Tumor-Induced Sentinel Lymph Node Lymphangiogenesis and Increased Lymph Flow Precede Melanoma Metastasis
2007; Elsevier BV; Volume: 170; Issue: 2 Linguagem: Inglês
10.2353/ajpath.2007.060761
ISSN1525-2191
AutoresMaria I. Harrell, Brian M. Iritani, Alanna Ruddell,
Tópico(s)Angiogenesis and VEGF in Cancer
ResumoLymphangiogenesis is associated with human and murine cancer metastasis, suggesting that lymphatic vessels are important for tumor dissemination. Lymphatic vessel alterations were examined using B16-F10 melanoma cells implanted in syngeneic C57Bl/6 mice, which form tumors metastasizing to draining lymph nodes and subsequently to the lungs. Footpad tumors showed no lymphatic or blood vessel growth; however, the tumor-draining popliteal lymph node featured greatly increased lymphatic sinuses. Lymph node lymphangiogenesis began before melanoma cells reached draining lymph nodes, indicating that primary tumors induce these alterations at a distance. Lymph flow imaging revealed that nanoparticle transit was greatly increased through tumor-draining relative to nondraining lymph nodes. Lymph node lymphatic sinuses and lymph flow were increased in mice implanted with unmarked or with foreign antigen-expressing melanomas, indicating that these effects are not due to foreign antigen expression. However, tumor-derived immune signaling could promote lymph node alterations, as macrophages infiltrated footpad tumors, whereas lymphocytes accumulated in tumor-draining lymph nodes. B lymphocytes are required for lymphangiogenesis and increased lymph flow through tumor-draining lymph nodes, as these alterations were not observed in mice deficient for B cells. Lymph node lymphangiogenesis and increased lymph flow through tumor-draining lymph nodes may actively promote metastasis via the lymphatics. Lymphangiogenesis is associated with human and murine cancer metastasis, suggesting that lymphatic vessels are important for tumor dissemination. Lymphatic vessel alterations were examined using B16-F10 melanoma cells implanted in syngeneic C57Bl/6 mice, which form tumors metastasizing to draining lymph nodes and subsequently to the lungs. Footpad tumors showed no lymphatic or blood vessel growth; however, the tumor-draining popliteal lymph node featured greatly increased lymphatic sinuses. Lymph node lymphangiogenesis began before melanoma cells reached draining lymph nodes, indicating that primary tumors induce these alterations at a distance. Lymph flow imaging revealed that nanoparticle transit was greatly increased through tumor-draining relative to nondraining lymph nodes. Lymph node lymphatic sinuses and lymph flow were increased in mice implanted with unmarked or with foreign antigen-expressing melanomas, indicating that these effects are not due to foreign antigen expression. However, tumor-derived immune signaling could promote lymph node alterations, as macrophages infiltrated footpad tumors, whereas lymphocytes accumulated in tumor-draining lymph nodes. B lymphocytes are required for lymphangiogenesis and increased lymph flow through tumor-draining lymph nodes, as these alterations were not observed in mice deficient for B cells. Lymph node lymphangiogenesis and increased lymph flow through tumor-draining lymph nodes may actively promote metastasis via the lymphatics. The contribution of the lymphatic system to tumor metastasis is being increasingly appreciated through studies of murine as well as human cancers.1Cao Y Emerging mechanisms of tumour lymphangiogenesis and lymphatic metastasis.Nat Rev Cancer. 2005; 5: 735-743Crossref PubMed Scopus (258) Google Scholar The discovery of the vascular endothelial growth factors VEGF-C and VEGF-D, which activate lymphatic vessel growth by stimulating VEGF receptor (VEGFR)-3 expressed on lymphatic endothelium, allowed examination of the role of lymphangiogenesis in tumor dissemination.2He Y Karpanen T Alitalo K Role of lymphangiogenic factors in tumor metastasis.Biochim Biophys Acta. 2004; 1654: 3-12PubMed Google Scholar, 3Stacker S Baldwin M Achen M The role of tumor lymphangiogenesis in metastatic spread.FASEB J. 2002; 16: 922-934Crossref PubMed Scopus (268) Google Scholar In mice, VEGF-C or VEGF-D overexpression promotes tumor lymphangiogenesis and lymph node (LN) metastasis,4Stacker