Table 1.
TLR4 activation of monocytes preferentially induces lymphatic differentiation.
Fig 1.
TLR4-induced myeloid-lymphatic transition is preceded by shift to vascular phenotype.
Human monocytes cultured for 10 days with CSF1-supplemented medium were seeded on slides and treated with (A) growth medium with CSF1 (labeled -LPS) or (B) medium containing CSF1 and 50 ng/ml of LPS (labeled +LPS). After 4 days of treatment, cells were stained for vascular endothelial markers VEGFR-3, VEGFR-2, NRP-2, and podocalyxin. All images were acquired at 400X magnification.
Fig 2.
TLR4-mediated activation of human monocytes induces a lymphatic endothelial phenotype.
CD14+ monocytes were isolated from the blood of healthy donors using anti-CD14 IgG conjugated magnetic beads. (A) Overlays of representative flow cytometry histograms demonstrating expression of lymphatic markers VEGFR-3, LYVE-1 and PDPN for freshly isolated cells stained with marker-specific antibodies (red line) or isotype controls (grey area). (B, C, D, E) Cells were cultured for 10 days with human CSF1 followed by 4 days with CSF1-supplemented medium with or without TLR4 ligands. Overlays of representative histograms demonstrate expression of VEGFR-3, LYVE-1 and PDPN in cells treated with CSF1 only (B) or CSF1 and (C) LPS (50 ng/mL), (D) HMGB1 (50 ng/mL), or (E) nab-PXL (30 nM). All analyses were performed in duplicate and reproduced using monocytes from three different donors. Representative histograms for each target, time point, and differentiation stimulus are shown.
Fig 3.
TLR4-reprogrammed monocytes upregulate LEC-specific proteins and downregulate some myeloid markers.
CD14+ monocytes seeded on slides were double-stained on Day 1 in culture or on Day 14 (10 days with CSF1 followed by 4 days LPS) for expression of myeloid markers (A, D) CD14, and (G, J) CD68, and lymphatic-specific proteins (B, E) PDPN, and (H, K) PROX1. Double-staining of myeloid and LEC markers (A to L) used FITC- and Cy3-conjugated secondary antibodies, respectively. Single staining with Cy3-conjugated secondary antibodies was performed on cells at day 1 and 14 to detect LYVE-1 (M, N) and ITGA9 (O, P). Merged images of myeloid and lymphatic proteins detected in the same cells are shown in panels C, F, I and L. All images were acquired at 200X magnification.
Fig 4.
Lymphatic reprogramming of human monocytes is accompanied by upregulation of inflammatory cytokines and receptors.
CD14+ monocytes were treated with CSF1 alone (control) or additionally differentiated with (A, D) LPS, (B, E) HMGB1 or (C, F) nab-PXL. After 4 days of treatment, expression profiles of cells in control and TLR4 ligand-treated groups were compared using PCR arrays. All experiments were reproduced twice with each target analyzed in triplicate and normalized to β-actin. Results for each target are reported as mean fold-change in treated cells compared with the control group ± S.E.M. Results for selected targets are depicted with collective data provided in S3 Table under sub-groups “Cytokines” and “Cytokine Receptors”.
Fig 5.
Cytokines upregulated by TLR4 signaling might play autocrine, paracrine and chemotactic roles.
Human monocytes differentiated by LPS, HMGB1 and nab-PXL were analyzed by RT-qPCR as described under Methods. Matched pairs of cytokines and receptors were grouped based on expression pattern suggestive of roles in (A, D, G) paracrine, (B, E, H) autocrine, or (C, F, I) chemotactic signaling pathways. Results are plotted as mean fold-change ± S.E.M in CD14+ cells treated with TLR4 ligands as compared with CSF1 alone.
Fig 6.
TLR4 activation induces lymphatic reprogramming of myeloid cells isolated from BM of immunocompetent and immunodeficient mice.
CD11b+ cells isolated from BM of C57BL/6 mice (n = 4) and CB-17/SCID mice (n = 3) were used to induce M-LECP differentiation using nab-PXL (30 nM). Control cells were treated for identical time with medium containing only CSF1. After 4 days of nab-PXL or control treatment, cells were used to extract RNA for RT-qPCR analysis of representative targets (n = 43) performed in triplicate. Heat-maps of transcriptional changes detected in individual C57BL/6 and CB-17/SCID mice are shown. Beta actin-normalized data reported as mean fold-change ± S.E.M are shown in S4 and S5 Tables for C57BL/6 and CB-17/SCID mice, respectively. Rows correspond to listed genes and columns to individual mice with mouse strains indicated at the top. Scale bar denotes relative downregulation, no change, and upregulation as indicated by green, white and red colors, respectively.
Fig 7.
Human and mouse myeloid cells share similar patterns of TLR4-induced lymphatic reprogramming.
