Nodal Lymphangiogenesis and Metastasis
2009; Elsevier BV; Volume: 175; Issue: 5 Linguagem: Inglês
10.2353/ajpath.2009.090420
ISSN1525-2191
AutoresSatoshi Hirakawa, Michael Detmar, Dontscho Kerjaschki, Shogo Nagamatsu, Keitaro Matsuo, Atsushi Tanemura, Nobuyuki Kamata, Koichiro Higashikawa, Hidenori Okazaki, Kenji Kameda, Hisayo Nishida‐Fukuda, Hideki Mori, Yasushi Hanakawa, Koji Sayama, Yuji Shirakata, Mikiko Tohyama, Sho Tokumaru, Ichiro Katayama, Koji Hashimoto,
Tópico(s)Hedgehog Signaling Pathway Studies
ResumoNodal lymphangiogenesis promotes distant lymph node (LN) metastasis in experimental cancer models. However, the role of nodal lymphangiogenesis in distant metastasis and in the overall survival of cancer patients remains unknown. Therefore, we investigated mechanisms that might facilitate regional and distant LN metastasis in extramammary Paget's disease (EMPD). We retrospectively analyzed the impact of tumor-induced lymphatic vessel activation on the survival of 116 patients, the largest cohort with EMPD studied to date. Nodal lymphangiogenesis was significantly increased in metastatic, compared with tumor-free, LNs (P = 0.022). Increased lymphatic invasion within regional LNs was significantly associated with distant metastasis in LN (P = 0.047) and organs (P = 0.003). Thus, invasion within regional LNs is a powerful indicator of systemic tumor spread and reduced patient survival in EMPD (P = 0.0004). Lymphatic vessels associated with tumors expressed stromal cell-derived factor-1 (SDF-1), whereas CXCR4 was expressed on invasive Paget cells undergoing epithelial-mesenchymal transition (EMT)-like process. A431 cells overexpressing Snail expressed increased levels of CXCR4 in the presence of transforming growth factor-β1. Haptotactic migration assays confirmed that Snail-induced EMT-like process promotes tumor cell motility via the CXCR4-SDF-1 axis. Sinusoidal lymphatic endothelial cells and macrophages expressed SDF-1 in subcapsular sinuses of lymph nodes before Paget cell arrival. Our findings reveal that EMT-related features likely promote lymphatic metastasis of EMPD by activating the CXCR4-SDF-1 axis. Nodal lymphangiogenesis promotes distant lymph node (LN) metastasis in experimental cancer models. However, the role of nodal lymphangiogenesis in distant metastasis and in the overall survival of cancer patients remains unknown. Therefore, we investigated mechanisms that might facilitate regional and distant LN metastasis in extramammary Paget's disease (EMPD). We retrospectively analyzed the impact of tumor-induced lymphatic vessel activation on the survival of 116 patients, the largest cohort with EMPD studied to date. Nodal lymphangiogenesis was significantly increased in metastatic, compared with tumor-free, LNs (P = 0.022). Increased lymphatic invasion within regional LNs was significantly associated with distant metastasis in LN (P = 0.047) and organs (P = 0.003). Thus, invasion within regional LNs is a powerful indicator of systemic tumor spread and reduced patient survival in EMPD (P = 0.0004). Lymphatic vessels associated with tumors expressed stromal cell-derived factor-1 (SDF-1), whereas CXCR4 was expressed on invasive Paget cells undergoing epithelial-mesenchymal transition (EMT)-like process. A431 cells overexpressing Snail expressed increased levels of CXCR4 in the presence of transforming growth factor-β1. Haptotactic migration assays confirmed that Snail-induced EMT-like process promotes tumor cell motility via the CXCR4-SDF-1 axis. Sinusoidal lymphatic endothelial cells and macrophages expressed SDF-1 in subcapsular sinuses of lymph nodes before Paget cell arrival. Our findings reveal that EMT-related features likely promote lymphatic metastasis of EMPD by activating the CXCR4-SDF-1 axis. The metastatic spread of cancer cells from a primary site generally occurs in sentinel lymph nodes (LNs). Thus, the presence and extent of LN metastasis determines staging and prognosis in most human malignancies and often guides therapeutic decisions.1Van Trappen PO Pepper MS Lymphatic dissemination of tumour cells and the formation of micrometastases.Lancet Oncol. 