SA Caesar C Baldwin ME Thornton GE Williams RA Prevo R Jackson DG Nishikawa S Kubo H Achen MG VEGF-D promotes the metastatic spread of tumor cells via the lymphatics.Nat Med. 2001; 7: 186-191Crossref PubMed Scopus (1058) Google Scholar, 5Skobe M Hawighorst T Jackson DG Prevo R Janes L Velasco P Riccardi L Alitalo K Claffey K Detmar M Induction of tumor lymphangiogenesis by VEGF-C promotes breast cancer metastasis.Nat Med. 2001; 7: 192-198Crossref PubMed Scopus (1498) Google Scholar, 6Mandriota SJ Jussila L Jeltsch M Compagni A Baetens D Prevo R Banerji S Huarte J Montesano R Jackson DG Orci L Alitalo K Christofori G Pepper MS Vascular endothelial growth factor-C-mediated lymphangiogenesis promotes tumour metastasis.EMBO J. 2001; 20: 672-682Crossref PubMed Scopus (836) Google Scholar whereas inhibition of VEGFR-3 signaling blocks lymphatic vessel growth and tumor dissemination.7He Y Kozaki K Karpanen T Koshikawa K Yla-Herttuala S Takahashi T Alitalo K Suppression of tumor lymphangiogenesis and lymph node metastasis by blocking vascular endothelial growth factor receptor 3 signaling.J Natl Cancer Inst. 2002; 94: 819-825Crossref PubMed Scopus (451) Google Scholar, 8He Y Rajantie I Pajusola K Jeltsch M Holopainen T Yla-Herttuala S Harding T Jooss K Takahashi T Alitalo K Vascular endothelial cell growth factor receptor 3-mediated activation of lymphatic endothelium is crucial for tumor cell entry and spread via lymphatic vessels.Cancer Res. 2005; 65: 4739-4746Crossref PubMed Scopus (343) Google Scholar In human cancers, increased VEGF-C or VEGF-D expression is often associated with metastasis or poor prognosis.2He Y Karpanen T Alitalo K Role of lymphangiogenic factors in tumor metastasis.Biochim Biophys Acta. 2004; 1654: 3-12PubMed Google Scholar, 3Stacker S Baldwin M Achen M The role of tumor lymphangiogenesis in metastatic spread.FASEB J. 2002; 16: 922-934Crossref PubMed Scopus (268) Google Scholar Moreover, identification of tumor cells in tumor-draining sentinel LNs is increasingly used to diagnose metastatic cancers, including melanoma and breast cancer.9Turner RR Ollila DW Krasne DL Giuliano AE Histopathologic validation of the sentinel lymph node hypothesis for breast carcinoma.Ann Surg. 1997; 226: 271-278Crossref PubMed Scopus (586) Google Scholar, 10Morton DL Hoon DS Cochran AJ Turner RR Essner R Takeuchi H Wanek LA Glass E Foshag LJ Hsueh EC Bilchik AJ Elashoff D Elashoff R Lymphatic mapping and sentinel lymphadenectomy for early-stage melanoma: therapeutic utility and implications of nodal microanatomy and molecular staging for improving the accuracy of detection of nodal micrometastases.Ann Surg. 2003; 238: 538-549PubMed Google Scholar These findings indicate that the lymphatic system is involved in tumor dissemination to secondary organs, presumably by lymphatic delivery to the lymph nodes and to the systemic circulation via the thoracic duct. Abnormal blood vessel growth in tumors can alternatively promote tumor dissemination via the bloodstream, so that the lymphatic or the vascular systems can mediate metastasis depending on the particular type of cancer examined.1Cao Y Emerging mechanisms of tumour lymphangiogenesis and lymphatic metastasis.Nat Rev Cancer. 2005; 5: 735-743Crossref PubMed Scopus (258) Google Scholar Although the contribution of lymphatic vessels to tumor metastasis has been experimentally demonstrated, little is known yet about the mechanisms involved in tumor dissemination via the lymphatics. High tumor interstitial fluid pressure is thought to promote tumor cell entry into lymphatic vessels that have lower fluid pressure.11Jain RK Barriers to drug delivery in solid tumors.Sci Am. 1994; 271: 58-65Crossref PubMed Scopus (883) Google Scholar, 12Padera TP Kadambi A di Tomaso E Carreira CM Brown EB Boucher Y Choi NC Mathisen D Wain J Mark EJ Munn LL Jain RK Lymphatic metastasis in the absence of functional intratumor lymphatics.Science. 