Endothelial deficiency of L1 reduces tumor angiogenesis and promotes vessel normalization - PubMed (original) (raw)

. 2014 Oct;124(10):4335-50.

doi: 10.1172/JCI70683. Epub 2014 Aug 26.

Alessandra Villa, Francesca Angiolini, Andrea Doni, Giovanni Mazzarol, Noemi Rudini, Luigi Maddaluno, Mina Komuta, Baki Topal, Hans Prenen, Melitta Schachner, Stefano Confalonieri, Elisabetta Dejana, Fabrizio Bianchi, Massimiliano Mazzone, Ugo Cavallaro

Endothelial deficiency of L1 reduces tumor angiogenesis and promotes vessel normalization

Elena Magrini et al. J Clin Invest. 2014 Oct.

Abstract

While tumor blood vessels share many characteristics with normal vasculature, they also exhibit morphological and functional aberrancies. For example, the neural adhesion molecule L1, which mediates neurite outgrowth, fasciculation, and pathfinding, is expressed on tumor vasculature. Here, using an orthotopic mouse model of pancreatic carcinoma, we evaluated L1 functionality in cancer vessels. Tumor-bearing mice specifically lacking L1 in endothelial cells or treated with anti-L1 antibodies exhibited decreased angiogenesis and improved vascular stabilization, leading to reduced tumor growth and metastasis. In line with these dramatic effects of L1 on tumor vasculature, the ectopic expression of L1 in cultured endothelial cells (ECs) promoted phenotypical and functional alterations, including proliferation, migration, tubulogenesis, enhanced vascular permeability, and endothelial-to-mesenchymal transition. L1 induced global changes in the EC transcriptome, altering several regulatory networks that underlie endothelial pathophysiology, including JAK/STAT-mediated pathways. In particular, L1 induced IL-6-mediated STAT3 phosphorylation, and inhibition of the IL-6/JAK/STAT signaling axis prevented L1-induced EC proliferation and migration. Evaluation of patient samples revealed that, compared with that in noncancerous tissue, L1 expression is specifically enhanced in blood vessels of human pancreatic carcinomas and in vessels of other tumor types. Together, these data indicate that endothelial L1 orchestrates multiple cancer vessel functions and represents a potential target for tumor vascular-specific therapies.

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Figures

Figure 9

Figure 9. Vascular L1 is upregulated in various human tumor types.

TMAs containing different tumor types and their normal tissue counterparts were stained for PECAM-1 (A and C) and L1 (B and D). Examples of thyroid carcinoma (A and B) and gastric carcinoma (C and D) are shown. Scale bars: 200 μm. (E) Quantitation of L1-positive vessels in noncancerous and tumor tissue expressed as percentage of L1-positive over PECAM-1–positive vessels. **P < 0.002 (n = 5 for each tissue type).

Figure 8

Figure 8. L1 is upregulated in human PDAC-associated vessels.

(A) Consecutive sections from human noncancerous or PDAC tissue were stained for PECAM-1 (left panels) and L1 (right panels) in order to visualize L1-positive vessels (arrows). Scale bars: 100 μm. (B) Quantitation of L1-positive vessels in noncancerous (n = 11) and tumor tissue (n = 18), expressed as percentage of L1-positive over PECAM-1–positive vessels. **P < 0.01.

Figure 7

Figure 7. L1 regulates EC function via the IL-6/JAK/STAT3 pathway.

(A) Gene network of L1-regulated genes. In bold, IPA-predicted upstream modulators. Lines connect modulators to direct targets, and colors indicate the consistency with the predicted activity with the expression change observed in L1-overexpressing luECs (i.e., target expression). Orange, consistent predicted activation of TFs; blue, consistent predicted inhibition of TFs; yellow, inconsistent predicted activation of TFs; grey, not defined activity. (B) qRT-PCR analysis of the indicated genes in mock- and L1-transfected luECs. Transcript levels were normalized as described in Methods and are shown as fold changes in L1-transfected cells relative to mock-transfected cells (n = 3). (C) The amount of IL-6 released in the culture medium by mock- and L1-transfected luECs was quantified by ELISA. (D) Immunoblotting analysis of mock- and L1-transfected luECs for IL-6Rα, phosphorylated STAT3, and total STAT3. (E) Immunoblotting analysis for phosphorylated and total STAT3 in mock- and L1-transfected luECs, treated either with anti–IL-6Rα antibody or with control IgG. (F) Immunoblotting analysis for phosphorylated and total STAT3 in mock- and L1-transfected luECs, treated either with vehicle (DMSO) or with 20 μM JAKi. Actin in D–F served as loading control. (G) Proliferation curves of mock- and L1-transfected luECs treated either with vehicle (DMSO) or with the indicated concentration of JAKi. (H) Mock- and L1-transfected luECs treated either with vehicle (DMSO) or with 20 μM JAKi were subjected to 24-hour migration assays. Data in G and H represent the mean ± SD from a representative experiment performed in triplicate. **P < 0.01; ***P < 0.001.