2002; 3: 44-52Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar Although surgical resection of primary tumors and their regional LN metastases can cure several types of cancer, distant LN and organ metastases represent a significant therapeutic concern due to the absence of effective antimetastatic therapies. The mechanisms of tumor cell metastasis to regional and distant LNs have remained unclear, mainly due to the absence of lymphatic-specific markers and lack of insight into the molecular mechanisms mediating tumor cell entry and persistence within the lymphatic system. 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Thus, primary tumors in the skin induced LN lymphangiogenesis even before they metastasized, thereby preparing the lymphvascular niche, a tumor-conditioned microenvironment that serves as a future metastatic site within the regional LNs.18Hirakawa S Kodama S Kunstfeld R Kajiya K Brown LF Detmar M VEGF-A induces tumor and sentinel lymph node lymphangiogenesis and promotes lymphatic metastasis.J Exp Med. 2005; 201: 1089-1099Crossref PubMed Scopus (583) Google Scholar, 20Hirakawa S Brown LF Kodama S Paavonen K Alitalo K Detmar M VEGF-C-induced lymphangiogenesis in sentinel lymph nodes promotes tumor metastasis to distant sites.Blood. 2007; 109: 1010-1017Crossref PubMed Scopus (424) Google Scholar, 21Hirakawa S From tumor lymphangiogenesis to lymphvascular niche.Cancer Sci. 2009; 100: 983-989Crossref PubMed Scopus (76) Google Scholar Recent studies have shown that stromal cell-derived factor (SDF)-1, a ligand for the chemokine receptor CXCR4, is required for the formation of vascular niches that maintain hematopoietic stem cells in murine bone marrow.22Sugiyama T Kohara H Noda M Nagasawa T Maintenance of the hematopoietic stem cell pool by CXCL12-CXCR4 chemokine signaling in bone marrow stromal cell niches.Immunity. 2006; 25: 977-988Abstract Full Text Full Text PDF PubMed Scopus (1703) Google Scholar, 23Chiang AC Massague J Molecular basis of metastasis.N Engl J Med. 2008; 359: 2814-2823Crossref PubMed Scopus (805) Google Scholar, 24Kopp HG Avecilla ST Hooper AT Rafii S The bone marrow vascular niche: home of HSC differentiation and mobilization.Physiology. 2005; 20: 349-356Crossref PubMed Scopus (418) Google Scholar Furthermore, CXCR4 is induced in several types of invasive cancers. 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Increasing evidence indicates that tumor lymphangiogenesis arises in different types of human cancers, and that tumor-associated lymphatic vessel expansion is associated with enhanced rates of sentinel LN metastasis and reduced patient survival.28Rinderknecht M Detmar M Tumor lymphangiogenesis and melanoma metastasis.J Cell Physiol. 2008; 216: 347-354Crossref PubMed Scopus (125) Google Scholar, 29Dadras SS Lange-Asschenfeldt B Velasco P Nguyen L Vora A Muzikansky A Jahnke K Hauschild A Hirakawa S Mihm MC Detmar M Tumor lymphangiogenesis predicts melanoma metastasis to sentinel lymph nodes.Mod Pathol. 2005; 18: 1232-1242Crossref PubMed Scopus (280) Google Scholar, 30Dadras SS Paul T Bertoncini J Brown LF Muzikansky A Jackson DG Ellwanger U Garbe C Mihm MC Detmar M Tumor lymphangiogenesis: a novel prognostic indicator for cutaneous melanoma metastasis and survival.Am J Pathol. 2003; 162: 1951-1960Abstract Full Text Full Text PDF PubMed Scopus (446) Google Scholar However, the mechanisms of tumor lymphangiogenesis and its relative importance to cancer metastasis and the survival of patients with different types of cancer have remained controversial. Therefore, the function(s) of tumor-associated lymphatic vessels need to be clarified so that interactions between tumor-associated LECs and invasive tumor cells within primary sites and the subsequent formation of LN metastasis can be understood in more detail. Extramammary Paget's disease (EMPD) is a cutaneous adenocarcinoma that is characterized by the presence of vacuolated Paget cells.31Cooper SM WF Bolognia JL JJ Rapini RP Anogenital (Non-venereal) disease. MOSBY Elsevier, Edinburgh2008: 1059Google Scholar The condition usually develops in genital and/or axillary skin and appears as an erythematous plaque at the early stages that is characterized by slow intraepidermal growth. During tumor progression, EMPD can develop nodules and ulcerations associated with local tissue invasion. Subsequently, tumors metastasize to regional LNs and distant organs, leading to a poor outcome.32MacKie RM CE Burns TBS Cox N Griffiths C Tumours of the skin appendages. Blackwell Publishing, Malden2004: 37.31Google Scholar Although these clinical features indicate that EMPD progression is associated with tumor lymphangiogenesis and angiogenesis, the