2002; 296: 1883-1886Crossref PubMed Scopus (813) Google Scholar Intratumoral lymphatic vessel growth often correlates with metastasis of human melanoma, breast, or head and neck cancers,13Dadras S Lange-Aschenfeldt 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, 14Maula SM Luukkaa M Granman R Jackson D Jalkanen S Ristamaki R Intratumoral lymphatics are essential for the metastatic spread and prognosis in squamous cell carcinomas of the head and neck region.Cancer Res. 2003; 63: 1920-1926PubMed Google Scholar, 15Choi WW Lewis MM Lawson D Yin-Goen Q Birdsong GG Cotsonis GA Cohen C Young AN Angiogenic and lymphangiogenic microvessel density in breast carcinoma: correlation with clinicopathologic parameters and VEGF-family gene expression.Mod Pathol. 2005; 18: 143-152Crossref PubMed Scopus (207) Google Scholar where tumor cells can be observed within lymphatic vessels, suggesting that lymphatic vessel growth is important for tumor spread. However, studies of murine and human tumors indicated that intratumoral lymphatics are often nonfunctional,12Padera TP Kadambi A di Tomaso E Carreira CM Brown EB Boucher Y Choi NC Mathisen D Wain J Mark EJ Munn LL Jain RK Lymphatic metastasis in the absence of functional intratumor lymphatics.Science. 2002; 296: 1883-1886Crossref PubMed Scopus (813) Google Scholar whereas expansion of peritumoral lymphatic vessels could instead mediate metastasis.16Bjorndahl MA Cao R Burton JB Brakenhielm E Religa P Galter D Wu L Cao Y Vascular endothelial growth factor-A promotes peritumoral lymphangiogenesis and lymphatic metastasis.Cancer Res. 2005; 65: 9261-9268Crossref PubMed Scopus (156) Google Scholar Moreover, a recent study demonstrated that inhibition of lymphangiogenesis does not block metastasis to tumor-draining LNs in mice.17Wong SY Haack H Crowley D Barry M Bronson RT Hynes RO Tumor-secreted vascular endothelial growth factor-C is necessary for prostate cancer lymphangiogenesis, but lymphangiogenesis is unnecessary for lymph node metastasis.Cancer Res. 2005; 65: 9789-9798Crossref PubMed Scopus (131) Google Scholar Hence, much remains to be learned about how tumor cells enter and travel through lymphatic vessels and lymph nodes during metastasis to secondary organs. Diagnosis of cancer metastasis frequently involves examining tumor-draining sentinel LNs for cancer cells. However, an active role of sentinel LNs in tumor dissemination via the lymphatics has not been considered until recently. We discovered extensive lymphangiogenesis in LNs from Eμ-c-myc transgenic mice developing lymphomas18Ruddell A Mezquita P Brandvold KA Farr A Iritani BM B lymphocyte-specific c-Myc expression stimulates early and functional expansion of the vasculature and lymphatics during lymphomagenesis.Am J Pathol. 2003; 163: 2233-2245Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar even before the development of lymphomas, which could contribute to lymphoma dissemination. In this model, Myc-expressing immature B cells accumulate in LN before progressing to form a highly metastatic lymphoma.19Harris AW Pinkert CA Crawford M Langdon WY Brinster RL Adams JM The Em-c-myc transgenic mouse: a model for high-incidence spontaneous lymphoma and leukemia of early B cells.J Exp Med. 1988; 167: 353-371Crossref PubMed Scopus (323) Google Scholar LNs of Eμ-c-myc mice exhibit active lymphatic sinus growth at early stages of lymphoma formation, whereas lymphatic vessels in other organs are unaffected.18Ruddell A Mezquita P Brandvold KA Farr A Iritani BM B lymphocyte-specific c-Myc expression stimulates early and functional expansion of the vasculature and lymphatics during lymphomagenesis.Am J Pathol. 2003; 163: 2233-2245Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar Lymphangiogenesis within LNs from Eμ-c-myc mice is accompanied by a 23-fold increase in lymph flow through LNs, as determined by a footpad dye injection assay. These findings led us to propose that LN lymphangiogenesis and increased lymph flow could actively promote dissemination of lymphomas to secondary organs. Our discovery of LN lymphangiogenesis in mice developing metastatic B cell lymphomas suggested that the LN could also be involved in lymphatic dissemination of solid tumors. In this study, we used the B16-F10 metastatic melanoma model to examine whether alterations of tumor or LN lymphatic vessels arise in mice developing metastatic solid tumors. The B16-F10 melanoma cell line was derived from a spontaneous melanoma arising in a C57Bl/6 mouse.20Fidler IJ Selection of successive tumor lines for metastasis.Nat New Biol. 1973; 242: 148-149Crossref PubMed Scopus (1275) Google Scholar Footpad injection