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Metastasis is impaired by endothelial-specific Dll4 loss-of-function through inhibition of epithelial-to-mesenchymal transition and reduction of cancer stem cells and circulating tumor cells

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Abstract

Systemic inhibition of Dll4 has been shown to thoroughly reduce cancer metastasis. The exact cause of this effect and whether it is endothelial mediated remains to be clarified. Therefore, we proposed to analyze the impact of endothelial Dll4 loss-of-function on metastasis induction on three early steps of the metastatic process, regulation of epithelial-to-mesenchymal transition (EMT), cancer stem cell (CSC) frequency and circulating tumor cell (CTC) number. For this, Lewis Lung Carcinoma (LLC) cells were used to model mouse tumor metastasis in vivo, by subcutaneous transplantation into endothelial-specific Dll4 loss-of-function mice. We observed that endothelial-specific Dll4 loss-of-function is responsible for the tumor vascular regression that leads to the reduction of tumor burden. It induces an increase in tumoral blood vessel density, but the neovessels are poorly perfused, with increased leakage and reduced perivascular maturation. Unexpectedly, although hypoxia was increased in the tumor, the number and burden of macro-metastasis was significantly reduced. This is likely to be a consequence of the observed reduction in both EMT and CSC numbers caused by the endothelial-specific Dll4 loss-of-function. This multifactorial context may explain the concomitantly observed reduction of the circulating tumor cell count. Furthermore, our results suggest that endothelial Dll4/Notch-function mediates tumor hypoxia-driven increase of EMT. Therefore, it appears that endothelial Dll4 may constitute a promising target to prevent metastasis.

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Acknowledgements

The authors thank Dr. Hugo Pissarra for the assistance in the histological analysis of lung metastases.

Funding

This work was supported by the Portuguese Foundation for Science and Technology (FCT; http://www.fct.pt/index.phtml.en), Grants PTDC/SAU-ONC/116164/2009 and PTDC/SAU-ONC/121742/2010 to AT. CIISA has provided support through Project UID/CVT/276/2019, funded by FCT. LM is a PhD student supported by a studentship from FCT (Grant No. SFRH/BD/74229/2010). AT is a Postdoctoral Researcher supported by FCT (Grant No. SFRH/BPD/110174/2015). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Author notes

  1. Liliana Mendonça

    Present address: USF Vida + , ACES Cávado II Gerês/Cabreira, ARS Norte, Vila Verde, Braga, Portugal

  2. Marina Badenes

    Present address: Instituto Gulbenkian de Ciência, Oeiras, Portugal

  3. Catarina Carvalho

    Present address: Centro Champalimaud, Fundação Champalimaud, Lisbon, Portugal

  4. Joana Gigante

    Present address: Boehringer Ingelheim, Lisbon, Portugal

Authors and Affiliations

  1. CIISA – Centro de Investigação Interdisciplinar em Sanidade Animal, Faculdade de Medicina Veterinária, Universidade de Lisboa, Lisbon, Portugal

    Liliana Mendonça, Alexandre Trindade, Catarina Carvalho, Jorge Correia, Marina Badenes, Joana Gigante & António Duarte

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  1. Liliana Mendonça
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Correspondence to Alexandre Trindade.

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Mendonça, L., Trindade, A., Carvalho, C. et al. Metastasis is impaired by endothelial-specific Dll4 loss-of-function through inhibition of epithelial-to-mesenchymal transition and reduction of cancer stem cells and circulating tumor cells. Clin Exp Metastasis 36, 365–380 (2019). https://doi.org/10.1007/s10585-019-09973-2

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