Prospects for Vasculature Reorganization in Sentinel Lymph Nodes

[Cell Cycle 6:5, 514-517, 1 March 2007]; ©2007 Landes Bioscience

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Prospects for Vasculature Reorganization in Sentinel Lymph Nodes Abstract Primary tumors can induce vasculature and lymph channel reorganizations within sentinel lymph nodes before the arrival of cancer cells. The key blood vessels in such nodes that are remodeled are high endothelial venules (HEVs). The morphological altera‑ tion of HEVs in the presence of a cancer, coupled with the increased proliferation rate of the endothelial cells, results in a functional shifting of HEVs from immune response to blood‑flow carrier. This tumor‑induced reorganization is quite different from an endotoxin‑induced inflammatory vasculature alteration. We review some of the accepted doctrines on lymph flow and lymphatic metastasis in light of this reorganization. More investigations are needed to elucidate the molecular mechanisms underlying the morpho‑ logical and functional alteration of HEVs, the fluid exchanges between lymph and blood, the microenvironmental preparation for cancer cells to survive and expand in the lymph node, and the detail process of further distant dissemination of cancer cells from the lymph nodes. Further study of this pathological process may help to explain some clinical phenomena, and should aid in developing novel therapeutics and prevention strategies against cancer metastasis.

3Laboratory of Analytical, Cellular and Molecular Microscopy; Van Andel Research Institute; Grand Rapids, Michigan USA

*Correspondence to: Chao-Nan Qian; Laboratory of Cancer Genetics; Van Andel Research Institute; 333 Bostwick Avenue. NE; Grand Rapids, Michigan 49503 USA; Tel.: 616.234.5538; Fax: 616.234.5539; Email: [email protected]

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Original manuscript submitted: 01/22/07 Revised manuscript submitted: 02/01/07 Manuscript accepted: 02/02/07

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2Department of Nasopharyngeal Carcinoma; Sun Yat-sen University Cancer Center; 651 Dongfeng East Road; Guangzhou, China.

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1Laboratory of Cancer Genetics; Van Andel Research Institute; Grand Rapids, Michigan USA

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Chao-Nan Qian1,2 James H. Resau3 Bin Tean Teh1

Previously published as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=3931

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Tumor‑Induced Angiogenesis and Lymphangiogenesis within Metastatic Lymph Nodes In microscopic examination of a metastatic axillary lymph node from a typical breast cancer patient, one can easily identify large blood vessels nurturing the secondary tumor (Fig. 1A). Similar large blood vessels are also present in the same lymph node tissue section distant from the metastatic area (Fig. 1B), although they are less common in normal lymph node tissue. Moreover, these so‑called “mother vessels” within the secondary tumor nests are sometimes surrounded by remnant lymphoid tissues (dashed circle in Fig. 1A). These unique observations suggest that the mother vessels might not be de novo blood vessels generated after the arrival of cancer cells, but preexisting vessels remodeled before the establishment of the secondary tumor. We have recently reported that these mother vessels within secondary tumor sites are actually remodeled high endothelial venules (HEVs), which are preexistent in normal lymphoid tissue.1 In that study, we were the first to describe the transformation of HEVs from immune‑related vessels to tumor vasculature. The dramatic transformation of HEVs in the sentinel lymph node (SLN), coupled with the increased proliferation rate of HEV endothelial cells, occurs before the physical presence of metastatic tumor cells. The dilation of the lymphatic channel in the sentinel lymph node could also be induced by the primary tumor before metastasis. In animal models, the dilation of lymphatic channels before metastasis is positively correlated with primary tumor weight. Of particular interest for oncology is the observation that the tumor‑induced reorganization of lymph node vasculature is morphologically quite different from endotoxin‑induced inflammatory vasculature alteration.1 In endotoxin‑induced lymphadenopathy, the dilated lymph sinuses are full of lymphocytes; while the dilated lymph sinuses in tumor‑reactive lymphadenopathy are full of fluid with few cellular structures. The morphology of the HEVs is not altered at all in the endotoxin‑induced lymphadenopathy. Our findings imply that the mechanism of tumor‑induced vascular reorganization in the lymph node is not the same as that of inflammatory hyperemia, although they both could occur remote from the primary lesions.

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sentinel lymph node, tumor vasculature, high endothelial venule, angiogenesis, lymphangiogenesis, metastasis

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Acknowledgements

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We thank David Nadziejka, science editor of the Van Andel Research Institute, for critically reading this article. We also thank the Gerber Foundation, Hauenstein Foundation, Schregardus Family Foundation, and Amway Japan Limited for their financial support.

