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Skin is a highly ordered immune organ that coordinates rapid responses to external insult while maintaining self-tolerance. In healthy tissue, lymphatic vessels drain fluid and coordinate local immune responses; however, environmental factors induce lymphatic vessel dysfunction, leading to lymph stasis and perturbed regional immunity. These same environmental factors drive the formation of local malignancies, which are also influenced by local inflammation. Herein, we discuss clinical and experimental evidence supporting the tenet that lymphatic vessels participate in regulation of cutaneous inflammation and immunity, are important contributors to malignancy and potential biomarkers and targets for immunotherapy.
Skin is the largest organ in mammals, serving as a physical and immunological barrier to the external environment (1). As an immune organ, skin coordinates rapid responses to external challenge through constitutive immune surveillance involving both resident and recruited leukocytes (2). Exquisite spatiotemporal regulation of immune cells is required for homeostatic tissue maintenance - loss of function is associated with a variety of dermatopathologies including chronic infection, inflammation, autoimmunity and cancer (1). The interplay between local inflammation, tissue remodeling and anti-tumor immune responses are of particular interest given recent success of immunotherapies, namely blockade of immune checkpoint molecules (3). Since tumor microenvironments in part regulate anti-tumor immunity, these also present targets for release of local immunosuppression that may synergize with checkpoint therapies (4). While tumor-associated blood vessels are appreciated for their role in regulating leukocyte infiltration and function (5), tumor-associated lymphangiogenesis has lagged behind with respect to recognition of its role(s) tumor-associated inflammation and immunity. Recent revelations, however, have highlighted these important roles with respect to coordination of local inflammation (6) and immunity (7–11), indicating that their deregulation may similarly influence inflammation-induced tumor progression and metastasis.
In this review, we discuss clinical and experimental evidence revealing the significant inflammatory and immunomodulatory functions of lymphatic vasculature, and suggest how tumor-associated remodeling may alter regional inflammation and immunity, thereby contributing to disease progression and metastasis. In addition, we suggest that lymphatic vessel morphology and function may be a biomarker of cutaneous immune status, and discuss how lymphatic biomarkers may provide insight into diagnosis and therapy for cutaneous malignancy.
It is now well appreciated that environmental exposures account for occurrence of many different types of cancer, with 15–20% of cancer deaths can be linked to inflammation or infection (12). Environmental factors include infectious agents, physical trauma (non-healing scars, burns), industrial toxins (e.g. tar, arsenic, radiotherapy), as well as those associated with lifestyle (tobacco, alcohol, diet, ultraviolet exposure) (13). As one might expect, tumors whose etiology is associated with environment-induced inflammation often occur in tissues with greatest surface area including lung, skin and gastrointestinal tract (14). Inflammation is considered one of ten hallmarks of cancer and is increasingly appreciated for its complex role in tumor initiation, promotion, malignant conversion and metastasis (15). Furthermore, immune infiltrates provide prognostic biomarkers (16) and potent targets for cancer therapy (17). We are just beginning to understand, however, how complex interactions between environmental agents, genetic factors and tissue microenvironments coordinate local inflammation and subsequent tumorigenesis.
Skin is a stratified tissue composed of epidermal, dermal, and subcutaneous adipose layers functioning in concert as a barrier to the external environment (1). Keratinocytes are the primary cell type in epidermis and undergo continuous cycles of homeostatic proliferation and differentiation to maintain a cornified layer protecting against water loss and microbe penetration (18). Melanocytes reside in basal layers of epidermis. Dermis is largely composed of collagenous and non-collagenous (e.g. elastic fibers and structural proteoglycans) connective tissue components that provide substratum support for blood and lymphatic vessels, nerves, mesenchymal support cells (fibroblasts), and resident immune cells. The cellular components of these layers critically regulate immune surveillance to facilitate rapid responses to insult while importantly maintaining immune tolerance (2,19) - breakdown of this balance is associated with a diverse set of immune-mediated dermatopathologies (1).
There are four major types of skin cancer, basal cell carcinoma, squamous cell carcinoma, melanoma and non-epithelial skin cancers (e.g. Merkel Cell, Kaposi’s sarcoma and cutaneous lymphoma). Predisposition to skin cancer is associated with chronic cutaneous inflammation (e.g. discoid lupus erythematosus, chronic wounds, dystrophic epidermolysis bullosa), viral infections (e.g. human immunodeficiency virus, human herpes virus 8 and human papilloma virus) and inflammatory environmental agents (e.g. UVR, viral infection, aging, diet, smoking, radiotherapy, phototherapy, physical trauma) (13), with inflammation playing a well-documented role in regulating progression of these cutaneous malignancies (20), however, skin cancers are also associated with chronically injured or non-healing scars, burns and chronic friction, with incidence of malignancy in scar tissue being low (0.1–2.5%) (13).