occurrence and the pathogenetic role of vascular activation in EMPD have not been studied. Moreover, little is understood about the mechanisms through which Paget cells acquire the invasive phenotype that spreads to LNs and beyond. The epithelial-mesenchymal transition (EMT), which plays a key role in promoting embryonic development, induces the striking transformation of epithelial cells to adopt the features of mesenchymal cells such as the expression of N-cadherin and loss of E-cadherin.23Chiang AC Massague J Molecular basis of metastasis.N Engl J Med. 2008; 359: 2814-2823Crossref PubMed Scopus (805) Google Scholar, 33Thiery JP Epithelial-mesenchymal transitions in tumour progression.Nat Rev Cancer. 2002; 2: 442-454Crossref PubMed Scopus (5489) Google Scholar, 34Kang Y Massague J Epithelial-mesenchymal transitions: twist in development and metastasis.Cell. 2004; 118: 277-279Abstract Full Text Full Text PDF PubMed Scopus (1245) Google Scholar Recent studies have further proposed that cancer cells can acquire EMT-like phenotypes such as loss of cell polarity, loss of cell-cell adhesion, and/or loss of keratin expression and considerable expression of vimentin during tumor progression.35Lee JM Dedhar S Kalluri R Thompson EW The epithelial-mesenchymal transition: new insights in signaling, development, and disease.J Cell Biol. 2006; 172: 973-981Crossref PubMed Scopus (1709) Google Scholar, 36Klymkowsky MW Savagner P Epithelial-mesenchymal transition: a cancer researcher's conceptual friend and foe.Am J Pathol. 2009; 174: 1588-1593Abstract Full Text Full Text PDF PubMed Scopus (403) Google Scholar Among the molecular regulators of EMT-like processes in the tumor microenvironment, the transcription factor Snail initiates the down-regulation of E-cadherin expression in several tumor cells of epithelial origin, enabling these cells to detach from the tumor mass and to invade the surrounding stroma toward tumor-associated blood vessels to metastasize to distant organs.37Gupta GP Massague J Cancer metastasis: building a framework.Cell. 2006; 127: 679-695Abstract Full Text Full Text PDF PubMed Scopus (3299) Google Scholar The pleiotropic cytokine transforming growth factor (TGF)-β1 promotes EMT38Moustakas A Heldin CH Signaling networks guiding epithelial-mesenchymal transitions during embryogenesis and cancer progression.Cancer Sci. 2007; 98: 1512-1520Crossref PubMed Scopus (655) Google Scholar but whether EMT-like behavior by tumor cells enhances their invasion of lymphatic vessels in primary sites remains obscure. The present study investigates tumor angiogenesis, lymphangiogenesis, lymphatic invasion, and EMT-like phenotypes in 116 patients with EMPD, representing the largest cohort of this type analyzed in a single study. We investigated whether nodal lymphangiogenesis and/or lymphatic invasion within regional LNs promotes distant LN metastasis and examined the molecular mechanisms promoting lymphatic cancer spread in EMPD. Overall, we identified lymphatic invasion of regional LNs as a novel, significant prognostic indicator of metastasis and survival of patients with EMPD. Moreover, we found that invasive Paget cells undergoing EMT-like process express CXCR4, whereas its ligand SDF-1 is produced by tumor-associated lymphatic vessels and lymphatic sinuses of draining lymph nodes. Thus, the SDF1 axis plays an important role in mediating EMPD LN metastasis, and CXCR4 expression by Paget cells predicts tumor metastasis and patient survival. Patients with EMPD in the groin area were retrospectively identified through a review of survival data from the Graduate Schools of Medicine at Ehime and Osaka Universities. Surgical samples from primary skin tumors were collected from 116 patients and tissue samples of regional LNs were obtained from 45 of them. H&E staining of primary tumors in the skin identified carcinoma in situ (CIS) in 73 patients and invasive growth of primary tumors in the dermis of 43 of them. Regional LN metastasis was confirmed at the time of diagnosis in 20 of these patients and 23 had none. We also obtained representative sections from 17 patients with Bowen's disease, 42 patients with malignant melanoma in situ, and 15 normal skin samples from the surgical margin. This retrospective study was approved by the institutional review boards of the Graduate Schools of Medicine at Ehime and Osaka Universities. Table 1 shows the clinical and