), according to the manufacturer's instructions. Primers were obtained from Real Time Primers (Elkins Park, PA), and real-time quantitative PCR was performed on Applied BioSystems 7900HT (Applied Biosystems/Life Technology, Grand Island, NY). Glyceraldehyde-3-phosphate dehydrogenase–normalized mRNA for specific gene expression was calculated by the 2−ΔΔCT method using SDS software version 2.3 (Applied Biosystems/Life Technologies). Breast cancer xenografts were established by inoculation of human cancer cells (5 × 105 cells per site in 0.1 mL RPMI 1640 medium) into the thoracic or abdominal mammary glands of female nude mice at 7 to 10 weeks of age. For coinjection experiments, the inoculum was first incubated for 1 hour on ice in 0.1 mL RPMI 1640 medium containing 20 μg total polyclonal human IgG from either healthy donors or patients; alternatively, 9.5 μg affinity-purified IgG was used. For TF neutralization, the incubation was in the presence of 10 μg anti-human TF antibody (American Diagnostica-Sekisui, Stamford, CT). For experiments using i.p. administration of human IgGs (rather than coinjection), one injection of 80 mg/kg body weight (bw) human IgG was administrated i.p. at day 14 after grafting. Mice were monitored every 7 days for the formation of visible tumors. Tumor incidence was calculated as the percentage of grafted glands bearing visible tumors. Tumor volumes were measured with a caliper for longest (L) and shortest (S) length and thickness (Th) by a blinded observer (Y.-Y.W., A.V.N., or E.Y.L.), and total volume was calculated as LxSxTh. Tumor volume in grafts of GFP-expressing tumor cells was determined weekly using a whole body Xenogen IVIS imaging system (Caliper Life Sciences, Hopkinton, MA). Background signal was measured using parallel mammary glands injected with 5 × 105 non-transduced cells. Female PyMT mice at 6 weeks of age were administered one i.p. injection of approximately 80 mg/kg bw human IgG. Tumors in all thoracic and abdominal mammary glands were analyzed for tumor stage by a blinded observer (Y.-Y.W., A.V.N., or E.Y.L.) using criteria established and described in detail in previous reports.25Lin E.Y. Nguyen A.V. Russell R.G. Pollard J.W. Colony-stimulating factor 1 promotes progression of mammary tumors to malignancy.J Exp Med. 2001; 193: 727-740Crossref PubMed Scopus (1293) Google Scholar, 26Lin E.Y. Jones J.G. Li P. Zhu L. Whitney K.D. Muller W.J. Pollard J.W. Progression to malignancy in the polyoma middle T oncoprotein mouse breast cancer model provides a reliable model for human diseases.Am J Pathol. 2003; 163: 2113-2126Abstract Full Text Full Text PDF PubMed Scopus (769) Google Scholar Four mice per group were injected i.p. once with approximately 80 mg/kg bw aPL#1 IgG or alternatively coinjected with 1 × 105 MDA-MB-436 cells and 20 μg aPL#1 IgG into each of two mammary glands. Approximately 100 μL blood was collected from each mouse at the jugular vein every 7 days for 4 weeks (2 weeks for the coinjection group and 1 week for untreated controls). Control blood samples were collected from three untreated mice. The titers of aPS then were measured by the Immunodiagnostic Laboratory at MMC, as described above. For quantitation of tumor blood vessel density, vessels were labeled in vivo as described previously.27Lin E.Y. Li J.F. Gnatovskiy L. Deng Y. Zhu L. Grzesik D.A. Qian H. Xue X.N. Pollard J.W. Macrophages regulate the angiogenic switch in a mouse model of breast cancer.Cancer Res. 2006; 66: 11238-11246Crossref PubMed Scopus (814) Google Scholar Briefly, lysine-fixable, Texas Red–conjugated dextran (mol. wt.: 70,000) (Life Technologies), 6.2 mg/mL in PBS, was administrated i.v. (21 μg/gm bw) 5 to 7 minutes before tissue harvest. We demonstrated that this