Furthermore, cells in skin secrete a diversity of inflammatory mediators, including toll-like receptor (TLR) ligands, polypeptide growth factors e.g. tissue necrosis factor (TNF)α, as well as chemokines that direct trafficking and subsequent activation of recruited leukocytes; many of which have been implicated as mediators of tumor progression in skin (20). For example, mast cells foster skin carcinogenesis through release of pro-survival and pro-angiogenic polypeptide growth factors and matrix remodeling proteinases that activate proliferative programs in keratinocytes, fibroblasts and endothelial cells (21). B cells secrete (auto)antibodies that form immune complexes with complement proteins – these accumulate in skin and influence resident and recruited myeloid cells activation and function (20). Tumor-infiltrating leukocytes play an important role in regulating angiogenic responses, which in turn, regulate their entry (22), and thereby influence metastatic potential (23). While much focus has been placed on blood vasculature with respect to leukocyte infiltration and function, lymphatic endothelium may similarly participate in these complex interactions. In support of this, using a murine model of chemically-induced skin carcinogenesis, it was recently reported that lymphatic vessels and their subsequent drainage are required for induction of local leukocytic infiltration, and consequently their absence resulted in initiation of fewer tumors (6). Given our current understanding of how lymphatic vessels participate in regulating local inflammation and immunity, as described below, it is reasonable to hypothesize that tumor-associated lymphatic remodeling participates in shaping immune microenvironments of solid tumors.
Lymphatic vessel architecture dictates local flow and the unidirectional transport of peripheral information to the secondary lymphoid system (24). Lymphatic vessels develop as a hierarchical, one-way drainage system functioning to transport cells and fluid into the lymphoid system and as a result of this architecture, lymphatic vessels lie at the interface of tissue biology and immunology (24). In skin, lymphatic vessels form two plexuses, one superficial extending into dermal papillae, and the other near the subpapillary arterial network (Figure 1A). Initial lymphatic capillaries are small, blind-ended vessels with minimal basement membrane that directly attach to extracellular matrix (ECM) through anchoring filaments allowing for rapid response to changes in local interstitial fluid pressure; swelling of ECM due to edema pulls on these filaments opening the loose, button-like inter-endothelial junctions (Figure 1B) (25–28).
Lymphatic capillaries drain into collecting vessels characterized by a continuous basement membrane, smooth muscle coverage and a system of valves that prevent retrograde fluid flow (Figure 1C). Lymphatic dysfunction results in accumulation of protein-rich interstitial fluid (lymphedema) leading to progressive fibrosis, adipose deposition and inflammation (29). While lymphatic vessels are required for drainage of soluble antigen and trafficking of activated dendritic cells (DCs) to local lymph nodes, their contribution to other aspects of inflammation and immunity are just being explored. There have been several excellent reviews highlighting current knowledge of lymphatic vessels to inflammation (30) and tolerance (31). Herein, we highlight their roles in immune induction and resolution or tolerance (Figure 2A–D), and discuss how their dysfunction contributes to tumor progression and metastasis. Of note, as many of these studies were not performed in skin, it remains to be seen how many of these mechanisms will translate across tissue systems.
Lymphatic vessels in adult tissues are typically quiescent, but may become activated when inflamed, resulting in lymphatic vessel remodeling within peripheral tissue and draining lymph nodes (26). Lymphatic remodeling is defined as changes to lymphatic vessel structure and morphology either through mechanisms of proliferative lymphangiogenesis or lymphatic vessel enlargement (26). Lymphangiogenesis specifically describes the formation of new lymphatic vessels from pre-existing vessels and occurs in many experimental and clinical inflammatory diseases including transplant rejection (renal and cornea), inflammatory bowel disease, chronic airway inflammation, and psoriasis (26,32–35). Though associated with various inflammatory settings, it remains unclear as to whether lymphangiognesis is a driving force in generation of inflammation and pathology or instead is an active attempt to resolve the inflammatory process.
Vascular endothelial growth factors (VEGFs) and their receptors are largely responsible for development, sprouting and remodeling of lymphatic vessels (26). Both hematopoietic and non-hematopoietic cells in skin orchestrate lymphangiogenic responses during inflammation. Epithelial cells in skin and fibroblastic reticular cells in lymph nodes support VEGF-A-dependent lymphangiogenesis (34,36) while macrophages induce local lymphangiogenesis largely through secretion of VEGF-C, that in turn signals through vascular endothelial growth factor receptor (VEGFR) 3, also required for developmental lymphatic vessel growth (37–39). VEGF-A producing B cells are required for inflammatory lymphangiogenesis in draining lymph node (40), but can be compensated for by neutrophils, likely through modulation of VEGF-A bioavailability and some VEGF-D production (41). In contrast, T cells can inhibit de novo lymphangiogenesis in an interferon (IFN)-γ-dependent manner at least in lymph nodes (42). Importantly, at least in the respiratory tract, inflammatory lymphangiogenesis is characterized by remodeling of the loose, button-like interendothelial junctions into tight, zipper-like junctions, characterized by firm adhesion along the length of two neighboring cells (Figure 1A) that may have functional consequences for mechanisms of immune cell entry and fluid transport (25,28).
