New Model of Macrophage Acquisition of the Lymphatic Endothelial Phenotype

Macrophage-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.Time-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.We 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

Chemotherapy elicits pro-metastatic extracellular vesicles in breast cancer models

Cytotoxic chemotherapy is an effective treatment for invasive breast cancer. However, experimental studies in mice also suggest that chemotherapy has pro-metastatic effects. Primary tumours release extracellular vesicles (EVs), including exosomes, that can facilitate the seeding and growth of metastatic cancer cells in distant organs, but the effects of chemotherapy on tumour-derived EVs remain unclear. Here we show that two classes of cytotoxic drugs broadly employed in pre-operative (neoadjuvant) breast cancer therapy, taxanes and anthracyclines, elicit tumour-derived EVs with enhanced pro-metastatic capacity. Chemotherapy-elicited EVs are enriched in annexin A6 (ANXA6), a Ca2+-dependent protein that promotes NF-κB-dependent endothelial cell activation, Ccl2 induction and Ly6C+CCR2+ monocyte expansion in the pulmonary pre-metastatic niche to facilitate the establishment of lung metastasis. Genetic inactivation of Anxa6 in cancer cells or Ccr2 in host cells blunts the prometastatic effects of chemotherapy-elicited EVs. ANXA6 is detected, and potentially enriched, in the circulating EVs of breast cancer patients undergoing neoadjuvant chemotherapy

Resolvins suppress tumor growth and enhance cancer therapy

National Cancer Institute grants RO1 01CA170549-02 (to D. Panigrahy and C.N. Serhan), ROCA148633-01A4 (to D. Panigrahy), and GM095467 (to C.N. Serhan); the Stop and Shop Pediatric Brain Tumor Fund (to M.W. Kieran); the CJ Buckley Pediatric Brain Tumor Fund (to M.W. Kieran); Alex Lemonade Stand (to M.W. Kieran); Molly’s Magic Wand for Pediatric Brain Tumors (to M.W. Kieran); the Markoff Foundation Art-In-Giving Foundation (to M.W. Kieran); the Kamen Foundation (to M.W. Kieran); Jared Branfman Sunflowers for Life (to M.W.K.); and The Wellcome Trust program 086867/Z/08 (to M. Perretti)

Lymphatic endothelial progenitors originate from plastic myeloid cells activated by toll-like receptor-4

<div><p>Background</p><p>Myeloid-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.</p><p>Methodology/Principal findings</p><p>Human 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 <i>in vitro</i> were functional <i>in vivo</i> as demonstrated by significantly increased lymphatic vessel density and lymphatic metastasis detected in orthotopic breast cancer models.</p><p>Conclusions/Significance</p><p>We established a novel TLR4-dependent protocol for <i>in vitro</i> 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.</p></div

TLR4 activation of monocytes preferentially induces lymphatic differentiation.

<p>TLR4 activation of monocytes preferentially induces lymphatic differentiation.</p

Activation of NF-κB signaling precedes elevation of VEGFR-3 in LPS-treated RAW264.7 macrophages.

<p>(<b>A</b>) RT-qPCR analysis of NF-κB p50 and p65 expression in RAW264.7 macrophages treated with 100 ng/ml LPS for 0–24 hours (n = 6 per group). Relative transcript expression was normalized to β-actin. Data presented as β-actin normalized transcript expression ± SEM. The <i>P</i>-values represent *<0.05, **<0.01, and ***<0.001 versus control as determined by Student's unpaired <i>t</i> test. (<b>B</b>) Protein expression of NF-κB p50 phosphorylated on Ser-337 (phospho-p50), non-phosphorylated NF-κB p50, NF-κB p65 phosphorylated on Ser-276 (phospho-p65), non-phosphorylated NF-κB p65, VEGFR-3, and β-actin was determined by Western blot in RAW264.7 macrophages treated with 100 ng/ml of LPS for 0–48 hours. Representative blot from two independent experiments performed in triplicate wells is shown (total n = 6 per time point). (<b>C</b>) RT-qPCR analysis of LPS-induced VEGFR-3 expression in the presence of NF-κB inhibitor, leptomycin B. Relative transcript expression was normalized to β-actin. Data presented as β-actin normalized transcript expression ± SEM. CTRL (taken as 1) represents the mean basal VEGFR-3 expression in untreated RAW264.7 macrophages. The <i>P</i>-values represent *<0.05, **<0.01, and ***<0.001 <i>versus</i> control as determined by Student's unpaired <i>t</i> test.</p