method precisely marked endothelium-lined tumor vessels in mice.27Lin E.Y. Li J.F. Gnatovskiy L. Deng Y. Zhu L. Grzesik D.A. Qian H. Xue X.N. Pollard J.W. Macrophages regulate the angiogenic switch in a mouse model of breast cancer.Cancer Res. 2006; 66: 11238-11246Crossref PubMed Scopus (814) Google Scholar Tissues were fixed with 10% formalin, divided into sections, and counterstained with DAPI, and the entire tumor area was imaged. Vessel density was measured as the ratio of Texas Red–positive area/DAPI-positive area using WCIF ImageJ software version 1.37a (NIH, Bethesda, MD). For size matching of tumors, the longest diameters of tumors were determined from the tagged image file format images using Photoshop (Adobe, New York, NY). To obtain fluorescent images of tumor-associated macrophages, lysine-fixable fluorescein isothiocyanate–dextran (mol. wt.: 70,000) (Life Technologies), 6.2 mg/mL in PBS, was administrated i.v. (21 μg/g bw) 2 hours before tissue harvest. We have previously demonstrated that this method specifically labels macrophages within the tumor.27Lin E.Y. Li J.F. Gnatovskiy L. Deng Y. Zhu L. Grzesik D.A. Qian H. Xue X.N. Pollard J.W. Macrophages regulate the angiogenic switch in a mouse model of breast cancer.Cancer Res. 2006; 66: 11238-11246Crossref PubMed Scopus (814) Google Scholar To assess α-smooth muscle actin (SMA)–positive cells and their association with blood vessels, sections were simultaneously stained with a monoclonal anti–α-SMA antibody (Sigma-Aldrich) and rabbit anti–von Willebrand factor (Dako, Carpinteria, CA), followed by Alexa Fluor-488–conjugated goat anti-mouse IgG2α and Texas Red–conjugated goat anti-rabbit antibodies (Molecular Probes/Life Technologies). Confocal images were prepared by the Analytic Imaging Core Facility at Einstein. The antibodies used were rabbit anti-human annexin A5,28Rand J.H. Wu X.X. Guller S. Gil J. Guha A. Scher J. Lockwood C.J. Reduction of annexin-V (placental anticoagulant protein-I) on placental villi of women with antiphospholipid antibodies and recurrent spontaneous abortion.Am J Obstet Gynecol. 1994; 171: 1566-1572Abstract Full Text PDF PubMed Scopus (169) Google Scholar rat anti-F4/80 (Caltag/Life Technologies), rat anti-Ly6G (clone: RB6-8C5) (BD Pharmingen, San Jose, CA), goat anti-TF (American Diagnostic-Sekisui, Stamford, CT), and mouse monoclonal anti-VEGF (Santa Cruz Biotechnology, Inc., Dallas, TX). Immunohistochemistry (IHC) was performed following standard procedures. For quantitation of leukocyte infiltration, the entire tumor area was imaged, and the ratio of diaminobenzidine-positive area/hematoxylin counterstain was determined using WCIF ImageJ software. Human breast cancer cells (5 × 105) were incubated on ice with 20 μg control or aPL+ IgG, as in the coinjection procedure. BD Matrigel Matrix (Growth Factor Reduced; BD Biosciences, San Jose, CA) was added to a final concentration of approximately 70%, and the mixture was injected into mammary gland. To label blood vessels in the plug, Texas Red–conjugated dextran was administered before harvest, as described above. Plugs were fixed in 10% formalin or zinc fixative (BD Biosciences) at 4°C overnight and then paraffin embedded and divided into sections. Gross images of the plugs were obtained using a Zeiss STEMI (Zeiss, Thornwood, NY) at the Einstein Analytical Imaging Core Facility. The unpaired Student's t-test was used to assess significance for all data sets, with the exception of tumor incidence, PyMT tumor stage, and aPL incidence in breast cancer patients, which were analyzed using a Fisher's exact test. In view of previous reports of aPL occurrence in cancer across multiple tumor types,10Tincani A. Taraborelli M. Cattaneo R. Antiphospholipid antibodies and malignancies.Autoimmun Rev. 2010; 9: 200-202Crossref PubMed Scopus (58) Google Scholar, 11Font C. Vidal L. Espinosa G. Tassies D. Monteagudo J. Farrus B. Visa L. Cervera R. Gascon P. Reverter J.C. Solid cancer, antiphospholipid antibodies, and venous thromboembolism.Autoimmun Rev. 2011; 10: 222-227Crossref PubMed Scopus (33) Google Scholar, 12Reinstein E. Shoenfeld Y. Antiphospholipid syndrome and cancer.Clin Rev Allergy Immunol. 