Mobilization of DCs towards draining afferent lymphatic vessels is dependent on expression of the C-C chemokine receptor 7 (CCR7) that allows for active homing towards the C-C motif ligand 21 (CCL21) and CCL19-producing lymphatic vasculature (43). In addition to CCL21/CCL19, other chemokine signals required for cell entry into afferent lymph include chemokine C-X-C motif chemokine ligand (CXCL)12, sphingosine-1-phosphate (S1P), CX3CL1, the decoy receptor D6 (44). Also involved are adhesion molecules including intraceullar adhesion molecule 1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), L1CAM (CD171), ALCAM (CD166), C-type lectin receptor (CLEC-2), CD31, semaphorins, CD73, and the scavenger receptor CLEVER-1 (44,45). Whether integrins are absolutely required for DC transmigration is debated but is likely a function of inflammatory context and lymphatic endothelial cell (LEC) activation status (45–49). Cytokines, toll-like receptor (TLR) ligands and interstitial fluid flows all alter expression of adhesion molecules on LECs so as to promote leukocyte migration (44), and LECs derived from various inflammatory contexts acquire distinct transcriptional programs (50) indicating that LEC function and leukocyte trafficking patterns may be inflammation-specific. Furthermore, the endothelial glycoprotein PLVAP expressed by LECs controls entry of both lymphocytes and antigens into lymph nodes (51). The PVLAP protein acts as a diaphragm spanning transendothelial channels that transect sinus-lining LECs and confer selectivity of the sinus-parenchyma barrier (51). This selectivity may provide a way for the host to segregate small, likely inert proteins, which enter the lymph node conduit system (52) from larger agents that might cause damage if disseminated systemically.
One class of proteins that regulate or tune innate and adaptive immunity are the chemokine decoy receptors that scavenge and sequester chemokines to decrease local inflammatory signaling (53). This subfamily of “silent” chemokine receptors includes Duffy antigen receptor for chemokines (DARC), D6 (also known as CCBP2), and CCX-CKR (also known as CCRL1) and are strategically expressed in distinct cellular contexts (e.g. DCs and endothelial cells) where they regulate spatiotemporal inflammatory β-chemokine signaling (53). D6 is predominantly expressed by LECs (skin, gut, lung and syncytiotrophoblast layer of the placenta) - D6-null mice are unable to properly resolve local acute inflammation following dermal challenge due to exaggerated cutaneous inflammatory responses characterized by an accumulation of β-inflammatory chemokines at sites of inflammation (54), a process that may prevent further leukocyte recruitment. Furthermore, D6, induced during inflammation by interleukin-6 and IFNγ, facilitates selective presentation of homeostatic chemokines (e.g. CCL21) over inflammatory chemokines to prevent inappropriate inflammatory cell attachment to LECs and proper selection of mature over immature DCs (55). Extensive perilymphatic accumulation of leukocytes is observed in D6-null mice in both peripheral sites of inflammation and draining LNs, resulting in lymphatic congestion and impaired transport of antigen presenting cells and fluid (56). Importantly, D6 is downregulated in several human malignancies including Kaposi sarcoma, a cutaneous malignance of lymphatic endothelial origin, where low levels of D6 is associated with disease aggressiveness and infiltration of proangiogenic macrophages (57)
Lymphatic control of dendritic cell trafficking through both adhesion molecules and chemokines is an finely-regulated, multi-step process (45,49). Furthermore, given the intimate relationship peripheral afferent lymphatic vessels have with egressing leukocytes (45), it remains plausible that egressing leukocytes are educated by afferent lymphatic vessels as they exit peripheral tissue. The characterization of specialized three-dimensional structures at the interface of DC/LEC adhesions (58), intralymphatic crawling of DCs following entry into peripheral initial capillaries (59), acquisition of peptide:MHCII complexes by LECs from DCs (60) and LEC-mediated inhibition of DC maturation (61) all suggest that long-lived intercellular interactions between egressing leukocytes and peripheral afferent lymphatic vessels may be a novel control point for leukocyte function.
In addition to providing an initial route for entry of newly activated DCs into the afferent lymph, lymphatic vessels may also provide an important route of egress during resolution of acute and chronic inflammation. Consistent with this idea, induction of lymphatic vessel growth in skin is protective against acute and chronic skin inflammation (62,63), and inhibition of vessel growth in experimental inflammatory bowel disease exacerbates inflammation and disease progression (35). In murine inflammatory bowel disease macrophage egress from tissue in response to VEGF-C driven lymphangiogenesis is critical for limiting local inflammation (35). Similarly, in a mouse model of chronic respiratory tract infection, airway inflammation promoted bronchial lymphedema and airway obstruction when lymphangiogenesis was impaired (64). Furthermore, homeostatic gut leukocytes broadly migrate to draining LNs (65), whereas during cutaneous immunity regulatory T cells preferentially egress from skin (66).