Human and mouse cells were treated with specie-specific CSF1 followed by treatment with nab-PXL (30 nM) before RT-qPCR analysis. Data for all analyzed targets (n = 43) are shown in S3 and S4 Tables for human monocytes and mouse cells from immunocompetent C57BL/6 strain, respectively. Similarities in gene expression are shown by grouping targets based on properties such as (A) lymphatic-specific genes; (B) transcriptional regulators; (C) inflammatory modulators; and (D) relatively unchanged genes. Results are presented as mean fold change ± S.E.M. for human and mouse cells treated with nab-PXL compared with cells treated with CSF1 alone for identical period of time.
Fig 8.
NF-κB inhibitors significantly suppress MLT.
Four sets of human monocytes were differentiated with LPS, HMGB1 or nab-PXL as described under Methods. One set was treated with only TLR4 ligands and served as control (A). Three other sets were additionally treated with (B) 10 nM of isohelenin, (C) 5 μM of PDTC, or (D) 10 nM of leptomycin-B. After 4 days of treatment, mRNA was extracted and used to determine expression of the indicated lymphatic genes. The analyses for each condition and gene target were performed in triplicate. Results are presented as mean fold-changes of beta actin normalized values ± S.E.M. Note 30-fold difference in the scale of the Y-axis of control plotted data (A) and that from NF-κB inhibitors treated cells (B-D).
Fig 9.
Endogenous M-LECP promote inflammatory and tumor lymphangiogenesis in vivo.
(A, B) C57BL/6 female mice were subjected to a lethal dose of radiation and injected with BM cells from GFP transgenic mice. Once BM reconstitution was complete (4 weeks later), mice received daily injections of LPS (50 μg) of for two weeks to induce chronic peritonitis. At this time, diaphragms were removed and stained with antibodies to GFP and (A) Lyve-1 or (B) Meca-32. (C) EMT6-Luc tumor bearing mice were injected with 5x106 of GFP+/CD11b+/Pdpn- or GFP+/CD11b+/Pdpn+ BM cells (6–7 mice per group). EMT6-Luc tumors were harvested 14 days later and stained with antibodies to Lyve-1 and GFP. Images were acquired at 400X magnification. (D) Recruitment of CD11b+/Pdpn- and CD11b+/Pdpn+ monocytes to EMT6-Luc tumors was quantified by determining the number of GFP+ pixels per field from four fields per section. (E) Peritumoral LVD of EMT6-Luc tumors was calculated by counting all peritumoral Lyve-1+ vessels on a section and normalizing per section area. Square and dot symbols represent Pdpn-positive and Pdpn-negative CD11b+ cells, respectively. Each symbol represents a value obtained from an individual mouse and reports the percentage of field covered by GFP cells or the density of Lyve-1+ vessels, respectively. The black bar indicates the mean for each group. Statistical significance was calculated by Student’s t-test with P-value indicated above black bracket.
Fig 10.
TLR4-induced M-LECP differentiated in vitro promote tumor lymphangiogenesis in vivo.
CD11b+ cells isolated from BM of GFP transgenic mice were differentiated with LPS and injected into tumor-bearing mice. (A) Overlays of representative histograms demonstrating expression of Vegfr-3, Lyve-1 and Pdpn for cells treated with CSF1 only (control, grey area) or CSF1 plus LPS to induce lymphatic differentiation (red lines). The mean % of positive cells for LEC markers from two independent experiments ± S.E.M. are shown. (B, C) Co-localization of GFP+ cells with Lyve-1+ (B) or Meca-32+ (C) vessels in a syngeneic breast cancer MMTV-PyMT tumors. (D, E) Co-localization of GFP+ cells with Lyve-1+ (D) or Meca-32+ (E) vessels in a human xenograft breast cancer ZR-75 model. All images were acquired at 400X magnification. (F) Quantification of blood and lymphatic vessels with integrated GFP. (G, H) Changes in peritumoral LVD in mice bearing MMTV-PyMT (G) or ZR-75 (H) tumors injected with M-LECP as compared with saline-injected controls.
Fig 11.
In vitro produced M-LECP increase lymphatic metastasis in vivo.
Renilla luciferase-tagged ZR-75 tumor cells were orthotopically implanted into mammary fat pad of immunodeficient SCID mice and allowed to reach 500 mm3 before injecting mice i.v. with 2x106 of in vitro LPS-differentiated mouse M-LECP or saline control (4 mice per group). (A) Tumor growth was monitored 2–3 times per week. Metastatic burden to lymph nodes (B) and lungs (C) was analyzed 45 days after M-LECP injection by measuring Renilla luciferase activity and normalizing per mg of total protein in tissue lysates. Each dot represents metastatic burden for an individual mouse expressed as RLU/mg of protein x 104. The black bracket indicates statistically significant difference between control and M-LECP treated groups (n = 4) determined by a Mann-Whitney test with P-value listed above the line.