Lymphangiogenesis and Tumor Metastasis: Myth Or Reality?1

462 Vol. 7, 462–468, March 2001 Clinical Cancer Research

Minireview Lymphangiogenesis and Tumor Metastasis: Myth or Reality?1

Michael S. Pepper2 lymphatics is the most common pathway of initial dissemina- Department of Morphology, University Medical Center, 1211 Geneva tion, with patterns of spread via afferent lymphatics following 4, Switzerland routes of natural drainage. Sentinel lymph nodes are a variable but limited set of nodes that are the first to receive drainage from any given location. As a rule, carcinomas preferentially metas- Abstract tasize to these lymph nodes, although intralymphatic tumor cells The metastatic spread of tumor cells is responsible for can pass directly into the blood vascular system through the majority of cancer deaths, and with few exceptions, all venolymphatic communications, and vice versa. Sentinel lymph cancers can metastasize. Clinical findings have long sug- node biopsy and histopathological examination improve tumor gested that by providing a pathway for tumor cell dissemi- staging and facilitate the planning of therapeutic strategies (re- nation, tumor-associated lymphatics are a key component of viewed in Refs. 1–3). metastatic spread. It is not known, however, whether pre- Despite these well-established principles, many key ques- existing vessels are sufficient to serve this function, or tions regarding the mechanisms of lymphatic tumor spread still whether tumor cell dissemination requires de novo lym- remain unanswered: phatic formation (lymphangiogenesis) or an increase in lym- (a) Does the de novo formation of lymphatic capillaries phatic size. Lymphangiogenesis has traditionally been over- (lymphangiogenesis) and/or lymphatic enlargement dilatation shadowed by the greater emphasis placed on the blood increase the probability of lymphatic tumor dissemination be- vascular system (angiogenesis). This is due in part to the lack yond that which would occur exclusively through preexisting of identification of lymphangiogenic factors, as well as suit- vessels? able markers that distinguish blood from lymphatic vascu- (b) What are the molecular mechanisms of lymphangio- lar endothelium. This scenario is changing rapidly after the genesis and lymphatic enlargement? identification of the first lymphangiogenic factor, vascular (c) Is the process of tumor cell intravasation into lymphat- endothelial growth factor C (VEGF-C). Increased expres- ics analogous to that which occurs in the blood vascular system? sion of VEGF-C in primary tumors correlates with in- (d) Is inhibition of lymphangiogenesis a realistic therapeu- creased dissemination of tumor cells to regional lymph tic strategy for inhibiting tumor cell dissemination and the nodes in a variety of human carcinomas. Here I will review formation of metastasis? what is known about the mechanisms of lymphangiogenesis, It has long been suggested that “A lymphatic system as an particularly in the context of metastatic tumor spread, and anatomical entity is not demonstrable in tumors” (4), and studies will critically examine the role of VEGF-C in this process. in human and animal tumors involving injection of tracers into However, despite recent progress in this field, it remains to lymphatics revealed that tumors do not have an intrinsic lym- be determined whether inhibition of lymphangiogenesis is a phatic vascular supply (see, for example, Refs. 5 and 6). How- realistic therapeutic strategy for inhibiting tumor cell dis- ever, it has been proposed that this reflects the collapse of semination and the formation of metastasis. lymphatics within the tumor due to mechanical stress generated by proliferating cancer cells, and that this in turn contributes to Introduction increased pressure within the tumor interstitium (7). Although With few exceptions, all cancers can metastasize. Metas- the lack of intratumoral lymphatics appears to be a consistent tasis unequivocally signifies that a tumor is malignant, and the feature, dilated and engorged lymphatics in peritumoral stroma, metastatic spread of tumor cells is responsible for the majority which occasionally penetrate into the tumor periphery, are fea- of cancer deaths. Tumor dissemination may occur through a tures that are observed with equal frequency (7, 8). It has been number of pathways: (a) local tissue invasion; (b) direct seeding suggested that these lymphatics are likely to be existing lym- 3 of body cavities or surfaces; (c) hematogenous spread; and (d) phatics that have enlarged in response to VEGF-C , rather than lymphatic spread. Clinical and pathological observations sug- new lymphatic vessels (lymphangiogenesis; Ref. 7). gest that for many carcinomas, transport of tumor cells via The presence of tumor cells in peri- or juxtatumoral lymphatics is not an uncommon feature for many primary tumors. Does lymphatic dissemination of tumor cells require active tumor cell intravasation into lymphatic vessels? In a detailed study on breast carcinoma, Hartveit (8) has described the pres- Received 11/2/00; revised 12/13/00; accepted 12/28/00. ence of an open lymphatic labyrinth in close association with the The costs of publication of this article were defrayed in part by the primary tumor and suggests that “Tumor cells lying free in the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. 1 Supported by grants from the Swiss National Science Foundation. 2 To whom requests for reprints should be addressed, at Department of Morphology, University Medical Center, 1 rue Michel Servet, 1211 3 The abbreviations used are: VEGF, vascular endothelial growth factor; Gene`ve 4, Switzerland. Phone: 4122-702-52-91; Fax: 4122-347-33-34; VEGFR, VEGF receptor; bFGF, basic fibroblast growth factor; TNF, E-mail: [email protected]. tumor necrosis factor. Downloaded from clincancerres.aacrjournals.org on September 28, 2021. © 2001 American Association for Cancer Research. Clinical Cancer Research 463

