Jacqueline D. Shields

Induction of Lymphoidlike Stroma and Immune Escape by Tumors That Express the Chemokine CCL21

Tolerating Tumors Successful tumor growth depends on the ability of the tumor to escape detection by the immune system. Human cancers that express the chemokine receptor CCR7 are associated with tumor metastasis and poor prognosis, suggesting that CCR7-dependent signaling might lead to an immunotolerant tumor microenvironment. Shields et al. (p. 749; published online 25 March; see the Perspective by Zindl and Chaplin) studied a mouse melanoma model in which the tumors expressed varying amounts of the CCR7 ligand, CCL21. Tumors expressing CCL21 exhibited more aggressive growth and attracted a class of suppressive, rather than pro-inflammatory, leukocytes. Furthermore, the tumor microenvironment was rich in immunosuppressive cytokines and exhibited lymph node–like features. These features were not present in tumors that expressed low amounts of CCL21. Thus, tumor CCL21 expression promotes an immunotolerant tumor microenvironment, which is permissive for tumor growth and spread. An immunotolerant microenvironment driven by chemokine expression contributes to tumor growth and spread. Tumor manipulation of host immunity is important for tumor survival and invasion. Many cancers secrete CCL21, a chemoattractant for various leukocytes and lymphoid tissue inducer cells, which drive lymphoid neogenesis. CCL21 expression by melanoma tumors in mice was associated with an immunotolerant microenvironment, which included the induction of lymphoid-like reticular stromal networks, an altered cytokine milieu, and the recruitment of regulatory leukocyte populations. In contrast, CCL21-deficient tumors induced antigen-specific immunity. CCL21-mediated immune tolerance was dependent on host rather than tumor expression of the CCL21 receptor, CCR7, and could protect distant, coimplanted CCL21-deficient tumors and even nonsyngeneic allografts from rejection. We suggest that by altering the tumor microenvironment, CCL21-secreting tumors shift the host immune response from immunogenic to tolerogenic, which facilitates tumor progression.

Vascular Endothelial Growth Factor-C and C-C Chemokine Receptor 7 in Tumor Cell–Lymphatic Cross-talk Promote Invasive Phenotype

Most carcinomas spread to distant sites through lymphatic vessels. Several preclinical and clinical studies have shown a positive correlation between the incidence of lymph node metastasis and secretion of the lymphatic growth factor vascular endothelial growth factor-C (VEGF-C) by tumor cells, suggesting tumor lymphangiogenesis as an escape mechanism. However, recent evidence has shown VEGF receptor-3 (VEGFR-3) expression on tumor cells and autocrine signaling, which increase metastatic potential. Furthermore, there is growing evidence implicating lymphatic-homing chemokine receptors, particularly C-C chemokine receptor 7 (CCR7), in lymph node metastasis. We report here that expressions of VEGF-C and CCR7 by tumor cells act synergistically to promote their invasion toward lymphatics. First, VEGF-C acts to increase lymphatic secretion of CCL21, which in turn drives CCR7-dependent tumor chemoinvasion toward lymphatics. Second, VEGF-C acts in an autocrine fashion to increase tumor invasiveness by increasing the proteolytic activity and motility of tumor cells in a three-dimensional matrix. Both of these effects are VEGFR-3 dependent and evident only in three-dimensional environments. We further verified that VEGF-C induces lymphatic CCL21 up-regulation in vivo by direct injection of VEGF-C protein intradermally in the mouse. Taken together, these results bridge the prometastatic functions of CCR7 and VEGF-C in tumors and show that, beyond lymphangiogenesis, VEGF-C promotes tumor invasion toward lymphatics by both autocrine and CCR7-dependent paracrine signaling mechanisms, which may be a significant cause of lymph node metastasis.

Transmural Flow Modulates Cell and Fluid Transport Functions of Lymphatic Endothelium

Rationale: Lymphatic transport of peripheral interstitial fluid and dendritic cells (DCs) is important for both adaptive immunity and maintenance of tolerance to self-antigens. Lymphatic drainage can change rapidly and dramatically on tissue injury or inflammation, and therefore increased fluid flow may serve as an important early cue for inflammation; however, the effects of transmural flow on lymphatic function are unknown. Objective: Here we tested the hypothesis that lymph drainage regulates the fluid and cell transport functions of lymphatic endothelium. Methods and Results: Using in vitro and in vivo models, we demonstrated that lymphatic endothelium is sensitive to low levels of transmural flow. Basal-to-luminal flow (0.1 and 1 &mgr;m/sec) increased lymphatic permeability, dextran transport, and aquaporin-2 expression, as well as DC transmigration into lymphatics. The latter was associated with increased lymphatic expression of the DC homing chemokine CCL21 and the adhesion molecules intercellular adhesion molecule-1 and E-selectin. In addition, transmural flow induced delocalization and downregulation of vascular endothelial cadherin and PECAM-1 (platelet/endothelial cell adhesion molecule-1). Flow-enhanced DC transmigration could be reversed by blocking CCR7, intercellular adhesion molecule-1, or E-selectin. In an experimental model of lymphedema, where lymphatic drainage is greatly reduced or absent, lymphatic endothelial expression of CCL21 was nearly absent. Conclusions: These findings introduce transmural flow as an important regulator of lymphatic endothelial function and suggest that flow might serve as an early inflammatory signal for lymphatics, causing them to regulate transport functions to facilitate the delivery of soluble antigens and DCs to lymph nodes.