Taken all together, dynamic patterns of leukocyte infiltration and egress appear to contribute to disease and control over leukocyte trafficking and tissue egress may be a novel point of immune regulation (67). Such a strategy is utilized by viruses to mediate immune escape (67). IFNα production following viral infection (68) and local skin irradiation (69) drive accumulation of cells in lymphoid organs, resulting in decreased peripheral blood and thoracic duct lymphocytes and overall immune suppression. Egress from peripheral tissues appears tightly controlled by a combination of CCR7 and sphingosine-1-phosphate (S1P) signaling, though coordination of these signals is poorly understood. In models of lung and skin infection, T cell egress from acutely inflamed peripheral tissue is dependent on CCL21/CCR7 chemotactic signals (70,71), while chronic inflammation adopts CCR7-independent mechanisms for tissue exit (72). Regulated tissue exit is likely required for recirculation of non-antigen specific T cells recruited sites of inflammation. Consistent with this, an interplay exists between antigen recognition and egress, where T cell receptor (TCR) stimulation results in downregulation of CCR7 and tissue retention (67). In solid tumors, at least in some cases, where antigen recognition can be limited due to physical constraints of tumor microenvironments as well as lack of potent neo-epitopes for TCR-recognition (73), cellular egress from tissues may correlate with poor local immunogenicity.
S1P gradients also importantly regulate tissue entry and exit throughout the body (e.g. blood to lymphoid tissue to lymph to peripheral tissue) (74). Lymphatic vessels express sphingosine kinase and maintain high levels of S1P in lymph generating gradients to direct tissue egress at steady state (75). Increased levels of S1P are present in inflamed peripheral tissues and this high tissue concentration may play a role in T cell retention (76). The S1P receptor, S1P1, inhibits migration of T lymphocytes into afferent lymphatic vessels both at steady state and during inflammation, resulting in their retention in non-lymphoid tissues at least partially through LFA-1/ICAM-1 and VLA-4/VCAM-1 interactions (76). The interplay between these signals provides a novel mechanism of control over leukocyte accumulation within peripheral tissue and subsequent disease progression. All together, these mechanisms suggest that lymphatic vessel function influences accumulation and/or retention of leukocytes within inflamed tissue thereby actively contributing to disease progression.
While the role of the lymphatic vasculature in cellular trafficking is appreciated, novel functions of lymphatic vessels and endothelial cells comprising their structure are emerging and indicate a role for lymphatic vessels in local immunmodulation and tolerance (24). LECs inhibit T cell activation and expansion through both antigen-dependent (7–10) and independent (77) mechanisms. While central mechanisms of tolerance eliminate self-reactive T cell clones during development in the thymus, education of the immune system is incomplete. As a consequence, tolerance must be maintained in the periphery to both environmental and self-antigens to prevent allergy and autoimmunity, respectively. Reminiscent of thymic epithelium, which deletes auto-reactive T cells in the thymus, LECs express multiple peripheral tissue antigens and directly present to auto-reactive T cells, albeit by a mechanism independent of the autoimmune regulator Aire (9). Direct presentation of the melanocyte-specific protein tyrosinase on major histocompatibility complex I (MHCI) by lymph node LECs mediates deletion of tyrosinase-specific T cells (10). T cell tolerance, in this context, is mediated by expression of the immune checkpoint molecule programmed death ligand 1 (PD-L1) on LECs and lack of costimulatory molecules; loss of PD-L1 activates tyrosinase-specific CD8+ T cells responses resulting in experimental vitiligo (10). While hyper-activation of melanocyte-specific T cells results in autoimmunity, vitiligo is prognostic for effective immunity during spontaneous regression of melanoma and following immunotherapy in melanoma patients (78). Induction of anti-self responses, in either an autoimmune or neoplastic context, requires a break in natural mechanisms of peripheral tolerance in which lymphatic vessels and their expression of PD-L1 play at least some role. Overexpression of PD-L1 is a hallmark of adaptive immune resistance in melanoma and a potential barrier to immunotherapy (79). While expression of PD-L1 is largely attributed to tumor cells, a wide variety of stromal cells also express PD-L1, and it remains to be determined how these players may contribute to immune suppression in tumor microenvironments (17).
In addition to direct presentation of peripheral antigens, LECs are phagocytic, scavenge lymph-borne antigen and cross-present to MHCI in a transport associated with antigen processing 1 (Tap1)-dependent manner (7,8). Again, presentation in this context results in lack of costimulation and dysfunctional T cell activation characterized by reduced IFNγ production, high-levels of PD-1 expression and enhanced apoptosis (7,8). Furthermore, overexpression of VEGF-C in murine melanomas results in immune protection against induced immunity, and specific cross-presentation of exogenous, tumor-associated antigens (ovalbumin) (7). In both the tumor context and at steady state, presentation of the CD8 epitope for ovalbumin by tumor-educated LECs results in dysfunctional activation of antigen-specific CD8 T cells (7,8). Interestingly, phagocytic activity of lymph node-resident LECs has recently been reported in a vaccine model, where LECs were long-term depots for vaccine antigen (80). While in these studies the authors did not demonstrate cross-presentation of antigen by LECs, this antigen-capture and archiving provided enhanced protection against infection (80). Understanding the contextual cues driving LEC decision making with respect to antigen capture and presentation will be of undeniable importance moving forward with respect to vaccination strategies and understanding the role of persisting and chronic antigen exposure during vaccination, infection and tumorigenesis.
