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A function-blocking CD47 antibody modulates extracellular vesicle-mediated intercellular signaling between breast carcinoma cells and endothelial cells

Abstract

Tumor cells release extracellular vesicles (EVs) into the tumor microenvironment that may facilitate malignant progression and metastasis. Breast carcinoma EVs express high levels of the thrombospondin-1 and signal regulatory protein-α receptor CD47, which is the target of several experimental therapeutics currently in clinical trials. We analyzed changes in gene expression and function in human umbilical vein endothelial cells (HUVEC) induced by treatment with EVs derived from breast carcinoma cells and the effects of the function-blocking CD47 antibody B6H12 on the resulting intercellular communication. CD47+ EVs exhibited greater uptake by HUVEC compared to CD47 EVs, but the CD47 antibody did not inhibit their uptake. Global and targeted analyses of transcripts demonstrated that treatment of HUVEC with EVs derived from MDA-MB-231 breast carcinomas cells altered pathways associated with tumor necrosis factor-α signaling, angiogenesis, lymphangiogenesis, endothelial-mesenchymal transition, and extracellular matrix. EVs from triple-negative MDA-MB-231 cells were more active than EVs from less metastatic breast carcinoma cell lines. Treatment with MDA-MB-231 EVs down-regulated VEGFR2 mRNA expression and tyrosine phosphorylation while enhancing phosphorylation of the tyrosine phosphatase SHP2. VEGFR2 expression and phosphorylation in HUVEC was further inhibited by the CD47 antibody. Consistent with the observed changes in endothelial-mesenchymal transition genes and SHP2, treatment with MDA-MB-231-derived EVs decreased Zeb1 protein levels in HUVEC, whereas the CD47 antibody increased Zeb1 levels. The induction of E-selectin and other known targets of tumor necrosis factor-α signaling by EVs was also enhanced by the CD47 antibody, and E-selectin was the most up-regulated transcript following CD47 antibody treatment alone. These studies reveal several mechanisms by which therapeutics targeting CD47 could modulate tumor growth by altering the cross talk between cancer-derived EVs and nonmalignant cells in the tumor stroma.

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Abbreviations

bCSC:

Breast cancer stem cells

ECM:

Extracellular matrix

EMT:

Epithelial-mesenchymal transition

EnMT:

Endothelial-mesenchymal transition

EVs:

Extracellular vesicles

HUVEC:

Human umbilical vein endothelial cells

LYVE1:

Lymphatic vessel endothelial hyaluronan receptor 1

SHP2:

Tyrosine-protein phosphatase non-receptor type 11

SIRPα:

Signal regulatory protein-α

TNFα:

Tumor necrosis factor-α

TSP1/THBS1 :

Thrombospondin-1 protein/gene

TXNIP:

Thioredoxin-interacting protein

VEGF:

Vascular endothelial growth factor

VEGFR2:

Vascular endothelial growth factor receptor-2

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Acknowledgements

This work was supported by the Intramural Research Program of the National Institutes of Health, National Cancer Institute, Center for Cancer Research (DDR). We thank Kavya Ravikanti, Marlen Castro Hernandez, Jana’e Waugh and Weiwei Wu for technical assistance.

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DDR and SK designed the research, SK, AG, SPS, AA performed experiments. SK, DDR, SPS and AG analyzed and interpreted the data. SK and DDR wrote the manuscript.

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Correspondence to David D. Roberts.

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The authors declare that they have no competing interests.

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Kaur, S., Elkahloun, A.G., Singh, S.P. et al. A function-blocking CD47 antibody modulates extracellular vesicle-mediated intercellular signaling between breast carcinoma cells and endothelial cells. J. Cell Commun. Signal. 12, 157–170 (2018). https://doi.org/10.1007/s12079-017-0428-0

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Keywords

  • Extracellular vesicles
  • Tumor microenvironment
  • Endothelial-mesenchymal transition
  • CD47
  • Angiogenesis
  • Tumor necrosis factor-α