periductal lymphatic spaces will be washed with the tide of ency to retract or undergo changes in size or form. Although tissue fluid into the labyrinth through the sinuses and on into the temporary lymphedema occurs after lymphatic disruption, this lymphatic drainage channels. There is no need to postulate usually resolves due to spontaneous regeneration or reconnec- active lymphatic invasion.” Evidence in favor of this hypothesis tion of lymphatics (18–20). Therefore, despite microsurgical or the alternative hypothesis of active tumor cell intravasation is advances in the repair of severed arteries, veins, and nerves, still lacking in the lymphatic system. disrupted lymphatics are usually not identified or reconnected during surgical intervention. In a study on incisional and punch biopsy skin wounds, Paavonen et al. (21) have confirmed that Mechanisms of Lymphangiogenesis in Vivo new lymphatic vessels appear a few days after blood vessels in Before considering the mechanisms of lymphangiogenesis, the peripheral granulation tissue. They also found that in resolv- it is necessary to recall the morphological features that distin- ing inflammation at the end of the wound-healing process, guish lymphatic capillaries from capillaries of the blood vascu- lymphatic capillaries tended to involute more rapidly than blood lar system. Like the latter, lymphatic capillaries are lined by a capillaries. Extensive lymphatic proliferation has also been ob- single layer of endothelial cells. However, unlike blood capil- served in acute and chronic inflammation outside of the wound- lary endothelium, lymphatic endothelial cells have poorly de- healing process (22). Newly formed lymphatics regress on res- veloped junctions with frequent large interendothelial gaps. olution of the inflammatory process. Lymphatic endothelial cells are also devoid of fenestrations. Lymphangiogenesis has traditionally been overshadowed Because pressure within lymphatic capillaries is only slightly by the greater emphasis placed on the blood vascular system higher than the interstitium, lumen patency is maintained by (angiogenesis). This is due in part to the lack of identification of anchoring filaments that connect the abluminal surfaces of en- lymphangiogenic factors, as well as suitable markers with which dothelial cells to the perivascular extracellular matrix. In con- to distinguish blood from lymphatic vascular endothelium. trast to capillary blood vessels, lymphatic capillaries do not However, this scenario is changing rapidly following the dis- possess a continuous basement membrane, and they are devoid covery of the first lymphangiogenic factor, VEGF-C (23, 24). of pericytes. However, like blood vascular endothelium, lymphatic endothelial cells contain Weibel-Palade bodies, and they are immunoreactive for von Willebrand factor and CD31 (plate- VEGF-C let/endothelial cell adhesion molecule 1). VEGF-C is a member of the VEGF family of growth During early development of the blood vascular system factors, which are highly conserved secreted glycoproteins that (vasculogenesis), new capillaries arise either de novo from regulate vasculogenesis, hematopoiesis, angiogenesis, lym- mesoderm-derived angioblasts or by sprouting from newly phangiogenesis, and vascular permeability and are implicated in formed vessels. How do new lymphatic capillaries arise during many physiological and pathological processes. To date, the embryogenesis? Studies on lymphatic ontogeny performed al- VEGF family is comprised of VEGF-A, -B, -C, and -D and Orf most 100 years ago, in which injection techniques were com- virus VEGFs (also called VEGF-E). Of the three VEGF tyrosine bined with serial sectioning, led to the hypothesis that lymphat- kinase receptors identified thus far (VEGFR-1, -2, and -3), ics arise through progressive sprouting from veins (see, for VEGFR-1 binds VEGF-A and –B, VEGFR-2 binds VEGF-A, example, Refs. 9 and 10). However, recent studies have revealed -C, -D, and -E, and VEGFR-3 binds VEGF-C and -D. VEGFRs that lymphatic capillaries also develop de novo from putative differ with respect to mechanisms of regulation and patterns of lymphangioblasts (11), as originally proposed at the beginning expression. For example, VEGFR-1 and -2 are expressed almost of the last century (12, 13). Two theories have therefore exclusively by vascular endothelial cells and hematopoietic pre- emerged regarding the mechanisms of vasculogenesis (forma- cursors, whereas VEGFR-3 is widely expressed in the early tion of the blood vascular tree during development) and embry- embryonic vasculature but becomes restricted to lymphatic en- onic lymphangiogenesis: (a) local de novo differentiation of dothelium at later stages of development and in postnatal life blood or lymphatic endothelium from angioblasts or lym- (reviewed in Refs. 25 and 26). phangioblasts, respectively; and (b) sprouting from preexisting VEGF-C displays a high degree of similarity to VEGF-A, blood capillaries (vasculogenesis/angiogenesis) or veins [lym- including conservation of the eight cysteine residues involved in phangiogenesis (reviewed in Refs. 14 and 15)]. intra- and intermolecular disulfide bonding. The cysteine-rich Exquisitely detailed descriptive studies have revealed the COOH-terminal half increases the length of the VEGF-C mechanisms of lymphangiogenesis in adult tissues, although polypeptide relative to other members of this family. Like very little is known about the molecules involved. Thus, during VEGF-A, both human and murine VEGF-C are alternatively wound healing, lymphatic capillaries grow by sprouting as ex- spliced (24). In addition, VEGF-C mRNA is first translated into tensions from preexisting lymphatics, much in the same way as a precursor from which the mature ligand is derived by cell- new blood capillaries arise by sprouting from preexisting cap- associated proteolytic processing after secretion (27). Unproc- illaries or postcapillary venules during angiogenesis. The ap- essed VEGF-C binds to VEGFR-3. Removal of its NH2- and pearance of new lymphatic capillaries is always secondary to COOH-terminal extensions increases the affinity of VEGF-C that of blood capillaries. Regeneration of severed lymphatics is for VEGFR-3 by approximately 400-fold. Processing also al- therefore slower than that of blood capillaries, although linear lows VEGF-C to bind to VEGFR-2. Processed VEGF-C induces growth occurs at a comparable speed (16, 17). Lymphatic cap- endothelial cell proliferation and migration, as well as increased illaries are less labile than blood capillaries: they send out fewer vascular permeability in the Miles assay. However, the respec- sprouts, anastomose less frequently, and show much less tend- tive roles of VEGFR-2 and -3 in mediating the biological effects