Under homeostatic conditions, lymphatic vessels facilitate acute immune induction followed by appropriate resolution as described above. However, environmental risk factors for malignancy (ultraviolet radiation, infection, and sustained trauma) also induce lymphatic vessel dysfunction and may disturb this critical immune balance in tissue (Figure 3). In this way, regions of skin can acquire sectorial immune dysfunction, e.g. immune compromised districts, over time due to accumulation of environmental insults even in the absence of systemic immune disorder (81). These ‘districts’ exhibit enhanced risk for recapitulation of cutaneous disorders specifically at sites of trauma or insult (e.g. herpetic infections, irradiation, and burns) (81), or instead can conversely be spared from systemic immune pathology (81), and thus establishing regional discrepancies in immune function. How localized immune dysfunction may contribute directly to malignancy remains unclear, however, the coordinated dysfunction induced by these agents on both cells that acquire tumorogenic capacity and surrounding tissue stroma (including lymphatic vessels) is interesting to consider.
Ultraviolet radiation (UVR) is the most significant environmental risk factor for development of cutaneous malignancies (13). In addition to direct roles in initiating oncogenic mutations, UVR drives leukocyte infiltration of tissues, suppresses local T cell function, and induces lymphatic vessel dysfunction (82). Clinically, lymphatic vessels are reduced in number and dilated in sun-damaged skin as compared to non-sun exposed areas (83), and pathologically-dilated lymphatic vessels are associated with severe photoaging (84). Similarly, in mice, acute exposure to UVB radiation induces leukocyte infiltration of skin associated with epidermal hyperplasia, erythema and increased lymphatic vessel leakiness leading to edema (85,86). Maintenance of lymphatic vessel integrity following UVR is at least in part due to tight junction molecules (claudin-5 and ZO-1) and administration of angiopoietin 1 (Ang1) improves integrity (85,87). Disruption of claudin-5 junctions exacerbates not only edema associated with UVB exposure, but also accompanying leukocytic infiltrate (85), while overexpression of Ang1 is instead protective (87). Overexpression of VEGF-C attenuates UVB-induced edema and leukocytic infiltrate through the promotion of local lymphangiogenesis (63).
A second environmental influence on lymphatic vessels is infection. Recurrent herpetic infections are a clinical cause of secondary lymphedema and are often associated with localized immune dysfunction (88). The majority of experimental data exploring neovascularization responses to viral infection in mice have focused on the cornea, a normally alymphatic tissue, where local induction of lymphangiogenesis is associated with wounding, graft rejection and loss of immune privilege (32). In particular, herpes simplex virus 1 (HSV-1) infection induces genesis of lymphatic vessels into cornea in a TLR-independent, VEGF-A-dependent manner (89). In this model lymphangiogenesis precedes angiogenesis and is maintained at later time points by VEGF-C produced by local T cells (90). In addition to herpetic infections, skin infection by human papilloma virus (HPV) similarly results in microscopic regions of lymphatic dysfunction marked by increased number and severe dilation of local vessels (29). These regions are also associated with immune dysfunction and emergence of dermatopathology (clinical warts) associated with latent HPV infection following local trauma (29). HPV16-associated cancers exhibit lymphangiogenesis as well, and both remodeling of lymphatic vessels and expression of VEGF-C are associated with increased risk and lymph node metastasis (91). In humans, lymphatic vessel density in squamous cell carcinomas is increased immediately adjacent to tumor nests (91). Similarly, in a model of murine de novo squamous cell carcinogenesis driven by HPV16 oncoproteins, pre-malignant lymphatic vessels are structurally altered but functional, and progressively lose drainage function in central regions of tumors resulting in disruption of tissue hemodynamics (92).
Finally, surgical or other physical trauma result in disruption of regional lymphatic vessel networks (e.g. following radical mastectomy and lymph node resection) thereby leading to local lymphedema. The frequency of extremity (arm) lymphedema following surgical intervention for breast cancer ranges from 21% to 40% depending on both the type of surgery, and use of adjuvant radiotherapy (29). Localized lymphedema in scar and adjacent tissues has been observed, and high incidence of bacterial and fungal infections at sites of amputation is indicative of impaired local immunity, with stump skin found to exhibit an altered microbiome as compared to a non-amputated regions (88). The incidence of malignancy in scar tissue is 0.1–2.5% (13), however, the drivers of malignant conversion in these injured tissues and what role if any lymphatic function may play remains unknown. Interestingly, VEGF-C therapy may restore lymphatic function at least in some contexts to prevent surgery-associated lymphedema. Administration of growth factors along with lymph node transfer in pigs promoted robust growth of lymphatic vessels helping to preserve transferred node structure and normalize lymphatic vascular anatomy (93).