Cooperative and redundant roles of VEGFR-2 and VEGFR-3 signaling in adult lymphangiogenesis

Activation of vascular endothelial growth factor (VEGF) receptor‐3 (VEGFR‐3) by VEGF‐C initiates lymphangiogenesis by promoting lymphatic proliferation and migration. However, it is unclear whether VEGFR‐3 signaling is required beyond these initial stages, namely during the organization of new lymphatic endothelial cells (LECs) into functional capillaries. Furthermore, the role of VEGFR‐2, which is also expressed on LECs and binds VEGF‐C, is unclear. We addressed these questions by selectively neutralizing VEGFR‐3 and/or VEGFR‐2 for various time periods in an adult model of lymphangiogen‐esis in regenerating skin. While blocking either VEGFR‐2 or VEGFR‐3 with specific antagonist mAbs (DC101 and mF4–31C1, respectively) prior to lymphatic migration prevented lymphangiogenesis, blocking VEGFR‐3 subsequent to migration did not affect organization into functional capillaries, and VEGFR‐2 blocking had only a small hindrance on organization. These findings were confirmed in vitro using human LECs and anti‐human antagonist mAbs (IMC‐1121a and hF4–3C5): both VEGFR‐2 and ‐3 signaling were required for migration and proliferation, but tubulogenesis in 3D cultures was unaffected by VEGFR‐3 blocking and partially hindered by VEGFR‐2 blocking. Furthermore, both in vitro and in vivo, while VEGFR‐3 blocking had no effect on LEC organization, coneutralization of VEGFR‐2, and VEGFR‐3 completely prevented lymphatic organization. Our findings demonstrate that cooperative signaling of VEGFR‐2 and ‐3 is necessary for lymphatic migration and proliferation, but VEGFR‐3 is redundant with VEGFR‐2 for LEC organization into functional capillaries.—Goldman, J., Rutkowski, J. M., Shields, J. D., Pasquier, M. C., Cui, Y., Schmökel, H. G., Willey, S., Hicklin, D. J., Pytowski, B., Swartz, M. A. Cooperative and redundant roles of VEGFR‐2 and VEGFR‐3 signaling in adult lymphangiogenesis. FASEB J. 21, 1003–1012 (2007)

Lymphatic Physiology and Function in Healthy Tissue and Cancer

The lymphatic system is the primary route of metastasis for many cancers, and it is this spread through the lymphatic vessels to lymph nodes and on to distant organs that is responsible for the majority of cancer-related deaths. Lymphatics, moreso than blood vessels, are thought to provide an overall favorable route for the survival and dissemination of tumor cells due to their anatomical features and low shear stress environment, but the mechanisms and physiological parameters governing lymphatic metastasis are only beginning to be understood. How cancer cells affect and gain access to local lymphatic vessels, travel within the vessels, and enter into the lymph nodes are all topics of recent research efforts, alongside questions of how tumor cells might mimic immune cells and escape the host adaptive immune response. In this chapter we cover the basic anatomy and physiology of the lymphatic system and how it relates to cancer metastasis through the lymphatics.

Abstract 298: Tumor-associated lymphatic vessels: Hijacking mechanisms of peripheral tolerance

Lymphatic vessels drain fluid, antigens, and immune cells from the periphery to the lymph nodes (LNs). In addition to transporting activated dendritic cells to mount adaptive immune responses in the LN, lymphatic drainage is brings soluble antigens from the periphery to LN-resident immature dendritic cells and B cells. It is also the most common site for cancer metastasis. Despite its importance, the role of tumor-associated lymphatic vessels and their drainage to the LN in regulating host immune responses to the tumor is poorly understood. We show that tumor expression of VEGF-C, the most potent lymphatic growth factor, promotes pro-tumor immune tolerance by several mechanisms. For one, it enhances drainage to the draining LN, where tumor antigens along with suppressive cytokines bathe the LN and could affect B and T cell education there. Second, tumor VEGF-C upregulates CCL21 in the tumor stroma and surrounding lymphatic vessels, which itself promotes T cell infiltration and suppression. Third, VEGF-C drives peritumoral and LN lymphangiogenesis, which can modulate the myeloid cell repertoire towards more suppressive phenotypes. These changes resulted in increased infiltrations of regulatory T cells and myeloid-derived suppressor cells, and increased levels of regulatory cytokines. Interestingly, VEGF-C-expressing tumors were impervious to prior immunization against tumor antigen, unlike control-transfected tumors, which were hindered by the vaccine-induced immune response. Together, these findings suggest that lymphatic drainage serves to maintain peripheral tolerance and that tumors may hijack such mechanisms to escape host immunity. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 298. doi:1538-7445.AM2012-298