pathological features used to diagnose EMPD. N-factors categorized no evident metastasis as N0, unilateral regional LN metastasis as N1, or bilateral regional LN metastasis as N2. We specifically categorized M-factors as no distant metastasis evident (M0), metastasis in distant LN(s) beyond regional LN(s), or metastasis in visceral organs (lung, liver, bone). Distant LN and organ metastases were detected by computer-assisted tomography, ultrasound examination, or skeletal scintigraphy. The Tumor Necrosis Metastasis classification for EMPD was designed and standardized by the Japanese Skin Cancer Society.39The Japanese Skin Cancer Society Extramammary Padget's disease.in: Saida T General rules for clinical and pathological studies on malignant neoplasms of the skin. Kanehara & Co. Ltd., Tokyo2002: 58Google ScholarTable 1Clinical and Pathological Characteristics of Patients with EMPDCategoryNonmetastaticMetastaticNo. of patients9620 Male6619 Female301Age at diagnosis (yr) Mean71.371.1 Range45–9244–82Stage IA, T1N0M0720 IB, T2N0M0210 II, T3N0M020 III, T4N0M0, anyTN1M018 IV, any TN2M0, any T any NMLN, any T any NMVO012T-factor T1, Carcinoma in situ720 T2, Microinvasive212 T3, Invasive without vascular invasion24 T4, Invasive with vascular invasion114N-factor N0, Metastasis undetectable960 N1, Unilateral regional LN metastasis011 N2, Bilateral regional LN metastasis09M-factor M0, Distant metastasis undetectable1000 MLN, Metastasis in distant LN beyond regional LN015 MVO, Metastasis in visceral organs (lung, liver, bone)014Units, except for age, are shown as numbers at time of diagnosis. Open table in a new tab Units, except for age, are shown as numbers at time of diagnosis. Primary tumors or LNs were fixed in buffered formalin, or embedded in OCT compound (Sakura Finetek, Torrance, CA) and snap-frozen. Paraffin (5 μm) or cryostat sections were immunostained as previously described,18Hirakawa S Kodama S Kunstfeld R Kajiya K Brown LF Detmar M VEGF-A induces tumor and sentinel lymph node lymphangiogenesis and promotes lymphatic metastasis.J Exp Med. 2005; 201: 1089-1099Crossref PubMed Scopus (583) Google Scholar, 20Hirakawa S Brown LF Kodama S Paavonen K Alitalo K Detmar M VEGF-C-induced lymphangiogenesis in sentinel lymph nodes promotes tumor metastasis to distant sites.Blood. 2007; 109: 1010-1017Crossref PubMed Scopus (424) Google Scholar using the primary antibodies shown in Table 2. The respective secondary antibodies were labeled with Alexa Fluor 488 or 594 (Molecular Probes, Eugene, OR). Antigens were usually retrieved in paraffin sections by incubation with citrate buffer (pH 6.0 for 30 minutes at 95°C) before immunostaining. Nuclei were counterstained with 4′,6′-diamidino-2-phenylindole (DAPI) (Molecular Probes). Sections were also immunohistochemically stained using a 3-amino-9-ethylcabazole peroxidase substrate kit (Vector Laboratories, Burlingame, CA). Respective control IgG was stained as a specificity control. Sections were examined, and digital images were captured using a confocal laser scanning microscope LSM510 (Carl Zeiss, Jena, Germany) or A1 (Nikon, Tokyo, Japan).Table 2Antibodies Used for Immunofluorescence and ImmunohistochemistryAntibodyStaining forSourceTypeNZ-1 for podoplaninLymphaticsY KatoRat monoclonal, IgG2aD2–40 for podoplaninLymphaticsNichirei BiosciencesMouse monoclonal, IgG1Prox1LymphaticsRELIATECRabbit polyclonalLYVE-1LymphaticsRELIATECRabbit polyclonalVEGFR-3LymphaticsR&D SystemsGoat polyclonalNeuropilin-2LymphaticsR&D SystemsGoat polyclonalvon Willebrand factorPanvascularDakoRabbit polyclonalMIB-1 for Ki-67Proliferating cellsDakoMouse monoclonal, IgG1Cytokeratin 7Paget cellsDakoMouse monoclonal, IgG1VEGF-APaget cells, immune cellsThermo Fisher Scientific Inc.Rabbit polyclonalVEGF-CPaget cellsImmuno-Biological LaboratoriesRabbit polyclonalE-cadherinPaget cellsEpitomicsRabbit polyclonalN-cadherinPaget cells (EMT-like)UpstateMouse monoclonal, IgG1VimentinPaget cells (EMT-like)DakoMouse monoclonal, IgG1CXCR4Invasive Paget cellsR&D SystemsMouse monoclonal, IgG2aSDF-1Tumor-associated lymphaticsR&D SystemsMouse monoclonal, IgG1KP1 for CD68MacrophagesDakoMouse monoclonal, IgG1HAM56MacrophagesDakoMouse monoclonal, IgM Open table in a new tab Representative sections obtained from 116 patients with primary EMPD, 17 with Bowen's disease, 42 with malignant melanoma in situ, and from 15 samples of normal skin were double-stained by differential