Lymphatic vessels have been evaluated in the context of many solid tumors for their ability to predict locoregional, lymph node metastasis and overall survival (94). We limit our discussion here to the interplay between inflammation and tumor-associated lymphangiogenesis and direct the reader to recent reviews that cover mechanisms of lymphangiogenesis, lymphogenous tumor spread and seeding in more detail (26,27). The major molecular drivers of tumor-associated lymphangiogenesis are VEGF-C and -D, produced both by tumor and infiltrating myeloid cells (26). VEGF-C and -D exert their biological effects through binding to VEGFR-3 and VEGFR-2, leading to activation of receptor-tyrosine kinase activity through auto-phosphorylation, which in turn activates aspects of LEC function, and vessel formation (95,96). VEGF-C induces endothelial cell migration, permeability, proliferation, vessel enlargement and enhanced trafficking of neoplastic cells to lymph nodes, largely via VEGFR-3-dependent mechanisms (96–100). VEGFR-3 expression correlates with lymphatic metastasis in some human tumors and high VEGF-C and -D expression is associated with increased lymphatic vessel density and lymphovascular invasion in both human tumors and animal models (97,99–102), and consequently inhibition of either VEGFR-3 or its coreceptor neuropilin 2 reduces incidence of lymph node metastasis (97,103–105).
In skin cancer the overwhelming majority of lymphatic vessel biomarker studies have focused on metastatic melanoma (94), where increased lymphatic vessel density correlates with VEGF-C expression in tumor microenvironments, lymphatic vessel invasion and lymph node metastasis (39,106,107). While several studies have demonstrated a positive correlation between lymphatic markers and survival (26,106) others have failed to demonstrate a correlation (108,109), thus generating some controversy over the true independent prognostic value of lymphatic vessel density and invasion biomarkers. In addition to melanoma however, recent studies have reported increased lymphangiogenesis in dermis adjacent to tumor nests in squamous cell carcinoma that also exhibit enhanced levels of VEGF-C produced by CD163+CD68+ macrophages (91). Similarly, an increase in the absolute numbers of lymphatic capillaries was observed in Merkel cell carcinoma, likely driven again by CD68+CD163+ tumor-associated macrophages (110), and increased levels of VEGFR3+ cells (both tumor and stroma) and lymphatic (podoplanin+) vessels correlated with disease progression in one study of Sézary syndrome, an aggressive subtype of cutaneous T-cell lymphoma (111).
In addition to remodeling of vessels in the primary tumor, significant enlargement of lymph sinuses in tumor-reactive lymph nodes is observed before arrival of metastatic cells (112). Distal remodeling of lymphatic vessels, both structural and functional, is mediated at least in part by VEGF-C and neuropilin-2 (98,113,114) and these changes may play a role in establishing pre-metastatic niches for locregional dissemination. Importantly, lymphatic remodeling induced by these signals is also associated with altered fluid flows that in turn correlates with lymph node metastasis (97–100,115). Dynamic lymphoscintography that traces accumulation of radionucleotide over time in draining LNs reveals that increased rates of lymphatic flow associate with increased incidence of metastasis and lymphatic vessel density in primary tumors (116). Moreover, VEGF-C overexpression further promotes lymph flow (116), and exogenous application of VEGF-C reduces tumor interstitial fluid pressures (117).
It was previously thought that functional lymphatic vessels were excluded from solid tumor parenchyma, thus negating their role in tumor progression. However, neither intratumoral lymphangiogenesis nor intratumoral lymphatic vessels are required in all cases for lymph node metastasis (118,119). Furthermore, an impaired capacity of lymphatic vessels to transport fluid does not necessarily correlate with their role in transporting cells, and instead, remodeling of peritumoral lymphatic vessels and activation of existing vessels is sufficient for altered homing properties and metastatic potential (98,118,119). The mechanisms by which lymphatic vessels actively recruit and promote seeding of tumor cells has been reviewed elsewhere (26). It is of note, however, that direct evidence for a sequential model of metastasis whereby lymphogenous spread precedes hematogenous colonization of distal organs is lacking (26,120); thus the relative contribution of tumor-associated lymphatic remodeling and metastasis to overall survival and disease progression remains somewhat unclear.