of VEGF-C are incompletely understood. Unlike VEGF-A, gene product involved in regulating early lymphatic develop- VEGF-C expression does not appear to be regulated by hypoxia ment (44); and (c) LYVE-1, a lymphatic endothelial receptor for (28). the extracellular matrix/lymphatic fluid glycosaminoglycan, Gene deletion studies on VEGF-C have not been published hyaluronan (45). To date, none of these markers have been to date. However, in a manner that is unprecedented for a gene shown to retain their lymphatic specificity in the tumor setting. that does not undergo imprinting, heterozygous inactivation of With regard to VEGFR-3, although it is expressed exclusively the VEGF-A gene is embryonic lethal (29, 30). The observation by lymphatics in normal adult tissues, the fact that it is widely that the phenotype of VEGF-AϪ/Ϫ mice is more severe than expressed in embryonic blood vascular endothelium and reex- that of VEGF-Aϩ/Ϫ mice demonstrates the existence of a pressed in tumor blood vessels (39, 46, 47) complicates its use dose-dependent requirement for VEGF-A during embryogene- in studies on tumor lymphangiogenesis. sis. VEGFR-2-deficient mice die at an earlier stage in embry- 5Ј-Nucleotidase has also been used successfully to distin- onic development than mice deficient in VEGF-A (31). This guish lymphatic from blood vascular endothelium (48, 49), and suggests that other VEGFR-2 ligands may compensate for the several apparently lymphatic-specific monoclonal antibodies loss of VEGF-A. It remains to be determined whether this ligand have been reported (50, 51), although these antibodies do not is VEGF-C, or whether VEGF-A and -C are redundant in early appear to have found widespread use. Finally, because lym- vasculogenesis. phatic capillaries lack a continuous basement membrane, immu- Based on its expression profile and its binding to nohistochemistry for extracellular matrix components has also VEGFR-3, VEGF-C has been implicated in the development of been used to distinguish them from capillaries of the blood the lymphatic system (32). In addition, transgenic overexpres- vascular system (52, 53). However, this is unlikely to be reliable sion of VEGF-C using the keratin 14 promoter induces lym- in tumors because angiogenic blood vessels appear to be par- phatic vessel enlargement in the skin (33), and recombinant tially or completely devoid of a basement membrane. VEGF-C induces lymphangiogenesis in the chick chorioallantoic membrane (34). However, VEGF-C also stimulates angiogenesis under certain experimental conditions (35, 36). Further- Lymphangiogenesis in Vitro more, studies in VEGFR-3-null mice have revealed that this Numerous attempts have been made since 1984 to isolate receptor plays an important role in the development of the early and culture endothelial cells from lymphatic vessels in a variety blood vascular system, before the emergence of lymphatic ves- of species (human, bovine, canine, ovine, rat, and murine; Refs. sels (37). VEGFR-3 is also expressed in endothelial cells of 54–70). However, with the exception of cells isolated from tumor blood vessels, where it is thought to play a role in tumor human lymphangiomas (54, 58), all previous studies describe angiogenesis (7, 38, 39). In wound healing, however, VEGFR-3 the isolation of cells from mesenteric collecting or thoracic is confined to lymphatic endothelium (quiescent and regenerat- ducts, i.e., they are of large vessel origin. To date, there are no ing) and is not expressed in blood vessels (21). reports describing isolation of endothelial cells from lymphatic VEGF-D, an additional member of the VEGF family, con- capillaries. tains the eight conserved cysteine residues characteristic of the In the adult organism, lymphatic endothelium expresses VEGF family and has a cysteine-rich COOH-terminal extension VEGFR-2 and -3. To date, cultured large vessel lymphatic similar to VEGF-C. Like VEGF-C, VEGF-D is proteolytically endothelial cells of bovine or canine origin have been found to processed after secretion, and it binds to and activates VEGFR-2 express either VEGFR-1 and -2 (71) or VEGFR-1 and -3 (70), and -3 (40–42). Clearly, this VEGFR-3 ligand could also play respectively. Thus, the characteristic in vivo pattern of VEGFR a role in (tumor) lymphangiogenesis. expression in large vessel lymphatic endothelial cells appears to In summary, in the normal adult organism, VEGF-C ap- be lost in culture. (Alternatively, there may be species differ- pears to be a lymphangiogenic factor, and VEGFR-3 is re- ences with respect to VEGFR expression in vivo.) Whether it is stricted to lymphatic endothelium. VEGFR-2, in contrast, is possible to maintain the characteristic in vivo pattern of VEGFR expressed by both blood vascular and lymphatic endothelium. expression in tissue culture remains to be determined. However, VEGF-C also induces the formation of new blood Cultured large vessel lymphatic endothelial cells have been vessels, but this appears to be restricted to early development used to study angiogenesis in vitro. Thus, spontaneous reorga- and certain pathological settings such as tumorigenesis; in both nization into a branching and anastomizing network of capil- of these settings, VEGFR-3 is also expressed by blood vascular lary-like tubes was observed in planar (two-dimensional) cul- endothelium. Although there is currently no explanation for the tures of bovine and ovine lymphatic endothelial cells (64). selective effects of VEGF-C on lymphangiogenesis versus an- However, these tubes were inside-out i.e., their lumina con- giogenesis, this may depend on the repertoire of VEGFRs ex- tained extracellular matrix and cell debris. Reorganization was pressed (including heterodimers between the different VEGFRs) accelerated by addition of type I collagen or gelatin to the or on the extent of VEGF-C proteolytic processing. cultures (i.e., to the apical surface of the cells). Spontaneous sprouting has been observed from aggregates of cultured bovine lymphatic endothelial cells in three-dimensional collagen gels Markers of Lymphatic Endothelium in Vivo (65). In a three-dimensional collagen gel model of in vitro The second major advance in the field of lymphangiogen- angiogenesis (72), the same cells were induced to invade in esis has come with the discovery of lymphatic endothelium- response to VEGF, VEGF-C, and bFGF and to form capillary- specific markers. These include: (a) podoplanin, a glomerular like tubular structures; in this assay, synergism was observed podocyte membrane mucoprotein (43); (b) Prox-1, a homeobox between bFGF and VEGF (65, 71).

