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PML–RARa modulates the vascular signature of extracellular vesicles released by acute promyelocytic leukemia cells

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Abstract

Oncogenic transformation is believed to impact the vascular phenotype and microenvironment in cancer, at least in part, through mechanisms involving extracellular vesicles (EVs). We explored these questions in the context of acute promyelocytic leukemia cells (NB4) expressing oncogenic fusion protein, PML–RARa and exquisitely sensitive to its clinically used antagonist, the all-trans retinoic acid (ATRA). We report that NB4 cells produce considerable numbers of EVs, which are readily taken up by cultured endothelial cells triggering their increased survival. NB4 EVs contain PML–RARa transcript, but no detectable protein, which is also absent in endothelial cells upon the vesicle uptake, thereby precluding an active intercellular trafficking of this oncogene in this setting. ATRA treatment changes the emission profile of NB4-related EVs resulting in preponderance of smaller vesicles, an effect that occurs in parallel with the onset of cellular differentiation. ATRA also increases IL-8 mRNA and protein content in NB4 cells and their EVs, while decreasing the levels of VEGF and tissue factor (TF). Endothelial cell uptake of NB4-derived EVs renders these cells more TF-positive and procoagulant, and this effect is diminished by pre-treatment of EV donor cells with ATRA. Profiling angiogenesis-related transcripts in intact and ATRA-treated APL cells and their EVs reveals multiple differences attributable to cellular responses and EV molecular packaging. These observations point to the potential significance of changes in the angiogenic signature and activity associated with EVs released from tumor cells subjected to targeted therapy.

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Abbreviations

APL:

Acute promyelocytic leukemia

ATO:

Arsenic trioxide

ATRA:

All-trans retinoic acid

EVs:

Extracellular vesicles

IL-8:

Interleukin 8

NTA:

Nanoparticle tracking analysis

PML–RARa:

Promyelocytic leukemia–retinoic acid receptor alpha

TF:

Tissue factor

TF-PCA:

TF-dependent procoagulant activity

VEGF:

Vascular endothelial growth factor

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Acknowledgments

We thank our McGill colleagues for their assistance: J. Mui (Electron Microscopy) and M. Fu (Confocal Microscopy). The project was supported by operating grants to J. R. from the Canadian Institutes of Health Research (CIHR Foundation Grant; MOP 111119, 133424). The infrastructure support was provided by Fonds de Recherche en Santé du Québec (FRSQ). J. R. is Jack Cole Chair in Pediatric Hematology/Oncology. Y. F. was supported by Postdoctoral Fellowship from the Cole Foundation, National Natural Science Foundation of China for Young Scientists (30801062), China Scholarship Council (2009831055) and Scholarship of Renji Hospital Sponsored Overseas Project; DG was supported by Michael Whitehead Fellowship Endowment to Montreal Children’s Hospital Foundation, and EDA was a recipient of the FRSQ Doctoral Studentship and Piccoli Research Fund.

Contributions

YF—designed, conducted and interpreted the experiments, and wrote the manuscript. This included culturing and analysis of NB4 and HUVEC cells, Western blotting ELISA, NTA and other assays. DG, THL, EDA, LM and BM—established the assays, conducted experiments provided intellectual input and revised the manuscript. This included calibration of NTA assays, RNA and Western protocols, EV transfer experiments, FACS and other experiments; JR—conceived and designed the study, interpreted the data, administered and supervised the project.

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Authors and Affiliations

  1. The Research Institute of the McGill University Health Centre, Montreal Children’s Hospital, McGill University, 1001 Decarie Blvd, Montreal, QC, H4A 3J1, Canada

    Yi Fang, Delphine Garnier, Tae Hoon Lee, Esterina D’Asti, Laura Montermini, Brian Meehan & Janusz Rak

  2. Department of Hematology, Renji Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, 200127, China

    Yi Fang

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  1. Yi Fang
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  2. Delphine Garnier
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Correspondence to Janusz Rak.

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J.R is the inventor on a pending patent application describing identification of oncogenes in the cargo of extracellular vesicles. Otherwise authors declare no competing interests.

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Fang, Y., Garnier, D., Lee, T.H. et al. PML–RARa modulates the vascular signature of extracellular vesicles released by acute promyelocytic leukemia cells. Angiogenesis 19, 25–38 (2016). https://doi.org/10.1007/s10456-015-9486-1

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