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Prospects for Vasculature Reorganization in Sentinel Lymph Nodes

the secondary tumors in the involved lymph nodes usually grow faster than primary tumors. On the other hand, the ample blood supply to the lymph node presumably ensures that the secondary tumor has a limited number of hypoxic cancer cells. The fewer hypoxic cancer cells in the involved lymph nodes will then result in the more sensitivity of the secondary tumor to radiotherapy and chemotherapy. Moreover, the increased blood supply in the lymph node may also result in better distribution of chemotherapeutic agents and suitable local drug concentration, which in turn contributes to better drug response. Further rigorous studies are required to support these assumptions.

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Revisit of Some Traditional Concepts about Lymph Flow and Lymphatic Metastasis

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In our study, the absence of dilation of the lymph sinus in the second lymph node station strongly indicates that the lymph produced by the primary tumor may return into the blood circulation after arrival at the sentinel lymph node. The complicated vascular and lymphatic networks in the sentinel lymph node under cancerous conditions may facilitate this kind of lymph flow shortcut. In Figure 2, we show functioning blood vessels hanging within the dilated lymph sinus in the sentinel lymph node before metastasis, providing a physical capability for the exchange of fluid between the two systems. It has been believed for a long time that after cancer cells arrive at the lymph node, the further dissemination most commonly relies on the cancer cells traveling through the final common lymphatic conduit (such as the thoracic duct or the right lymphatic duct) and then into the subclavian vein.7 However, in our study, the induction of enriched functional vasculature by the primary tumor in the sentinel lymph node provides a physical route for the cancer cells to directly enter the blood circulation and avoid the long journey through the thoracic duct. Real-time monitoring of the intravasation of cancer cells from sentinel lymph nodes may help to uncover whether this actually occurs.

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Figure 1. A tissue section of human axillary lymph node from a breast cancer patient was stained with a monoclonal antibody against von Willebrand factor, which is a blood vessel marker. (A) Inside the metastatic tumor lesion, besides the numerous capillary blood vessels, there are two large blood ves‑ sels (stars) surrounded by remnant lymphoid tissue (circled by dashed line). (B) Inside the lymphoid tissue from the same section of A but distant from the metastatic tumor lesion, there are also some large blood vessels (stars) similar to those found in the metastatic tumor lesion.

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Possible Consequences of Vasculature Reorganization in Metastatic Lymph Nodes

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Metastatic breast cancer lesions in regional lymph nodes are usually more sensitive to radiotherapy or induction chemotherapy when compared with the primary tumor.2‑4 The smaller tumor volume in regional lymph nodes relative to primary tumor volume has been considered the source of the differences in the therapy responses. However, in patients with nasopharyngeal carcinoma metastasized to the cervical lymph nodes, in which the secondary tumor volumes are frequently larger than the primary tumor volumes, similar observations have also been made: the tumors in the nodes have a faster and better response to induction chemotherapy or radiotherapy than the primary nasopharyngeal tumors.5,6 Obviously, tumor volume is not the main reason for the differing response rates. Our recent findings on the reorganization of the vasculature in the lymph node may help to explain these clinical phenomena. The tumor vasculature adapted from HEVs presumably provides an ample blood supply for accelerating tumor growth. As a consequence, www.landesbioscience.com

Issues and Challenges When one makes new observations, there are always many new and unanswered questions. The vasculature reorganization in sentinel lymph nodes before and after the arrival of metastatic cancer cells is such a situation. Further studies on this emerging field are necessary to elucidate the following issues. (1) The molecular mechanisms underlying the alterations of HEVs and lymph channels before metastasis. The known lymphangiogenesis inducers include VEGF‑A, VEGF‑C, VEGF‑D, FGF‑2, angiopoietin‑1, HGF, IGF and PDGF‑BB.8,9 In animal models, VEGF‑A and VEGF‑C have been reported to be able to induce lymphangiogenesis in sentinel lymph nodes.10,11 In our study, reorganization of the lymphatic channels and vascular system was not accomplished by injecting dead cells or plasma from tumor‑bearing mice.1 This fact, combined with the absence of any alteration of HEVs or lymph channels in the further lymph node stations, suggest that the inducer may not be a soluble molecule traveling in blood stream, but instead a larger lymph‑borne molecule that can accumulates in the sentinel lymph node via lymph flow. (2) The importance of the lymph flow shortcut into the circulation in sentinel lymph nodes. The confirmation of this shortcut and the exploration of the related molecular mechanism of establishing the shortcut will broaden our

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Figure 3. The tissue sections of a mouse popliteal lymph node were immuno‑ fluorescently stained with anti‑CD31 (red) coupled with anti‑desmin (green) or anti‑collagan IV (green) antibodies. The nuclei were stained with DAPI (blue).