When segments of rat thoracic duct were cultured in a (a) Does increased VEGF-C expression promote an in- plasma clot or collagen gel, lymphatic-like capillary channels crease in lymphatic vessel density (lymphangiogenesis) and/or arose spontaneously from ductal endothelium (73). Newly size? formed channels containing patent lumina were clearly visible (b) If so, is this sufficient to increase the rate of metastasis after 10–14 days and were surrounded by highly attenuated to lymph nodes? endothelium with abluminal anchoring filaments. (c) Are newly formed lymphatic vessels phenotypically Angiogenesis (and presumably lymphangiogenesis) is different from established lymphatics, and if so, does this make characterized by the triad of endothelial proliferation, migration, new lymphatic vessels more prone to promoting metastases? and protease activity. With respect to proliferation, this was (d) Are there other functions of VEGF-C that account for regulated positively in bovine large vessel lymphatic endothelial the increase in lymph node metastasis? These might include the cells by bFGF, epidermal growth factor, and transforming production of trophic, mitogenic, or chemotactic factors for growth factor-␣, whereas TNF-␣ and interleukin-1 were inhib- tumor cells by VEGF-C-stimulated lymphatic endothelium. itory (68). With respect to migration, bFGF stimulated migra- (e) Are lymphatics passive conduits for tumor cells or can tion of bovine large vessel lymphatic endothelial cells, whereas their adhesive and transport capacities be modulated? TNF-␣ inhibited it (68). Finally, with respect to protease activ- It has recently been reported that lymphatics surrounding a ity, VEGF, VEGF-C, and bFGF stimulate expression of uroki- VEGF-C-overexpressing tumor are enlarged, and it has been nase-type plasminogen activator, urokinase-type plasminogen suggested that the increase in lymphatic diameter may be suf- activator receptor, tissue-type plasminogen activator, and plas- ficient to increase metastasis (82). However, the respective roles minogen activator inhibitor 1 in bovine large vessel lymphatic of lymphangiogenesis and lymphatic hyperplasia in tumor dis- endothelial cells (65, 68, 71). Tissue-type plasminogen activator semination remain to be determined. An important function of and plasminogen activator inhibitor 1 were also stimulated by VEGF-A, besides those listed above, is to up-regulate expres- TNF-␣ (60, 68). sion of adhesion molecules in the vasculature (83, 84). Whether As indicated above, to date all in vitro studies have been other members of the VEGF family affect adhesion is not performed on large vessel lymphatic endothelial cells. However, known. postnatal lymphangiogenesis occurs by sprouting from preexisting lymphatic capillaries (see above). In the future, working Lymphangiogenesis and Other Human Pathologies with primary endothelial cells from lymphatic capillaries will be With respect to human pathology, the importance of the of paramount importance. In addition to in vitro studies similar lymphatic system is not restricted to tumor metastasis. Other to those described above, lymphatic capillary endothelial cells clinical settings in which the lymphatic system is involved/ would be useful (a) for the identification of molecules involved perturbed include: (a) congenital hypoplasia leading to in adhesive interactions with other cells (e.g., lymphocytes and lymphedema [inactivating VEGFR-3 mutations (85, 86)]; (b) tumor cells) and (b) for application of techniques of differential lymphedema due to impaired lymphatic drainage caused by gene expression to identify molecular differences between blood inflammatory or neoplastic obstruction [this includes ascites due and lymphatic capillary endothelial cells. The utility of these to lymphatic obstruction in peritoneal carcinomatosis (ovarian techniques in identifying gene expression profiles in normal carcinoma), edema of the arm after surgery or radiotherapy for versus tumor endothelial cells has recently been demonstrated breast cancer; elephantiasis due to massive fibrosis in the ingui- (74). nal region of patients with the parasitic infection filariasis]; and (c) abnormal proliferation in lymphangioma, lymphangiosar- VEGF-C, Lymphangiogenesis, and coma, and Kaposi’s sarcoma. Tumor Metastasis Much attention is currently being devoted to the regulation of angiogenesis in acute and chronic ischemia and to the regu- A number of reports have recently described a correlation lation of angiogenic growth factor gene expression by hypoxia. between VEGF-C expression, tumor lymphangiogenesis, and In addition, therapeutic angiogenesis is currently being tested as the formation of metastasis in regional lymph nodes. Thus, a an alternative therapeutic strategy for chronic cardiac or periph- significant correlation has been described in a variety of carci- eral ischemia. However, lymphangiogenesis has never been nomas (thyroid, prostate, gastric, colorectal, and lung carcino- studied in setting of ischemia. Likewise, no evidence exists at mas) between VEGF-C levels in primary tumors and lymph present concerning the possible role of hypoxia in the regulation node metastases (75–80). One study has described a strong of this process. correlation between lymphatic vessel density and VEGF-C expression (81). However, in this study, no correlation was observed between lymphatic vessel density and lymph node me- Summary and Perspectives tastases. Despite these highly suggestive correlative clinical Clinicopathological findings have long suggested that by findings, a direct role for VEGF-C in tumor lymphangiogenesis providing a pathway for tumor cell dissemination, lymphatics and subsequent metastasis has yet to be demonstrated, and to are a key component of metastatic spread. Tumor cell metastasis date there is no animal model in which these phenomena can be to regional lymph nodes is thus an early event in metastatic explored. In addition, these observations raise a number of spread, and this parameter is frequently used to predict disease questions regarding the mechanisms by which increased expres- outcome or to determine therapeutic strategies. However, as sion of VEGF-C in primary tumors results in an increase in Wilting et al. (15) recently pointed out: “A completely open lymph node metastases: question is that of the development of lymphatics in tumors and