Figure 6

Figure 6. L1 regulates EC transcriptome.

(A) Hierarchical clustering of genes differentially expressed in L1-transfected versus control luECs. Three independent experimental replicates of L1-overexpressing and control cells were screened by gene expression microarray. Data were log2 transformed before clustering analysis. Red, upregulated genes; blue, downregulated genes. A total of 361 upregulated and 580 downregulated genes (i.e., 496 and 743 probe sets, respectively) were identified in L1-overexpressing cells. (B) qRT-PCR analysis of the indicated genes in mock- and L1-transfected luECs. Transcript levels were normalized as described in Methods and are shown as fold changes in L1-transfected cells relative to mock-transfected cells (n = 3). (C) qRT-PCR analysis of the indicated genes in luECs transfected either with 2 different L1 siRNAs (A and B) or with a control siRNA. Transcript levels are shown as fold changes in L1 siRNA–transfected cells relative to control cells (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001. (D) IPA analysis of L1-regulated genes. The enriched biofunctions were selected based on their significance (P < 0.05; Benjamini-Hochberg correction). Blue bars indicate the –log(P value) of enrichment.

Figure 5

Figure 5. L1 promotes EndMT.

(A) Immunoblotting analysis of mock- and L1-transfected luECs for S100A4/FSP1, Id1, KLF4, CD44, N-cadherin, fibronectin, and collagen IV. Actin, tubulin, and vinculin served as loading controls. (B) qRT-PCR analysis of the indicated genes in mock- and L1-transfected cells. Transcript levels were normalized as described in Methods and are shown as fold changes in L1-transfected cells relative to mock-transfected cells (n = 3). *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 4

Figure 4. L1 confers an angiogenic phenotype to ECs and enhances endothelial permeability.

(A) Proliferation curves of mock- and L1-transfected luECs. (B) Growth curves of hemangiomas formed by mock- or L1-transfected luECs injected subcutaneously into nude mice, as determined by volume measurement at the indicated time points. Representative images of hemangiomas explanted are shown (insets). (C) Weight of hemangiomas explanted 33 days after injection of mock- or L1-transfected luECs. (D) Migration assays of mock- and L1-transfected luECs were performed as described in Methods. (E) Matrigel-based tube formation assays of mock- and L1-transfected luECs were performed as described in Methods. (F) Mock- or L1-transfected luECs were stained for PECAM-1 (red) or VE-cadherin (green) prior to confocal analysis. Scale bars: 10 μm. (G) qRT-PCR analysis of claudin-5 mRNA in mock- and L1-transfected luECs. Transcript levels were normalized as described in Methods and are shown as fold changes in L1-transfected cells relative to mock-transfected cells (n = 3). (H) FITC-dextran permeability assays were performed on monolayers of mock- and L1-transfected luECs as described in Methods. Data in A, D, E, and H represent the mean ± SD from a representative experiment performed at least in triplicate. Data in B and C represent mean ± SEM from 10 to 12 mice per group. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 3

Figure 3. Treatment with anti-L1 antibodies reduces tumor growth and angiogenesis while increasing pericyte coverage in tumor vessels.

The volume (A) and weight (B) of pancreatic tumors from mice treated with anti-L1 antibodies (n = 6) or control (ctrl) IgG (n = 6) were recorded 14 days after Panc02 injection. Data represent means ± SEM. (C) Images of explanted tumors. (D) Quantitation of vessel density in tumors from mice treated with anti-L1 antibodies (n = 5) or control IgG (n = 5). (E) Quantitation of pericyte coverage in tumor vessels from mice treated with anti-L1 antibodies (n = 6) or control IgG (n = 5). *P < 0.05.

