Lipoxygenase mediates invasion of intrametastatic lymphatic vessels and propagates lymph node metastasis of human mammary carcinoma xenografts in mouse
Dontscho Kerjaschki, … , Dieter Steinhilber, Georg Krupitza
Dontscho Kerjaschki, … , Dieter Steinhilber, Georg Krupitza
Published April 11, 2011
Citation Information: J Clin Invest. 2011;121(5):2000-2012. https://doi.org/10.1172/JCI44751.
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Lipoxygenase mediates invasion of intrametastatic lymphatic vessels and propagates lymph node metastasis of human mammary carcinoma xenografts in mouse

Abstract

In individuals with mammary carcinoma, the most relevant prognostic predictor of distant organ metastasis and clinical outcome is the status of axillary lymph node metastasis. Metastases form initially in axillary sentinel lymph nodes and progress via connecting lymphatic vessels into postsentinel lymph nodes. However, the mechanisms of consecutive lymph node colonization are unknown. Through the analysis of human mammary carcinomas and their matching axillary lymph nodes, we show here that intrametastatic lymphatic vessels and bulk tumor cell invasion into these vessels highly correlate with formation of postsentinel metastasis. In an in vitro model of tumor bulk invasion, human mammary carcinoma cells caused circular defects in lymphatic endothelial monolayers. These circular defects were highly reminiscent of defects of the lymphovascular walls at sites of tumor invasion in vivo and were primarily generated by the tumor-derived arachidonic acid metabolite 12S-HETE following 15-lipoxygenase-1 (ALOX15) catalysis. Accordingly, pharmacological inhibition and shRNA knockdown of ALOX15 each repressed formation of circular defects in vitro. Importantly, ALOX15 knockdown antagonized formation of lymph node metastasis in xenografted tumors. Furthermore, expression of lipoxygenase in human sentinel lymph node metastases correlated inversely with metastasis-free survival. These results provide evidence that lipoxygenase serves as a mediator of tumor cell invasion into lymphatic vessels and formation of lymph node metastasis in ductal mammary carcinomas.

Authors

Dontscho Kerjaschki, Zsuzsanna Bago-Horvath, Margaretha Rudas, Veronika Sexl, Christine Schneckenleithner, Susanne Wolbank, Gregor Bartel, Sigurd Krieger, Romana Kalt, Brigitte Hantusch, Thomas Keller, Katalin Nagy-Bojarszky, Nicole Huttary, Ingrid Raab, Karin Lackner, Katharina Krautgasser, Helga Schachner, Klaus Kaserer, Sandra Rezar, Sybille Madlener, Caroline Vonach, Agnes Davidovits, Hitonari Nosaka, Monika Hämmerle, Katharina Viola, Helmut Dolznig, Martin Schreiber, Alexander Nader, Wolfgang Mikulits, Michael Gnant, Satoshi Hirakawa, Michael Detmar, Kari Alitalo, Sebastian Nijman, Felix Offner, Thorsten J. Maier, Dieter Steinhilber, Georg Krupitza

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Figure 2

MCF7 cell spheroids induce CCIDs in lymphatic endothelial cell monolayers and disrupt VE-cadherin at the CCIDs.

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MCF7 cell spheroids induce CCIDs in lymphatic endothelial cell monolayer...
(A) Spheroid of HLFs fails to induce any defects in a monolayer of human lymphatic endothelial cells (LECs, Cytotracker tagged in green) after 4 hours of cocultivation. (B) MCF7 spheroids induce circular CCIDs. (C) A MCF7 spheroid–induced CCID is outlined when the LEC borders are stained for CD31 (red, confocal image). Inset, LECs (demarcated with CD31 in red) at the margin of the CCIDs show expression of PPP1R12A (MYPT1) (green) indicating cell mobility (confocal image). (D) MCF7 spheroids induce CCIDs preferentially in LEC monolayers (left bar), but significantly less (14.3% of lymphatics; *P = 0.0047) in monolayers of microvascular blood endothelial cells (BECs, right bar). (E) When compared with spheroids of MCF7 cells (left bar, 100%), CCID formation in lymphatic monolayers is marginally induced by nonmalignant human breast epithelial cells MCF-10A (9.6% of MCF7 spheroids), and HLFs (11.3%). Data are presented as mean ± SEM. *P < 0.0001. (F and G) Tracings of LEC migration (red lines; starting positions are marked by circles) beneath a spheroid during a 4-hour coincubation. (H) Confocal image shows continuous LEC junctions of VE-cadherin (VE Cad) at distance from a spheroid. (I) At the spheroid’s margin, the VE-cadherin pattern is disrupted. (J) Confluent LECs were incubated with 1 μM 12(S)-HETE for 15 and 30 minutes or with solvent (0), and cell lysates were immunoblotted with antibodies to VE-cadherin or β-actin. As controls, VE-cadherin– MCF7 cells were used. Lanes were run on the same gel but are noncontiguous. Scale bars: 100 μm (A, B, F, and G); 25 μm (C); 50 μm (H and I).