of these cells produces metastatic melanoma detected in the tumor-draining LN and subsequently in the lungs.21Giavazzi R Garofalo A B16 melanoma metastasis.in: Brooks SA Schumacher U Methods in Molecular Medicine. Humana Press, Inc., Totowa, NJ2001: 223-229Google Scholar In this study, we identified extensive LN lymphangiogenesis and increased lymph flow through LNs draining B16 melanomas, which could promote dissemination of these tumors via the lymphatics. B16-F10 murine melanoma cells (American Type Culture Collection, Manassas, VA) were tested for mycoplasma or virus contamination before injection into mice (Research Animal Diagnostic Laboratory, University of Missouri, Columbia, MO). In some experiments, Anjou 293 packaging cells22Pear W Nolan G Scott M Baltimore D Production of high-titer helper-free retroviruses by transient transfection.Proc Natl Acad Sci USA. 1993; 90: 8392-8396Crossref PubMed Scopus (2301) Google Scholar were transiently transfected with the LXCG plasmid, a defective murine retroviral vector23Miller AD Miller DG Garcia JV Lynch CM Use of retroviral vectors for gene transfer and expression.Methods Enzymol. 1993; 217: 580-599Google Scholar encoding enhanced green fluorescent protein (GPF) (Clontech Laboratories, Inc., Mountain View, CA) downstream of the human cytomegalovirus immediate early promoter (generously provided by Dr. John Rasko, University of Sydney, Sydney, Australia). Viral supernatants were collected and incubated with B16 cells in 8 μg/ml Polybrene (Sigma-Aldrich, St. Louis, MO) for 2 days. Transduced cells were flow sorted for GFP positivity. Four- to 5-week-old C57BL/6J wild-type, homozygous C57BL/6J-TyrC-2J/J albino (carrying a spontaneous mutation in the tyrosinase gene), or homozygous μMT C57Bl/6 mice deficient for B lymphocytes were obtained from Jackson Laboratories (Bar Harbor, ME) and maintained in sterile microisolator rooms. Mice were injected in one hind footpad with 200,000 GFP-expressing or unmarked B16-F10 cells in a 50-μl volume of Hanks’ buffered saline solution,21Giavazzi R Garofalo A B16 melanoma metastasis.in: Brooks SA Schumacher U Methods in Molecular Medicine. Humana Press, Inc., Totowa, NJ2001: 223-229Google Scholar whereas the other footpad was injected with saline solution alone. After 18 to 22 days, mice were imaged under anesthesia for 30 minutes (see below) and euthanized with CO2, and tissues were dissected. Experimental methods involving animals were approved by the Fred Hutchinson Cancer Research Center Animal Care and Use Committee. Footpads or LN were serially sectioned to analyze the entire tissue. Eight-μm cryosections were fixed in acetone for 10 minutes, dried for 15 minutes, and formalin-fixed for 10 minutes, followed by 30 minutes of treatment with 0.3% hydrogen peroxide in methanol. Sections were immunostained with the following antibodies: MECA-32 (BD Biosciences, Bedford, MA), 10.1.1,24Farr A Nelson A Hosier S Kim A A novel cytokine-responsive cell surface glycoprotein defines a subset of medullary thymic epithelium in situ.J Immunol. 1993; 150: 1160-1171PubMed Google Scholar 8.1.1,24Farr A Nelson A Hosier S Kim A A novel cytokine-responsive cell surface glycoprotein defines a subset of medullary thymic epithelium in situ.J Immunol. 1993; 150: 1160-1171PubMed Google Scholar, 25Schacht V Ramirez MI Hong YK Hirakawa S Feng D Harvey N Williams M Dvorak AM Dvorak HF Oliver G Detmar M T1 alpha/podoplanin deficiency disrupts normal lymphatic vasculature formation and causes lymphedema.EMBO J. 2003; 22: 3546-3556Crossref PubMed Scopus (559) Google Scholar CD31 (BD Biosciences), or LYVE-1 (Upstate, Temecula, CA). Immunostaining was detected using horseradish peroxidase-labeled secondary antibodies with Vector VIP followed by methyl green counterstaining (Vector Laboratories, Burlingame, CA). Lymphatic vessel area was measured in 616 × 484-μm fields of 100× magnification images of 10.1.1 antibody-stained LN sections, using the NIH ImageJ program (National Institutes of Health, Bethesda, MD). Blood vessel area or density was similarly measured in LN sections immunostained with MECA-32 antibody. Individual lymphatic or blood vessels in the footpad were visually hand-counted by direct examination of microscope fields at ×100 magnification. Statistical