What may contribute to the association of lymphatic remodeling with poor prognosis is its relationship with local inflammation. Clinical and histopathological studies have demonstrated a correlation between cyclooxygenase (COX)-2 expression, lymphatic vessel density and lymph node metastasis in human malignancy (121–124). Current data indicates that the prostaglandin pathway influences lymphangiogenesis through upregulation of VEGF-C and -D expression in tumor-associated macrophages (125), as well as in neoplastic cells (126); however, the role of prostaglandins in lymphangiogenesis is not tumor-specific. In a subcutaneous matrigel plug assay of granuloma formation, lymphangiogenesis was also COX-2 dependent, requiring the prostaglandin E receptor (EP) 3 and 4 and macrophage production of VEGF-C (125). Similarly, COX-2 in the microenvironment of involuting mammary glands contributes to normal involution-associated lymphangiogenesis as well as mammary tumor-associated lymphangiogenesis, tumor cell invasion into lymphatic vessels and distal metastasis (127). Interestingly, it was recently reported that LECs regulate prostaglandin degradation downstream of VEGF-D/VEGFR-3/2 signaling resulting in the accumulation of tissue PGE2 and dilation of collecting lymphatic vessels draining the tumor (128). This dilation is associated with VEGF-D expression and enhanced LN metastasis in both human and mouse models (128). Specifically in skin, UVR stimulates COX-2 expression in epidermis (129) - the EP1, 2 and 4 receptors have been linked to UV-induced carcinogenesis (130), and notably inhibition of COX-2 via administration of celecoxib, prevents both squamous and basal cell carcinoma development in mice and humans (131,132).
Targeting of tumor-associated lymphangiogenesis as a potential therapy for solid tumors has been attempted in both pre-clinical and clinical trials (27,94). It is difficult, however, to separate effects on the lymphatic vasculature from hematogenous vessels given overlap in mechanisms required for their remodeling and growth, namely VEGFR3 (104,133,134). Either way, direct targeting of tumor vasculature in the clinic with FDA-approved drugs such as bevacizumab, sorafenib and sutinib that target VEGF-A and kinase activity of VEGF family receptors (all of which may also interfere with signaling on tumor-associated lymphatic endothelium) provide only transient benefit and ultimately tumors adapt and regrow (133).
Alternatively, it has been suggested that indirect inhibition of vascular remodeling may be induced through repolarization of immune microenvironments (135). Numerous studies have revealed that recruitment of myeloid cells facilitates the angiogenic switch through expression of VEGF and liberation of pro-angiogenic molecules from ECM (20,21). Targeting recruitment of these cells into tissue parenchyma results not only in release of local immunosuppression (i.e. increased effector T cell activity), but also a simultaneous normalization of angiogenic vasculature (135). Given the role local inflammation also plays in lymphangiogenesis it is reasonable to speculate that these strategies may similarly affect lymphatic vasculature. Use of non-steroidal anti-inflammatories (NSAIDs) for chemoprevention of cancer support this claim, as discussed above, where COX-2-dependent tumor formation is also associated with lymphangiogenesis in tumor microenvironments and LN metastasis (128,136,137).
Consequently, prophylactic treatment with inhibitors of COX-1 and 2 may halt tumor progression through simultaneous inhibition of local inflammation, lymphangiogenesis and angiogenesis (Figure 4A and B) (130,137). One caveat to direct or indirect anti-lymphanigogenic therapy, however, is evidence indicating that lymphatic vessels are resistant to ‘normalization’ therapy. Experimental evidence for this is provided by studies of mycoplasma pulmonis infection of murine respiratory tracts where angiogenesis and lymphangiogenesis are associated with infiltrating leukocytes (34). Resolution of inflammation through dexamethasone administration resolved the angiogenic responses but failed to resolve changes in lymphatic vessels, indicating that aberrant lymphatic vessel responses are long-lived and do not require continued maintenance. Only when dexamethasone was administered prophylactically, was local lymphatic vessel growth inhibited (34). Along these lines, in a corneal wounding model, lymphatic vessel regrowth kinetics accelerated with sequential challenges, indicating potential memory responses facilitating rapid recall growth in cornea (138). While it remains to be seen if similar observations can be made outside respiratory tract and cornea, these reports present the very interesting idea that long-lived lymphatic vessel aberrancies may exist, and may predict functional consequences for future inflammation, therapeutic resistance and recurrence. Clearly, if indeed refractory to normalization strategies, lymphatic vessels present an interesting challenge to clinical therapy, particularly with respect to therapeutic timing, that should be more systematically explored.
Recent interest in the role of the immune system in tumor progression has justified a series of studies evaluating the potential for local infiltrates to provide prognostic value for risk stratification (16). Melanoma was one of the first tumors to be associated with spontaneous anti-tumor immunity leading to regression - appearance of autoimmune vitiligo (139), presence of intralesional tumor infiltrating lymphocytes (140), expression of lymphocyte chemotactic factors (141) and a type I interferon transcriptional profile (142) enrich for a subset of clinical responders in this context. This idea has been formalized into a quantitative metric reflecting immune histopathology of tumors and is a significant prognostic indicator for disease-free survival and overall survival for colorectal cancer (16,143). Evaluation of immune contexture of tumors, e.g. ‘Immunoscore’, includes quantification of the density of CD8+ and CD45RO+ effector and memory T cells, as a function of their location in central tumor regions as compared to invasive margins (143). Recognition that Immunoscore significantly reflects a prognostic biomarker for tumor progression and patient outcome has yielded a broader awareness of the importance of CD8+ T cell infiltration as an indicator of an immunologically reactive tumor (4). What may further inform this type of immune-based stratification strategy is now an intense area of study and efforts to include various suppressive components of tumor microenvironments might provide added value to current approaches. These efforts typically focus on infiltrating immune cells of myeloid origin, however, the potential role for non-hematopoeitic stromal components such as vasculature, both blood and lymph, deserves attention. In fact, it may be the close-association between tumor-associated lymphangiogenesis and infiltrating myeloid cells underlying controversy in clinical lymphatic vessel biomarker studies (39,91). This, together with evidence that the degree of both intratumoral and peritumoral lymphatic vessel density associated with the presence of tumor-associated macrophages, more advanced stage (i.e. increased tumor thickness, mitotic count and ulceration) (39) and decreased numbers of infiltrating lymphocytes (144), indicates that correlations between lymphatic vasculature and local immunity in clinical samples is significant to consider.
