Lymphatic metastasis is the main prognostic factor for survival of patients with breast cancer and other epithelial malignancies. Mounting clinical and experimental data suggest that migration of tumor cells into the lymph nodes is greatly facilitated by lymphangiogenesis, a process that generates new lymphatic vessels from pre-existing lymphatics with the aid of circulating lymphatic endothelial progenitor cells. The key protein that induces lymphangiogenesis is vascular endothelial growth factor receptor-3 (VEGFR-3), which is activated by vascular endothelial growth factor-C and -D (VEGF-C and VEGF-D). These lymphangiogenic factors are commonly expressed in malignant, tumor-infiltrating and stromal cells, creating a favorable environment for generation of new lymphatic vessels. Clinical evidence demonstrates that increased lymphatic vessel density in and around tumors is associated with lymphatic metastasis and reduced patient survival. Recent evidence shows that breast cancers induce remodeling of the local lymphatic vessels and the regional lymphatic network in the sentinel and distal lymph nodes. These changes include an increase in number and diameter of tumor-draining lymphatic vessels. Consequently, lymph flow away from the tumor is increased, which significantly increases tumor cell metastasis to draining lymph nodes and may contribute to systemic spread. Collectively, recent advances in the biology of tumor-induced lymphangiogenesis suggest that chemical inhibitors of this process may be an attractive target for inhibiting tumor metastasis and cancer-related death. Nevertheless, this is a relatively new field of study and much remains to be established before the concept of tumor-induced lymphangiogenesis is accepted as a viable anti-metastatic target. This review summarizes the current concepts related to breast cancer lymphangiogenesis and lymphatic metastasis while highlighting controversies and unanswered questions.

The concept of inflammation-induced lymphangiogenesis (ie, formation of new lymphatic vessels) has long been recognized, but the molecular mechanisms remained largely unknown. The 2 primary mediators of lymphangiogenesis are vascular endothelial growth factor receptor-3 (VEGFR-3) and Prox1. The key factors that regulate inflammation-induced transcription are members of the nuclear factor-kappaB (NF-B) family; however, the role of NF-B in regulation of lymphatic-specific genes has not been defined. Here, we identified VEGFR-3 and

Emerging evidence suggests that cytotoxic therapy may actually promote drug resistance and metastasis while inhibiting the growth of primary tumors. Work in preclinical models of breast cancer have shown that acquired chemoresistance to the widely used drug paclitaxel (PXL) can be mediated by activation of the Toll-like receptor TLR4 in cancer cells. In this study, we determined the pro-metastatic effects of tumor-expressed TLR4 and PXL therapy and we investigated the mechanisms mediating these effects. While PXL treatment was largely efficacious in inhibiting TLR4-negative tumors, it significantly increased the incidence and burden of pulmonary and lymphatic metastasis by TLR4-positive tumors. TLR4 activation by PXL strongly increased the expression of inflammatory mediators, not only locally in the primary tumor microenvironment but also systemically in the blood, lymph nodes, spleen, bone marrow and lungs. These pro-inflammatory changes promoted the outgrowth of Ly6C+ and Ly6G+ myeloid progenitor cells and their mobilization to tumors, where they increased blood vessel formation but not invasion of these vessels. In contrast, PXL-mediated activation of TLR4-positive tumors induced de novo generation of deep intratumoral lymphatic vessels that were highly permissive to invasion by malignant cells. These results suggest that PXL therapy of patients with TLR4-expressing tumors may activate systemic inflammatory circuits that promote angiogenesis, lymphangiogenesis and metastasis, both at local sites and premetastatic niches where invasion occurs in distal organs. Taken together, our findings suggest that efforts to target TLR4 on tumor cells may simultaneously quell local and systemic inflammatory pathways that promote malignant progression, with implications for how to prevent tumor recurrence and the establishment of metastatic lesions, either during chemotherapy or after it is completed.