the(ir) relation to the formation of lymphogenic metastases.” 7. Leu, A. J., Berk, D. A., Lymboussaki, A., Alitalo, K., and Jain, R. K. This opinion has also been expressed by others (87, 88). Absence of functional lymphatics within a murine sarcoma: a molecular Increased expression of VEGF-C in spontaneously arising and functional evaluation. Cancer Res., 60: 4324–4327, 2000. human tumors has recently been reported to correlate with 8. Hartveit, F. Attenuated cells in breast stroma: the missing lymphatic system of the breast. Histopathology, 16: 533–543, 1990. increased lymphangiogenesis and dissemination of tumor cells 9. Ranvier, L. De´veloppement des vaisseaux lymphatiques. C. R. Acad. to regional lymph nodes. However, it is not known whether Sci., 121: 1105–1109, 1895. preexisting lymphatics are sufficient to serve this function, or 10. Sabin, F. R. On the origin and development of the lymphatic system whether metastasis requires the de novo formation of lymphatic from the veins and the development of the lymph hearts and thoracic capillaries (lymphangiogenesis) or lymphatic enlargement. duct in the pig. Am. J. Anat., 1: 367–389, 1902. Some authors have suggested that it is not necessary to invoke 11. Schneider, M., Othman-Hassan, K., Christ, B., and Wilting, J. lymphangiogenesis in this setting and that preexisting peritu- Lymphangioblasts in the avian wing bud. Dev. Dyn., 216: 311–319, 1999. moral lymphatics that enlarge in response to VEGF-C in tumors 12. Huntington, G. S. The genetic interpretation of the development of will suffice (7). If this hypothesis is correct, it may be necessary the lymphatic system. Anat. Rec., 2: 19–46, 1908. to invoke nonlymphangiogenic functions of VEGF-C to account 13. Kampmeier, O. F. The value of the injection method in the study of for the increase in lymph node metastasis. This might include the lymphatic development. Anat. Rec., 6: 223–233, 1912. the production of trophic, mitogenic, or chemotactic factors for 14. McClure, C. F. W. The endothelial problem. Anat. Rec., 22: 219– tumor cells by VEGF-C-stimulated lymphatic endothelium or 237, 1921. alterations in lymphatic endothelial-tumor cell adhesion. To 15. Wilting, J., Neeff, H., and Christ, B. Embryonic lymphangiogen- date, nothing is known about these potential interactions. esis. Cell Tissue Res., 297: 1–11, 1999. Finally, in contrast to its sister field, angiogenesis (re- 16. Clark, E. R. Reaction of experimentally isolated lymphatic capillaries in the tails of amphibian larvae. Anat. Rec., 24: 181–191, 1922. viewed in Refs. 89–92), very little is known about the mecha- 17. Clark, E. R., and Clark, L. C. Observations on the new growth of nisms and mediators of lymphangiogenesis. With regard to lymphatic vessels as seen in transparent chambers introduced into the angiogenesis, an extensive effort is currently being directed rabbit ear. Am. J. Anat., 51: 49–87, 1932. worldwide to identify antiangiogenic agents, particularly for use 18. Reichert, F. L. The regeneration of the lymphatics. Arch. Surg., 13: in anticancer therapy, and many potentially useful compounds 871–881, 1926. have progressed beyond preclinical studies into the early phases 19. Anthony, J. P., Foster, R. D., Price, D. C., Mahdavian, M., and of clinical trials. This is largely due to the identification of key Inoue, Y. Lymphatic regeration following microvascular limb replanta- molecular mediators. The recent, almost explosive interest in tion: a qualitative and quantitative animal study. J. Reconstr. Micro- surg., 13: 327–330, 1997. lymphangiogenesis, after many decades of dormancy, will un- 20. Slavin, S. A., Van den Abbeele, A. D., Losken, A., Swartz, M. A., doubtedly ensure that we move rapidly to attain similar objec- and Jain, R. K. Return of lymphatic function after flap transfer for acute tives in the lymphatic system. However, it still remains to be lymphedema. Ann. Surg., 229: 421–427, 1999. determined whether inhibition of lymphangiogenesis is a real- 21. Paavonen, K., Puolakkainen, P., Jussila, L., Jahkola, T., and Alitalo, istic therapeutic strategy for inhibiting tumor cell dissemination K. Vascular endothelial growth factor receptor-3 in lymphangiogenesis and the formation of metastases. in wound healing. Am. J. Pathol., 156: 1499–1504, 2000. 22. Pullinger, B. D., and Florey, H. W. Proliferation of lymphatics in inflammation. J. Pathol. Bacteriol., 45: 157–170, 1937. ACKNOWLEDGMENTS 23. Joukov, V., Pajusola, K., Kaipainen, A., Chilov, D., Lahtinen, I., Kukk, E., Saksela, O., Kalkkinen, N., and Alitalo, K. A novel vascular I am grateful to Drs. Roberto Montesano and Jonathan Sleeman for endothelial growth factor, VEGF-C, is a ligand for the Flt4 (VEGFR-3) their helpful comments. and KDR (VEGFR-2) receptor tyrosine kinases. EMBO J., 15: 290– 298, 1996. 24. Lee, J., Gray, A., Yuan, J., Luoh, S. M., Avraham, H., and Wood, REFERENCES W. I. Vascular endothelial growth factor-related protein: a ligand and 1. Fidler, I. J. Molecular biology of cancer: invasion and metastasis. In: specific activator of the tyrosine kinase receptor Flt4. Proc. Natl. Acad. V. T. DeVita, S. Hellman, and S. A. Rosenberg (eds.), Cancer: Princi- Sci. USA, 93: 1988–1992, 1996. ples and Practice of Oncology, 5th ed., pp. 135–152. Philadelphia: 25. Olofsson, B., Jeltsch, M., Eriksson, U., and Alitalo, K. Current Lippincott-Raven Publishers, 1997. biology of VEGF-B and VEGF-C. Curr. Opin. Biotechnol., 10: 528– 2. Cotran, R. S., Kumar, V., and Tucker, C. Neoplasia. In: Robbins 535, 1999. Pathologic Basis of Disease, 6th ed., pp. 268–271. Philadelphia: W. B. 26. Veikkola, T., Karkkainen, M., Claesson-Welsh, L., and Alitalo, K. Saunders Company, 1999. Regulation of angiogenesis via vascular endothelial growth factor re- 3. Sleeman, J. P. The lymph node as a bridgehead in the metastatic ceptors. Cancer Res., 60: 203–212, 2000. dissemination of tumors. Recent Results Cancer Res., 157: 55–81, 27. Joukov, V., Sorsa, T., Kumar, V., Jeltsch, M., Claesson-Welsh, L., 2000. Cao, Y., Saksela, O., Kalkkinen, N., and Alitalo, K. Proteolytic proc- 4. Gullino, P. M. Extracellular compartments of solid tumors. In: F. F. essing regulates receptor specificity and activity of VEGF-C. EMBO J., Becker (ed.), Cancer: A Comprehensive Treatise, Vol. 3, pp. 327–354. 16: 3898–3911, 1997. New York: Plenum Press, 1975. 28. Enholm, B., Paavonen, K., Ristima¨ki, A., Kumar, V., Gunji, Y., 5. Zeidman, I., Copeland, B. E., and Warren, S. Experimental studies on Klefstrom, J., Kivinen, L., Laiho, M., Olofsson, B., Joukov, V., Eriks- the spread of cancer in the lymphatic system. II. Absence of a lymphatic son, U., and Alitalo, K. Comparison of VEGF, VEGF-B, VEGF-C and supply in carcinoma. Cancer (Phila.), 8: 123–127, 1955. Ang-1 mRNA regulation by serum, growth factors, oncoproteins and 6. Tanigawa, N., Kanazawa, T., Satomura, K., Hikasa, Y., Hashida, M., hypoxia. Oncogene, 14: 2475–2483, 1997. Muranishi, S., and Sezak, I. H. Experimental studies on lymphatic 29. Carmeliet, P., Ferreira, V., Breier, G., Pollefeyt, S., Kieckens, L., vascular changes in the development of cancer. Lymphology, 14: 149– Gertsenstein, M., Fahrig, M., Vandenhoeck, A., Harpal, K., Eberhardt, 154, 1981. C., Declercq, C., Pawling, J., Moons, L., Collen, D., Risau, W., and