Figure 2

Figure 2. Endothelial L1 deficiency results in reduced tumor angiogenesis and in vessel normalization.

(A) Representative images of Panc02 tumor sections stained for PECAM-1 (red) to visualize vessels. Scale bars: 50 μm. (B) Quantitation of vessel density in tumors from L1floxed (n = 9) and Tie2-Cre;L1floxed mice (n = 5). (C) Representative images of Panc02 tumor sections costained for PECAM-1 (red) and the pericyte marker NG-2 (green) to visualize pericyte coverage. Scale bars: 50 μm. (D) Quantitation of pericyte coverage in tumor vessels from L1floxed (n = 8) and Tie2-Cre;L1floxed mice (n = 5). (E) Representative images of Panc02 tumor sections costained for the endothelial apical marker podocalyxin (cyan), the junctional marker VE-cadherin (yellow), and the basement membrane marker collagen IV (red). Arrows indicate the localization of VE-cadherin at cell-cell contact in Tie2-Cre;L1floxed tumor vessels (right), which is lost or dramatically reduced in L1floxed vessels (left). Scale bars: 10 μm. (F) Quantitation of collagen IV deposition in tumor vessels from L1floxed (n = 3) and Tie2-Cre;L1floxed mice (n = 3). (G) Representative images of Panc02 tumor sections from mice injected with Texas red–dextran. Sections were costained for Texas red (red) and PECAM-1 (green). Scale bars: 50 μm. (H) Quantitation of vascular permeability, expressed as the percentage of vessels showing extravasated dextran in tumors from L1floxed (n = 3) and Tie2-Cre;L1floxed mice (n = 3). *P < 0.05; ***P < 0.0005.

Figure 1

Figure 1. Ablation of endothelial L1 in Tie2-Cre;L1floxed mice and related effects on tumor development and mouse survival.

(A) Sections of normal pancreas and Panc02 tumors from L1floxed and Tie2-Cre;L1floxed mice were costained for L1 (green) and the vascular marker PECAM-1 (red), followed by confocal analysis. Arrows indicate vessels coexpressing PECAM-1 and L1; arrowheads indicate L1-positive nerves that served as internal control. Scale bars: 10 μm. (B and C) The volume (B) and weight (C) of pancreatic tumors from L1floxed and Tie2-Cre;L1floxed mice were recorded 14 days after Panc02 injection. Data represent mean ± SEM from 10 mice per group. (D) Images of explanted tumors. (E) Survival rates in Panc02 tumor–bearing L1floxed (n = 14) and Tie2-Cre;L1floxed mice (n = 13). **P < 0.01; ***P < 0.001.

References

    1. Ziyad S, Iruela-Arispe ML. Molecular mechanisms of tumor angiogenesis. Genes Cancer. 2011;2(12):1085–1096. doi: 10.1177/1947601911432334. - DOI - PMC - PubMed
    1. Ebos JM, Lee CR, Cruz-Munoz W, Bjarnason GA, Christensen JG, Kerbel RS. Accelerated metastasis after short-term treatment with a potent inhibitor of tumor angiogenesis. Cancer Cell. 2009;15(3):232–239. doi: 10.1016/j.ccr.2009.01.021. - DOI - PMC - PubMed
    1. Paez-Ribes M, et al. Antiangiogenic therapy elicits malignant progression of tumors to increased local invasion and distant metastasis. Cancer Cell. 2009;15(3):220–231. doi: 10.1016/j.ccr.2009.01.027. - DOI - PMC - PubMed
    1. Goel S, Wong AH, Jain RK. Vascular normalization as a therapeutic strategy for malignant and nonmalignant disease. Cold Spring Harb Perspect Med. 2012;2(3):a006486. - PMC - PubMed
    1. Moos M, Tacke R, Scherer H, Teplow D, Fruh K, Schachner M. Neural adhesion molecule L1 as a member of the immunoglobulin superfamily with binding domains similar to fibronectin. Nature. 1988;334(6184):701–703. doi: 10.1038/334701a0. - DOI - PubMed

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