Current immunohistochemical mapping of immune infiltrates and other components of tumor microenvironments are necessarily reliant on biopsies of primary or metastatic disease. Solid tumors, however, are known to be heterogeneous with respect to both distribution of intrinsic oncogenic signaling and activation of microenvironmental factors (145). Consequently, biopsies may not capture the full complexity within a developing tumor. While still the most valuable approach, identification of novel tissue biomarkers must be coupled to efforts to define correlative systemic biomarker signatures. The challenge in this approach is, however, exemplified by the fact that while VEGF-C is prognostic when detected in tissue by immunohistochemistry (101), few studies have demonstrated a correlation between serum VEGF A/C/D and LN metastasis (146). VEGF A/C/D produced at high concentrations in local tissue may become diluted or unstable as they circulate through the lymphoid system back to the thoracic duct due to their short half-lives in circulation, as a consequence it is not expected that serum levels will be necessarily diagnostic of LN metastases. A potential alternative source for biomarker identification is tumor interstitial fluid itself, which, unlike plasma, has a higher concentration of these proteins produced within tumor microenvironments and therefore may more reliably reflect altered biology within the tumor itself (147). Available methods for interstitial fluid isolation have been recently reviewed and include tissue centrifugation, tissue elution, capillary ultrafiltration and microdialysis (147). Enhanced levels of known cancer biomarkers, including VEGF, can be detected in interstitial fluid when compared to plasma (147), supporting the idea that tissue interstitial fluid, the contents of which are closer to lymph than plasma, may be a more robust source of biomarkers predicting primary tumor behavior.
Our understanding of the roles lymphatic vessels play in regulating tumor immune microenvironments in skin and other solid tumors is still in its infancy. The interplay between the vasculature (both hematogenous and lymphogenous), local inflammation and tumor progression sets up a far more complex story than that of nutrient delivery and waste removal (Figure 4A and B). The lymphatic vasculature coordinates local inflammation and immunity and its dysfunction may contribute to deregulated local inflammation, a hallmark of cancer as described by Hanahan and Weinberg (15). However, where lymphatic vessels really come into play in a developing cutaneous malignancy remains unclear. Whether their dysfunction early predisposes tissue to altered immunity thus leading to tumor immune escape or if their predominant role comes later during metastasis and spread remains an open question. Likely, lymphatic vessels participate in a continuous feedback loop, responding to microenvironmental change in peripheral tissue to propagate signal to lymph nodes that then alters host responses to tissue and cyclically continues. Our continued understanding of the complexities of lymphatic vessel function in the unique contexts of normal and diseased skin will provide a model to understand how regional immune dysfunction can participate in cutaneous malignancy and potential targets for prognostic and therapeutic strategies.
The tumor microenvironment and tumor-associated inflammation are now appreciated for not only their role in cancer progression but also response to therapy. The lymphatic vasculature is a less-appreciated component of this microenvironment that coordinates local inflammation and immunity and thereby critically shapes local responses. A mechanistic understanding for the complexities of lymphatic vessel function in the unique context of skin provides a model to understand how regional immune dysfunction drives cutaneous malignancies, and as such lymphatic vessels represent a biomarker of cutaneous immunity that may provide insight into cancer prognosis and effective therapy.
Disclosure Conflict of Interest: The authors have no conflicts of interest to disclose.
Financial Disclosure: The Lund Lab acknowledges support from the OHSU Knight Cancer Center (NIH P30-CA069533), the Collins Medical Trust of Oregon, the Medical Research Foundation, Cancer Research Institute and Department of Defense Peer Reviewed Cancer Research Program. TRM acknowledges support from the Cathy and Jim Rudd Career Development Award for Cancer Research, Medical Research Foundation, and the American Cancer Society – Friends of Rob Kinas. LMC acknowledges support from the NIH/NCI, DOD BCRP Era of Hope Scholar Expansion Award, Susan B. Komen Foundation, Breast Cancer Research Foundation, Stand Up To Cancer – Lustgarten Foundation Pancreatic Cancer Convergence Dream Team Translational Research Grant (SU2C-AACR-DT14-14), and Brenden-Colson Center for Pancreatic Health. The authors also acknowledge support from the OHSU Knight Cancer Institute. Stand Up to Cancer is a program of the Entertainment Industry Foundation administered by the American Association for Cancer Research.