Nagy, A. Abnormal blood vessel development and lethality in embryos 46. Kaipainen, A., Korhonen, J., Mustonen, T., van Hinsbergh, lacking a single VEGF allele. Nature (Lond.), 380: 435–439, 1996. V. W. M., Fang, G-H., Dumont, D., Breitman, M., and Alitalo, K. 30. Ferrara, N., Carver-Moore, K., Chen, H., Dowd, M., Lu, L., Expression of the fms-like tyrosine kinase 4 gene becomes restricted to O’Shea, K. S., Powell-Braxton, L., Hillan, K. J., and Moore, M. W. lymphatic endothelium during development. Proc. Natl. Acad. Sci. Heterozygous embryonic lethality induced by targeted inactivation of USA, 92: 3566–3570, 1995. the VEGF gene. Nature (Lond.), 380: 439–442, 1996. 47. Lymboussaki, A., Olofsson, B., Eriksson, U., and Alitalo, K. Vas- 31. Shalaby, F., Rossant, J., Yamaguchi, T. P., Gertsenstein, M., Wu, cular endothelial growth factor (VEGF) and VEGF-C show overlapping X-F., Breitman, M. L., and Schuh, A. C. Failure of blood-island forma- binding sites in embryonic endothelia and distinct sites in differentiated tion and vasculogenesis in Flk-1-deficient mice. Nature (Lond.), 376: adult endothelia. Circ. Res., 85: 992–999, 1999. 62–66, 1995. 48. Beckstead, J. H., Wood, G. S., and Fletcher, V. Evidence for the 32. Kukk, E., Lymboussaki, A., Taira, S., Kaipainen, A., Jeltsch, M., origin of Kaposi’s sarcoma from lymphatic endothelium. Am. J. Pathol., Joukov, V., and Alitalo, K. VEGF-C receptor binding and pattern of 119: 294–300, 1985. expression with VEGFR-3 suggests a role in lymphatic vascular devel- 49. Turner, R. R., Beckstead, J. H., Warnke, R. A., and Wood, G. S. opment. Development (Camb.), 122: 3829–3837, 1996. Endothelial cell phenotypic diversity. In situ demonstration of immu- 33. Jetlsch, M., Kaipainen, A., Joukov, V., Meng, X., Lakso, M., nologic and enzymatic heterogeneity that correlates with specific mor- Rauvala, H., Swartz, M., Fukumura, D., Jain, R. K., and Alitalo, K. phologic subtypes. Am. J. Clin. Pathol., 87: 569–575, 1987. Hyperplasia of lymphatic vessels in VEGF-C transgenic mice. Science 50. Sawa, Y., Mukaida, A., Suzuki, M., and Yoshida, S. Identification (Washington DC), 276: 1423–1425, 1997. of lymphatic vessels by using a monoclonal antibody specific for the 34. Oh, S. J., Jeltsch, M. M., Birkenhager, R., McCarthy, J. E., Weich, human thoracic duct. Microvasc. Res., 53: 142–149, 1997. H. A., Christ, B., Alitalo, K., and Wilting, J. VEGF and VEGF-C: 51. Ezaki, T., Matsuno, K., Fujii, H., Hayashi, N., Miyakawa, K., specific induction of angiogenesis and lymphangiogenesis in the differ- Ohmori, J., and Kotani, M. A new approach for identification of rat entiated avian chorioallantoic membrane. Dev. Biol., 188: 96–109, lymphatic capillaries using a monoclonal antibody. Arch. Histol. Cytol., 1997. 53 (Suppl.): 77–86, 1990. 35. Cao, Y., Linden, P., Farnebo, J., Cao, R., Eriksson, A., Kumar, V., Qi, J. H., Claesson-Welsh, L., and Alitalo, K. Vascular endothelial 52. Barsky, S. H., Baker, A., Siegal, G. P., Togo, S., and Liotta, L. A. growth factor C induces angiogenesis in vivo. Proc. Natl. Acad. Sci. Use of anti-basement membrane antibodies to distinguish blood vessel USA, 95: 14389–14394, 1998. capillaries from lymphatic capillaries. Am. J. Surg. Pathol., 7: 667–677, 1983. 36. Witzenbichler, B., Asahara, T., Murohara, T., Silver, M., Spyrido- poulos, I., Magner, M., Principe, N., Kearney, M., Hu, J. S., and Isner, 53. Nerlich, A. G., and and Schleicher, E. Identification of lymph and J. M. Vascular endothelial growth factor-C (VEGF-C/VEGF-2) pro- blood capillaries by immunohistochemical staining for various basement motes angiogenesis in the setting of tissue ischemia. Am. J. Pathol., 153: membrane components. Histochemistry, 96: 449–453, 1991. 381–394, 1998. 