analysis was performed by the two-tailed Student's t-test. Immunofluorescent staining with mitotic phosphohistone H3 (Upstate), 10.1.1, GFP (Molecular Probes, Eugene, OR), F4/80 (eBioscience, San Diego, CA), or control antibodies used acetone and formalin fixation and detection with fluorescein isothiocyanate (FITC)- or Alexa-labeled secondary antibodies (Molecular Probes) followed by mounting in DAPI-containing media (Vectashield; Vector Laboratories). Fixed sections were also directly immunostained with FITC-labeled MI/70 Mac-1 (BD Biosciences), rat IgG2b (BD Biosciences), or B220 antibodies (Caltag, Carlsbad, CA) after blocking with CD16/CD32 antibody to Fc receptor (Fc Block, BD Biosciences). The lymphocyte composition of LNs was determined by LN dissociation between frosted glass slides, nylon filtration, cell counting in a hemocytometer, and immunostaining with FITC-CD3 and PE-B220 (Caltag) followed by propidium iodide staining and flow cytometry. Real-time fluorescence images were obtained using a Xenogen IVIS Imaging System 100 equipped with a Cy5.5 filter set (Xenogen, Alameda, CA). Identical illumination settings (lamp voltage, filters) were used for all images, and fluorescence emission was normalized to fluorescent efficiency, where the value of each pixel in an efficiency image represents the fractional ratio of fluorescent emitted photons per incident excitation photon.26Troy T Jekic-McMullen D Sambucetti L Rice AB Quantitative comparison of the sensitivity of detection of fluorescent and bioluminescent reporters in animal models.Mol Imaging. 2004; 3: 9-23Crossref PubMed Scopus (329) Google Scholar Mice were anesthetized with 2 to 3% isoflurane, and were positioned supine in the Xenogen IVIS, with legs taped to expose the popliteal fossa. The dorsal toe of each hindfoot was then injected with 25 μl of Qtracker 705 nontargeted quantum dots (Qdot Corporation, Hayward, CA) or with 25 μl of Cy5.5-labeled magnetic nanoparticles (Nanocs, Inc., New York, NY), both diluted 1:1 in 0.3% Evans blue (Sigma-Aldrich) in saline. For determining lymph flow, total fluorescent efficiency of the popliteal LN area of the right and left leg of the animal were calculated over regions of interest using Living Image software (Xenogen) integrated with Igor (Wavemetrics, Lake Oswego, OR). Preinjection images were used to subtract background autofluorescence in the region of interest. Statistical analysis was performed with a two-tailed Student's paired t-test. The B16 melanoma cell line reliably undergoes metastasis to the tumor-draining popliteal LN and subsequently to the lungs within a few months after implantation in the footpad of syngeneic C57Bl/6 mice.21Giavazzi R Garofalo A B16 melanoma metastasis.in: Brooks SA Schumacher U Methods in Molecular Medicine. Humana Press, Inc., Totowa, NJ2001: 223-229Google Scholar The lymphatics of the foot drain directly through the popliteal LN,27Tilney NL Patterns of lymphatic drainage in the adult laboratory rat.J Anat. 1971; 109: 369-383PubMed Google Scholar which is advantageous for analysis of tumor spread through the draining LN. We used this model to characterize lymphatic and blood vessels in the primary tumor and draining popliteal LN as a first step to examine the involvement of the lymphatic system in dissemination of these tumors. Four-week-old albino C57Bl/6 mice were injected in one rear footpad with B16 cells, whereas the other rear footpad was injected with saline as an internal control. The melanoma cells were infected with a GFP-expressing retroviral vector to monitor tumor cell metastasis. Footpad tumors and LN were analyzed 18 to 22 days after tumor implantation, when tumors reached a 3- to 6-mm diameter. Serial sections of the resulting melanomas were analyzed by immunostaining with the 10.1.1 antibody, which specifically recognizes murine lymphatic endothelium.18Ruddell A Mezquita P Brandvold KA Farr A Iritani BM B lymphocyte-specific c-Myc expression stimulates early and functional expansion of the vasculature and lymphatics during lymphomagenesis.Am J Pathol. 2003; 163: 2233-2245Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 24Farr A Nelson A Hosier S Kim A A novel cytokine-responsive cell surface glycoprotein defines a subset of medullary thymic epithelium in situ.J Immunol. 