2007; 32: 184-187Crossref PubMed Scopus (24) Google Scholar we examined aPL status in newly diagnosed breast cancer patients at MMC (Table 1). Eight patients diagnosed with carcinoma in situ and 13 patients with invasive carcinoma were tested for seven classes of autoantibody frequently detected in APS patients. Of the 21 total patients, 6 (29%) had elevated aPL according to international consensus laboratory criteria for APS.22Miyakis S. Lockshin M.D. Atsumi T. Branch D.W. Brey R.L. Cervera R. Derksen R.H. DE Groot P.G. Koike T. Meroni P.L. Reber G. Shoenfeld Y. Tincani A. Vlachoyiannopoulos P.G. Krilis S.A. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS).J Thromb Haemost. 2006; 4: 295-306Crossref PubMed Scopus (5155) Google Scholar This value is consistent with previous reports of aPL prevalence in cancer.10Tincani A. Taraborelli M. Cattaneo R. Antiphospholipid antibodies and malignancies.Autoimmun Rev. 2010; 9: 200-202Crossref PubMed Scopus (58) Google Scholar, 11Font C. Vidal L. Espinosa G. Tassies D. Monteagudo J. Farrus B. Visa L. Cervera R. Gascon P. Reverter J.C. Solid cancer, antiphospholipid antibodies, and venous thromboembolism.Autoimmun Rev. 2011; 10: 222-227Crossref PubMed Scopus (33) Google Scholar, 12Reinstein E. Shoenfeld Y. Antiphospholipid syndrome and cancer.Clin Rev Allergy Immunol. 2007; 32: 184-187Crossref PubMed Scopus (24) Google Scholar Notably, 6 (46%) of 13 invasive cases were aPL positive, in comparison to 0 of 8 patients with noninvasive tumors (P < 0.05), consistent with a potential association of aPL with invasive malignancy.Table 1aPL Titer in Patients with Breast LesionsaPL statusCarcinoma in situ∗Carcinoma in situ includes both ductal and lobular carcinoma in situ.Invasive carcinoma†Invasive carcinoma includes invasive ductal or lobular carcinoma.TotalaPL+066aPL−8715Total81321aPL+ (% total)046‡P < 0.05 (Fisher's exact test).29aPL titer was determined by the Immunodiagnostic Laboratory at MMC. The test includes seven serological assays: anti-cardiolipin (IgM, IgG); anti-β-2-glycoprotein I (IgM, IgG, IgA); and anti-phosphatidylserine (IgM, IgG). Sera are classified as aPL positive if they are medium/high titer according to criteria established by international consensus.22Miyakis S. Lockshin M.D. Atsumi T. Branch D.W. Brey R.L. Cervera R. Derksen R.H. DE Groot P.G. Koike T. Meroni P.L. Reber G. Shoenfeld Y. Tincani A. Vlachoyiannopoulos P.G. Krilis S.A. International consensus statement on an update of the classification criteria for definite antiphospholipid syndrome (APS).J Thromb Haemost. 2006; 4: 295-306Crossref PubMed Scopus (5155) Google Scholar Samples with one or more positive outcomes were scored as aPL positive.∗ Carcinoma in situ includes both ductal and lobular carcinoma in situ.† Invasive carcinoma includes invasive ductal or lobular carcinoma.‡ P < 0.05 (Fisher's exact test). Open table in a new tab aPL titer was determined by the Immunodiagnostic Laboratory at MMC. The test includes seven serological assays: anti-cardiolipin (IgM, IgG); anti-β-2-glycoprotein I (IgM, IgG, IgA); and anti-phosphatidylserine (IgM, IgG). Sera are classified as aPL positive if they are medium/high titer according to criteria established by international consensus.22Miyakis S. Lockshin M.D. Ats
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