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basement membrane surrounding HEVs in the presence of cancer. Unlike other venules, HEVs have abundant supporting pericytes and thick, often multilayered basement membrane (Fig. 3).14 However, the existence and evolution of these supportive structures in a cancerous condition remain unexplored. Studies in this area will help in understanding the hypothesized fluid exchange between lymph and blood, and the hypothesized intravasation procedure of cancer cells in the lymph node. (5) The microenvironment of lymph nodes could be favorable for activation of dormant cancer cells. The activation of dormant metastatic solitary cancer cells, which are usually resistant the chemotherapy, has been regarded as the main reason for latent relapse in cancer patients.15,16 Since lymph node involvement is usually manifest early in patients with metastatic cancers, it will be important to investigate how the microenvironment of lymph nodes is more suitable than the microenvironments of other organs for the activation of dormant metastatic cancer cells, resulting in early onset of secondary tumors in the nodes. Gene expression profiles in head and neck cancers show that disseminated tumor cells in the lymph node do not need to undergo additional developmental changes to survive and proliferate.17 The fact that metastatic cancer cells can easily replace all of the lymphocytes and other normal cells in the lymph node but retain the preexisting vascular system for a longer time also strongly implies that the microenvironment in lymph node is favorable for the expansion of metastatic tumors. (6) The distant spread of cancer cells beyond the sentinel lymph node could result from endothelial remodeling in the sentinel lymph node. Despites the lack of convincing evidence showing that clustered cancer cells are more likely to form secondary tumors than single cancer cells, the endothelial‑covered tumor emboli have been found in a wide variety of human cancers to generate invasion‑independent metastases to distant organs.18,19 In this process, tumor nests are first surrounded by sinusoidal blood vessels and then enter the circulation as endothelium‑coated tumor cell emboli. The remodeled, enlarged HEVs and

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Figure 2. Twenty days after inoculation of human nasopharyngeal carcinoma cells (CNE‑2 cells) into the left hind footpad of a nude mouse, an intraventric‑ ular injection of 5 ml of 70‑kDa fluorescein‑dextran was carried out prior to sacrifice of the mouse and isolation of the popliteal sentinel lymph node. The lymph node was then fixed for 3 hrs on ice in PBS containing 4% paraformal‑ dehyde. Immunofluorescent staining of CD‑31 (using rhodamine‑conjugated secondary antibody) and nuclei (using DAPI) was performed in the lymph node sections. (A) A Normaski image of the tissue sections shows the dilated lymph sinuses before metastasis. (B) The merged fluorescent image of the same tissue area of A shows the blood vessels (red) in the tissue. Note that there are some functional blood vessels that have dextran (green) in their lumens. (C) merged image of A and B shows some blood vessels hanging inside the dilated lymph sinuses.

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knowledge about lymph circulation in cancerous conditions and may provide novel therapeutic targets. To study this shortcut, an effective evaluation system is needed, including appropriate tracer molecules, real‑time imaging technology for animal models, and quantitative analytic programs. (3) The adaptability of HEVs has been reported in noncancerous scenarios.12 However, the adaptation and differentiation of HEVs seem more dramatic in cancerous conditions, as indicated by morphological alterations and the absence of HEV marker MECA79.1 Under normal conditions, HEVs express numerous cytokines on their cell surface for lymphocyte homing.13 Clarification of the temporal sequence of cytokine expression during the functional shifting of HEVs from immune response to blood flow carrier will likely provide new prognostic markers and therapeutic targets. (4) Evaluating the structural alteration of pericytes and 516

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lymph sinuses in the sentinel lymph node provide a rich source of endothelia. It is therefore reasonable to hypothesize that the remodeled HEVs and lymph sinuses could facilitate distant metastases via the formation of such emboli. Confirmation of this hypothesis would undoubtedly suggest a novel therapeutic approach to preventing distant metastasis, by blocking the remodeling procedure of HEV and lymphatic channel in the lymph nodes. In summary, primary tumor‑induced preparation of the “soil” of the sentinel lymph node to facilitate metastatic tumor growth is an important tumor biological event. Understanding of this process will aid in developing novel prevention strategies for cancer metastasis and novel therapeutics.

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