1993; 150: 1160-1171PubMed Google Scholar The growing melanomas did not obviously alter the lymphatic vessels of the footpad. Lymphatic vessels are sparse in the control leg footpad and are also rarely observed within the footpad tumor (Figure 1A, arrows). Previous studies suggested that abnormal lymphatic vessels in the tumor periphery could mediate lymphatic metastasis.16Bjorndahl MA Cao R Burton JB Brakenhielm E Religa P Galter D Wu L Cao Y Vascular endothelial growth factor-A promotes peritumoral lymphangiogenesis and lymphatic metastasis.Cancer Res. 2005; 65: 9261-9268Crossref PubMed Scopus (156) Google Scholar However, we did not observe any increase in the number or size of occasional peritumoral lymphatic vessels. Immunostaining with the LYVE-1 antibody, which also recognizes lymphatic endothelium28Prevo R Banerji S Ferguson DJ Clasper S Jackson DG Mouse LYVE-1 is an endocytic receptor for hyaluronan in lymphatic endothelium.J Biol Chem. 2001; 276: 19420-19430Crossref PubMed Scopus (406) Google Scholar confirmed that lymphatic vessels are sparse in control footpads or in tumors (Figure 1B). These visual observations were confirmed by counting vessel density in regions spanning footpad tumors or in corresponding regions of the control leg footpad. Lymphatic density was similar in tumors and in control footpads (Figure 1E). These findings indicate that B16 melanoma growth within the footpad is not accompanied by intratumoral or peritumoral lymphatic vessel growth. Blood vessels in or around B16 footpad tumors were also unaffected by tumor growth, as shown by immunostaining with the vascular endothelial-specific MECA-32 antibody29Leppink DM Bishopp DK Sedmak DD Henry ML Ferguson RM Streeter PR Butcher EC Orosz CG Inducible expression of an endothelial cell antigen on murine myocardial vasculature in association with interstitial cellular infiltration.Transplantation. 1989; 48: 874-877Crossref PubMed Scopus (49) Google Scholar (Figure 1C, arrows). This finding was confirmed by immunostaining for CD31 (Figure 1D). Quantitation of MECA-32-positive vessels showed similar blood vessel density in tumors and in control footpads (Figure 1F). These findings indicate that B16 tumors do not obviously alter the vascular supply at early stages of tumor growth. Our finding that footpad melanomas fail to induce lymphatic or blood vessel growth suggested that some other mechanism promotes B16 tumor dissemination. The popliteal LN draining the footpad melanoma was examined to determine whether vessels in this lymphatic organ are altered by tumor growth. 10.1.1 immunostaining of the control leg popliteal LN showed sparse lymphatic sinuses restricted to the cortex (Figure 2A). However, the popliteal LN from the tumor-draining leg showed greatly increased and enlarged lymphatic sinuses distributed throughout the cortex and medulla (Figure 2, B and C). Lymphangiogenesis was consistently observed in the tumor-draining LNs from 10 mice whether the tumor was implanted in the left or right foot (data not shown). The LYVE-1 antibody, which also recognizes lymphatic endothelium,28Prevo R Banerji S Ferguson DJ Clasper S Jackson DG Mouse LYVE-1 is an endocytic receptor for hyaluronan in lymphatic endothelium.J Biol Chem. 2001; 276: 19420-19430Crossref PubMed Scopus (406) Google Scholar shows the expanded lymphatic sinuses (Figure 2H) in a pattern similar to that obtained with the 10.1.1 antibody (Figure 2, B and C). The 8.1.1 antibody to podoplanin expressed on lymphatic endothelium30Schacht V Dadras SS Johnson LA Jackson DG Hong YK Detmar M Up-regulation of the lymphatic marker podoplanin, a mucin-type transmembrane glycoprotein, in human squamous cell carcinomas and germ cell tumors.Am J Pathol. 