BackgroundMacrophage-derived lymphatic endothelial cell progenitors (M-LECPs) contribute to new lymphatic vessel formation, but the mechanisms regulating their differentiation, recruitment, and function are poorly understood. Detailed characterization of M-LECPs is limited by low frequency in vivo and lack of model systems allowing in-depth molecular analyses in vitro. Our goal was to establish a cell culture model to characterize inflammation-induced macrophage-to-LECP differentiation under controlled conditions.Methodology/Principal FindingsTime-course analysis of diaphragms from lipopolysaccharide (LPS)-treated mice revealed rapid mobilization of bone marrow-derived and peritoneal macrophages to the proximity of lymphatic vessels followed by widespread (∼50%) incorporation of M-LECPs into the inflamed lymphatic vasculature. A differentiation shift toward the lymphatic phenotype was found in three LPS-induced subsets of activated macrophages that were positive for VEGFR-3 and many other lymphatic-specific markers. VEGFR-3 was strongly elevated in the early stage of macrophage transition to LECPs but undetectable in M-LECPs prior to vascular integration. Similar transient pattern of VEGFR-3 expression was found in RAW264.7 macrophages activated by LPS in vitro. Activated RAW264.7 cells co-expressed VEGF-C that induced an autocrine signaling loop as indicated by VEGFR-3 phosphorylation inhibited by a soluble receptor. LPS-activated RAW264.7 macrophages also showed a 68% overlap with endogenous CD11b+/VEGFR-3+ LECPs in the expression of lymphatic-specific genes. Moreover, when injected into LPS- but not saline-treated mice, GFP-tagged RAW264.7 cells massively infiltrated the inflamed diaphragm followed by integration into 18% of lymphatic vessels.Conclusions/SignificanceWe present a new model for macrophage-LECP differentiation based on LPS activation of cultured RAW264.7 cells. This system designated here as the “RAW model” mimics fundamental features of endogenous M-LECPs. Unlike native LECPs, this model is unrestricted by cell numbers, heterogeneity of population, and ability to change genetic composition for experimental purposes. As such, this model can provide a valuable tool for understanding the LECP and lymphatic biology.

BackgroundMyeloid-derived lymphatic endothelial cells (M-LECP) are induced by inflammation and play an important role in adult lymphangiogenesis. However, the mechanisms driving M-LECP differentiation are currently unclear. We previously showed that activation of Toll-like receptor-4 (TLR4) induces myeloid-lymphatic transition (MLT) of immortalized mouse myeloid cells. Here the goals were to assess the potential of different TLR4 ligands to induce pro-lymphatic reprogramming in human and mouse primary myeloid cells and to identify transcriptional changes regulating this process.Methodology/Principal findingsHuman and mouse myeloid cells were reprogrammed to the lymphatic phenotype by TLR4 ligands including lipopolysaccharide (LPS), recombinant high mobility group box 1 protein (HMGB1), and paclitaxel. TLR4 induced similar MLT in cells from mice of different strains and immune status. Commonly induced genes were detected by transcriptional profiling in human and mouse myeloid cells from either immunocompetent or immunodeficient mice. Shared trends included: (1) novel expression of lymphatic-specific markers vascular endothelial growth factor receptor-3 (VEGFR-3), lymphatic vessel endothelial hyaluronan receptor-1 (LYVE-1) and podoplanin (PDPN) largely absent prior to induction; (2) lack of notable changes in blood vessel-specific markers; (3) transient expression of VEGFR-3, but sustained increase of vascular endothelial growth factor-C (VEGF-C) and a variety of inflammatory cytokines; (4) dependency of VEGFR-3 upregulation and other LEC genes on NF-κB; and (5) novel expression of lymphatic-specific (e.g., PROX1) and stem/progenitor (e.g., E2F1) transcription factors known for their roles in adult and embryonic vascular formation. M-LECP generated by TLR4 ligands in vitro were functional in vivo as demonstrated by significantly increased lymphatic vessel density and lymphatic metastasis detected in orthotopic breast cancer models.Conclusions/SignificanceWe established a novel TLR4-dependent protocol for in vitro production of functionally competent M-LECP from primary human or mouse myeloid cells and identified many potential regulators of this process. This information can be further exploited for research and therapeutic purposes.