54. Bowman, C. A., Witte, M. H., Witte, C. L., Way, D. L., Nagle, 37. Dumont, D. J., Jussila, L., Taipale, J., Lymboussaki, A., Mustonen, R. B., Copeland, J. G., and Daschbach, C. C. Cystic hygroma reconsid- T., Pajusola, K., Breitman, M., and Alitalo, K. Cardiovascular failure in ered: hamartoma or neoplasm? Primary culture of an endothelial cell mouse embryos deficient in VEGF receptor-3. Science (Washington line from a massive cervicomediastinal hygroma with bony lymphangio- DC), 282: 946–949, 1998. matosis. Lymphology, 17: 15–22, 1984. 38. Partanen, T. A., Alitalo, K., and Miettinen, M. Lack of lymphatic 55. Johnston, M. G., and Walker, M. A. Lymphatic endothelial and vascular specificity of vascular endothelial growth factor receptor-3 in smooth-muscle cells in tissue culture. In Vitro, 20: 566–572, 1984. 185 vascular tumors. Cancer (Phila.), 86: 2406–2412, 1999. 56. Gneep, D. R., and Chandler, W. Tissue culture of human and canine 39. Valtola, R., Salven, P., Heikkila, P., Taipale, J., Joensuu, H., Rehn, thoracic duct endothelium. In Vitro Cell. Dev. Biol., 21: 200–206, 1985. M., Pihlajaniemi, T., Weich, H., de Waal, R., and Alitalo, K. VEGFR-3 57. Gumkowski, F., Kaminska, G., Kaminska, M., Morrissey, L. W., and its ligand VEGF-C are associated with angiogenesis in breast and Auerbach, R. Heterogeneity of mouse vascular endothelium. Blood cancer. Am. J. Pathol., 154: 1381–1390, 1999. Vessels, 24: 11–23, 1987. 40. Orlandini, M., Marconcini, L., Ferruzzi, R., and Oliviero, S. Iden- 58. Way, D., Hendrix, M., Witte, M., Witte, C., Nagle, R., and Davis, tification of a c-fos-induced gene that is related to the platelet-derived J. Lymphatic endothelial cell line (CH3) from a recurrent retroperitoneal growth factor/vascular endothelial growth factor family. Proc. Natl. lymphangioma. In Vitro Cell. Dev. Biol., 23: 647–652, 1987. Acad. Sci. USA, 93: 11675–11680, 1996. 59. Jones, B. E., and Yong, C. J. Culture and characterization of bovine 41. Yamada, Y., Nezu, J., Shimane, M., and Hirata, Y. Molecular mesenteric lymphatic endothelium. In Vitro Cell. Dev. Biol., 23: 698– cloning of a novel vascular endothelial growth factor, VEGF-D. Genom- 706, 1987. ics, 42: 483–488, 1997. 60. Laschinger, C. A., Johnston, M. G., Hay, J. B., and Wasi, S. 42. Achen, M. G., Jeltsch, M., Kukk, E., Makinen, T., Vitali, A., Wilks, Production of plasminogen activator and plasminogen activator inhibitor A. F., Alitalo, K., and Stacker, S. A. Vascular endothelial growth factor by bovine lymphatic endothelial cells: modulation by TNF-␣. Thromb. D (VEGF-D) is a ligand for the tyrosine kinases VEGF receptor 2 (Flk1) Res., 59: 567–579, 1990. and VEGF receptor 3 (Flt4). Proc. Natl. Acad. Sci. USA, 95: 548–553, 61. Djoneidi, M., and Brodt, P. Isolation and characterization of rat 1998. lymphatic endothelial cells. Microcirc. Endothelium Lymphatics, 7: 43. Breiteneder-Geleff, S., Soleiman, A., Kowalski, H., Horvat, R., 161–182, 1991. Amann, G., Kriehuber, E., Diem, K., Weninger, W., Tschachler, E., 62. Yong, L. C., and Jones, B. E. A comparative study of cultured Alitalo, K., and Kerjaschki, D. Angiosarcomas express mixed endothe- vascular and lymphatic endothelium. Exp. Pathol., 42: 11–25, 1991. lial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium. Am. J. Pathol., 154: 385– 63. Leak, L. V., and Jones, M. Lymphatic endothelium isolation, char- 394, 1999. acterization and long term culture. Anat. Rec., 236: 641–652, 1993. 44. Wigle, J. T., and Oliver, G. Prox1 function is required for the 64. Leak, L. V., and Jones, M. Lymphangiogenesis in vitro: formation development of the murine lymphatic system. Cell, 98: 769–778, 1999. of lymphatic capillary-like channels from confluent monolayers of lym- 45. Banerji, S., Ni, J., Wang, S. X., Clasper, S., Su, J., Tammi, R., phatic endothelial cells. In Vitro Cell. Dev. Biol., 30A: 512–518, 1994. Jones, M., and Jackson, D. G. LYVE-1, a new homologue of the CD44 65. Pepper, M. S., Wasi, S., Ferrara, N., Orci, L., and Montesano, R. In glycoprotein, is a lymph-specific receptor for hyaluronan. J. Cell Biol., vitro angiogenic and proteolytic properties of bovine lymphatic endo- 144: 789–801, 1999. thelial cells. Exp. Cell Res., 210: 298–305, 1994.