immunofluorescence for podoplanin and von Willebrand factor, and then analyzed using a LSM 510 microscope (Carl Zeiss). Computer-assisted morphometric analyses of blood and lymphatic vessels on captured images proceeded using IP-LAB software (Scanalytics, Billerica, MA) as described previously.18Hirakawa S Kodama S Kunstfeld R Kajiya K Brown LF Detmar M VEGF-A induces tumor and sentinel lymph node lymphangiogenesis and promotes lymphatic metastasis.J Exp Med. 2005; 201: 1089-1099Crossref PubMed Scopus (583) Google Scholar, 20Hirakawa S Brown LF Kodama S Paavonen K Alitalo K Detmar M VEGF-C-induced lymphangiogenesis in sentinel lymph nodes promotes tumor metastasis to distant sites.Blood. 2007; 109: 1010-1017Crossref PubMed Scopus (424) Google Scholar Data are displayed as box and whisker plots. Primary human dermal LECs were isolated from neonatal foreskins as described previously.40Hirakawa S Hong YK Harvey N Schacht V Matsuda K Libermann T Detmar M Identification of vascular lineage-specific genes by transcriptional profiling of isolated blood vascular and lymphatic endothelial cells.Am J Pathol. 2003; 162: 575-586Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar LECs (1 × 104) were seeded onto triplicate fibronectin-coated culture dishes and propagated in endothelial cell growth medium40Hirakawa S Hong YK Harvey N Schacht V Matsuda K Libermann T Detmar M Identification of vascular lineage-specific genes by transcriptional profiling of isolated blood vascular and lymphatic endothelial cells.Am J Pathol. 2003; 162: 575-586Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar containing 0.5% fetal bovine serum (Invitrogen, Grand Island, NY). Conditioned media were collected at days 1, 3, or 5. Human SDF-1α or CCL21 levels were subsequently measured using enzyme-linked immunosorbent assays (ELISAs; Quantikine M; R&D Systems, Minneapolis, MN). Data were normalized to cell number at each time point. Recombinant human TGF-β1, human SDF-1α and anti-human CXCR4 monoclonal antibody 12G5 were purchased from R&D Systems. A431 cells stably transfected with mouse Snail or control pcDNA 3.1 (Invitrogen, Carlsbad, CA) vector were incubated with or without 10 ng/ml of recombinant human TGF-β1.41Yokoyama K Kamata N Fujimoto R Tsutsumi S Tomonari M Taki M Hosokawa H Nagayama M Increased invasion and matrix metalloproteinase-2 expression by Snail-induced mesenchymal transition in squamous cell carcinomas.Int J Oncol. 2003; 22: 891-898PubMed Google Scholar Single cell suspensions were prepared using cell dissociation buffer (Invitrogen). These cells were stained with the biotinylated anti-CXCR4 antibody 12G5, and then positively and negatively labeled with streptavidin-conjugated Alexa Fluor 488 (BD Biosciences Pharmingen) and propidium iodide, respectively. Stained cells (>10,000 cells/sample) were analyzed by flow cytometry using a FACScan and a FACSCalibur (BD Biosciences) and analyzed by FlowJo software (Tree Star, San Carlos, CA). For migration assays, cells were seeded in serum-free Dulbecco's modified Eagle's medium containing 0.2% delipidized bovine serum albumin into the upper chambers of 24-well FluoroBlok inserts (BD Biosciences) in the presence of human CXCR4-neutralizing monoclonal antibody 12G5 (10 μg/ml), or corresponding control IgG, and incubated for 3 hours at 37°C in the presence of SDF-1α in the bottom chambers. Cells on the undersides of inserts were stained with DAPI (Molecular Probes), and migrated cells were counted by computer-assisted image analysis of three random ×10 fields per well. Three independent experiments were performed for each assay. Across-group comparisons were performed by one-way analysis of variance when appropriate, followed by t-tests for pairwise comparisons. P values were adjusted using Bonferroni's method. Trends were analyzed by linear regression adjusted for age and gender. Overall survival after the date of surgery was defined as the primary endpoint. The Kaplan-Meier product limit method was applied and comparisons according to markers were examined using the log-rank test. We defined P < 0.05 as statistically significant. The macroscopic appearance of EMPD is characterized by a red patch, usually in the groin region, indicating a high level of vascularization (Figure 1A). Routine H&E stain showed a few number of small vessels in normal skin (Figure 1B). In
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