2005; 166: 913-921Abstract Full Text Full Text PDF PubMed Scopus (531) Google Scholar also recognized these lymphatic sinuses (data not shown), confirming that lymphatic endothelium is increased within these LNs. These findings indicate that the tumor somehow promotes expansion of lymphatic sinuses in the draining LN. Quantification of the area occupied by 10.1.1-positive lymphatic vessels in LNs by NIH ImageJ measurement of microscope images demonstrated a ninefold increase in lymphatic vessel area in the tumor-draining LNs relative to the control LNs (Figure 3A). These data confirm that lymphatic sinus expansion is consistently induced in the tumor-draining LN within 3 weeks after tumor implantation in the footpad. The effects of the melanomas on lymphatic vessels are restricted to the tumor-draining LNs, as control leg popliteal LNs from tumor-bearing mice showed the same sparse pattern of lymphatic sinuses (Figure 2A) as popliteal LNs from normal littermates (data not shown). Moreover, the nondraining mesenteric LN of tumor-bearing or control mice showed normal lymphatic sinuses (data not shown). The expansion of lymphatic sinuses in the tumor-draining LN could result from the proliferation of lymphatic endothelial cells. We tested whether lymphatic endothelial cell proliferation is involved by immunostaining LN with anti-phosphohistone H3 antibody that recognizes a phosphorylated serine 10 residue specific for mitotic cells.31Ajiro K Yoda K Utsumi K Nishikawa Y Alteration of cell cycle-dependent histone phosphorylations by okadaic acid.J Biol Chem. 1996; 271: 13197-13201Crossref PubMed Scopus (128) Google Scholar Punctate phosphohistone staining of mitotic nuclei was often detected in 10.1.1-positive lymphatic endothelial cells in tumor-draining LNs (Figure 2G, arrows), whereas phosphohistone staining was rarely observed in control LNs (data not shown). Actively dividing cell populations show 1 to 2% positivity for mitotic phosphohistone H3,32Brandvold KA Ewert DL Kent SC Neiman P Ruddell A Blocked B cell differentiation and emigration support the early growth of Myc-induced lymphomas.Oncogene. 2001; 20: 3226-3234Crossref PubMed Scopus (14) Google Scholar so that our frequent detection of mitotic lymphatic endothelium indicates that proliferation contributes at least in part to the expansion of lymphatic sinuses in tumor-draining LN. To determine whether the LN alterations are associated with invasion by melanoma cells, we analyzed tumor-draining LNs for the presence of melanoma cells. Sections throughout the entire LN were either directly examined for immunofluorescent GFP cells, were stained with GFP antibodies, or examined by light microscopy for the black-pigmented melanoma cells. The lungs of these animals did not yet contain metastases, in agreement with previous studies showing that lung metastases are not visible until several months after implantation.21Giavazzi R Garofalo A B16 melanoma metastasis.in: Brooks SA Schumacher U Methods in Molecular Medicine. Humana Press, Inc., Totowa, NJ2001: 223-229Google Scholar At 18 to 22 days after implant, only two of 10 LNs contained pigmented metastases that were GFP-positive (Figure 2I, arrow), whereas one LN contained a small cluster of GFP-positive cells (data not shown). The other seven nonmetastatic tumor-draining LNs showed no sign of melanin- or GFP-positive (Figure 2J) cells. The extent of lymphangiogenesis in LNs containing metastases (Figure 2B) was similar to that observed in LNs that did not yet contain melanoma cells (Figure 2C). These findings suggest that tumors in the foot act at a distance to induce lymphangiogenesis within the popliteal LN before melanoma cells are detectible within the LN. On the other hand, a small number of undetected tumor cells within the LN could potentially provoke this strong response. We tested whether the tumor-draining LN also undergo angiogenesis in response to tumor-derived signals. MECA-32 immunostaining identified capillaries and high endothelial venules throughout the cortex and medulla of the control leg popliteal LN (Figure 2D). The pattern of MECA-32 antibody immunostaining was similar in the tumor-draining popliteal LNs with (Figure 2E) or without (Figure 2F) metastasis. Quantitation of blood vessel area (Figure 3B) or density (Figure 3C) from microscope images using the NIH ImageJ program confirmed that there was no significant blood vessel growth in the tumor-draining popliteal LN. These findings indicate that B16 tumors activate lymphangiogenesis within the draining LN without inducing blood vessel growth. We previously found that LN lymphangiogenesis in Eμ-c-myc mice is accompanied by a 23-fold increase in lymph flow as measured by incorporation of TRITC dextran into draining poplite
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