66. Weber, E., Lorenzoni, P., Lozzi, G., and Sacchi, G. Culture of 79. Niki, T., Iba, S., Tokunou, M., Yamada, T., Matsuno, Y., and bovine thoracic duct endothelial cells. In Vitro Cell. Dev. Biol., 30A: Hirohashi, S. Expression of vascular endothelial growth factors A, B, C, 287–288, 1994. and D and their relationships to lymph node status in lung adenocarci- 67. Weber, E., Lorenzoni, P., Lozzi, G., and Sacchi, G. Cytochemical noma. Clin. Cancer Res., 6: 2431–2439, 2000. differentiation between blood and lymphatic endothelium: bovine blood 80. Ohta, Y., Nozawa, H., Tanaka, Y., Oda, M., and Watanabe, Y. and lymphatic large vessels and endothelial cells in culture. J. Histo- Increased vascular endothelial growth factor and vascular endothelial chem. Cytochem., 42: 1109–1115, 1994. growth factor-c and decreased nm23 expression associated with micro- 68. Liu, N-F., and He, Q-L. The regulatory effects of cytokines on dissemination in the lymph nodes in stage I non-small cell lung cancer. J. Thorac. Cardiovasc. Surg., 119: 804–813, 2000. lymphatic angiogenesis. Lymphology, 30: 3–12, 1997. 81. Ohta, Y., Shridhar, V., Bright, R. K., Kalemkerian, G. P., Du, W., 69. Leak, L. V., Yu, Z-X., Jones, M., and Ferrans, V. J. Characteriza- Carbone, M., Watanabe, Y., and Pass, H. I. VEGF and VEGF type C tion of a transformed ovine lymphatic endothelial cell line. Microcircu- play an important role in angiogenesis and lymphangiogenesis in human lation, 6: 63–73, 1999. malignant mesothelioma tumours. Br. J. Cancer, 81: 54–61, 1999. 70. Nojiri, H., and Ohhashi, T. Immunolocalization of nitric oxide 82. Padera, T. P., Yun, C., Kadambi, A., Mouta Carreira, C., and, Jain, synthase and VEGF receptors in cultured lymphatic endothelial cells. R. K. Local mechanics and VEGF-C alter peri-tumor lymphatic func- Microcirculation, 6: 75–78, 1999. tion. Proc. Am. Assoc. Cancer Res., 41: 88, 2000. 71. Pepper, M. S., Mandriota, S. J., Jeltsch, M., Kumar, V., and Alitalo, 83. Melder, R. J., Koenig, G. C., Witwer, B. P., Safabakhsh, N., Munn, K. Vascular endothelial growth factor (VEGF)-C synergises with basic L. L., and Jain, R. K. During angiogenesis, vascular endothelial growth fibroblast growth factor and VEGF in the induction of angiogenesis in factor and basic fibroblast growth factor regulate natural killer cell vitro, and alters endothelial cell proteolytic properties. J. Cell. Physiol., adhesion to tumor endothelium. Nat. Med., 2: 992–997, 1996. 177: 439–452, 1998. 84. Detmar, M., Brown, L. F., Schon, M. P., Elicker, B. M., Velasco, P., 72. Montesano, R., and Orci, L. Tumor-promoting phorbol esters in- Richard, L., Fukumura, D., Monsky, W., Claffey, K. P., and Jain, R. K. duce angiogenesis in vitro. Cell, 42: 469–477, 1985. Increased microvascular density and enhanced leukocyte rolling and 73. Nicosia, R. F. Angiogenesis and the formation of lymphaticlike adhesion in the skin of VEGF transgenic mice. J. Investig. Dermatol., channels in cultures of thoracic duct. In Vitro Cell. Dev. Biol., 23: 111: 1–6, 1998. 167–174, 1987. 85. Irrthum, A., Karkkainen, M. J., Devriendt, K., Alitalo, K., and 74. St Croix, B., Rago, C., Velculescu, V., Traverso, G., Romans, K. E., Vikkula, M. Congenital hereditary lymphedema caused by a mutation Montgomery, E., Lal, A., Riggins, G. J., Lengauer, C., Vogelstein, B., that inactivates VEGFR3 tyrosine kinase. Am. J. Hum. Genet., 67: and Kinzler, K. W. Genes expressed in human tumor endothelium. 295–301, 2000. Science (Washington DC), 289: 1197–1202, 2000. 86. Karkkainen, M. J., Ferrell, R. E., Lawrence, E. C., Kimak, M. A., 75. Bunone, G., Vigneri, P., Mariani, L., Buto, S., Collini, P., Pilotti, S., Levinson, K. L., McTigue, M. A., Alitalo, K., and Finegold, D. N. Pierotti, M. A., and Bongarzone, I. Expression of angiogenesis stimu- Missense mutations interfere with VEGFR-3 signalling in primary lators and inhibitors in human thyroid tumors and correlation with lymphedema. Nat. Genet., 25: 153–159, 2000. clinical-pathologic features. Am. J. Pathol., 155: 1967–1976, 1999. 87. Witte, M. H., Way, D. L., Witte, C. L., and Bernas, M. Lym- 76. Tsurusaki, T., Kanda, S., Sakai, H., Kanetake, H., Saito, Y., Alitalo, phangiogenesis: mechanisms, significance and clinical implications. K., and Koji, T. Vascular endothelial growth factor-C expression in EXS, 79: 65–112, 1997. human prostatic carcinoma and its relationship to lymph node metasta- 88. Wittekind, C. Diagnosis and staging of lymph node metastasis. sis. Br. J. Cancer, 80: 309–313, 1999. Recent Results Cancer Res., 157: 20–28, 2000. 77. Yonemura, Y., Endo, Y., Fujita, H., Fushida, S., Ninomiya, I., 89. Folkman, J. Antiangiogenic gene therapy. Proc. Natl. Acad. Sci. Bandou, E., Taniguchi, K., Miwa, K., Ohoyama, S., Sugiyama, K., and USA, 95: 9064–9066, 1998. Sasaki, T. Role of vascular endothelial growth factor C expression in the 90. Ferrara, N., and Alitalo, K. Clinical applications of angiogenic development of lymph node metastasis in gastric cancer. Clin. Cancer growth factors and their inhibitors. Nat. Med., 5: 1359–1364, 1999. Res., 5: 1823–1829, 1999. 91. Carmeliet, P., and Jain, R. K. Angiogenesis in cancer and other 78. Akagi, K., Ikeda, Y., Miyazaki, M., Abe, T., Kinoshita, J., Maehara, diseases. Nature (Lond.), 407: 249–257, 2000. Y., and Sugimachi, K. Vascular endothelial growth factor-C (VEGF-C) 92. Yancopoulos, G. D., Davis, S., Gale, N. W., Rudge, J. S., Wiegand, expression in human colorectal cancer tissues. Br. J. Cancer, 83: 887– S. J., and Holash, J. Vascular-specific growth factors and blood vessel 891, 2000. formation. Nature (Lond.), 407: 242–248, 2000.

Michael S. Pepper

Clin Cancer Res 2001;7:462-468.

Updated version Access the most recent version of this article at: http://clincancerres.aacrjournals.org/content/7/3/462

Cited articles This article cites 90 articles, 17 of which you can access for free at: http://clincancerres.aacrjournals.org/content/7/3/462.full#ref-list-1

Citing articles This article has been cited by 36 HighWire-hosted articles. Access the articles at: http://clincancerres.aacrjournals.org/content/7/3/462.full#related-urls

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://clincancerres.aacrjournals.org/content/7/3/462. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.