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Non-classical Notch signaling by MDA-MB-231 breast cancer cell-derived small extracellular vesicles promotes malignancy in poorly invasive MCF-7 cells


Aberrant Notch signaling is implicated in breast cancer progression, and recent studies have demonstrated links between the Notch pathway components Notch1 and Notch1 intracellular domain (N1ICD) with poor clinical outcomes. Growing evidence suggests that Notch signaling can be regulated by small extracellular vesicles (SEVs). Here, we used breast cancer cell models to examine whether SEVs are involved in functional Notch signaling. We found that Notch components are packaged into MDA-MB-231- and MCF-7-derived SEVs, although higher levels of N1ICD were detected in SEVs from the more aggressive MDA-MB-231 cell line than from poorly invasive MCF-7 cells. SEV-Notch components were functional, as SEVs cargo from MDA-MB-231 cells induced the expression of Notch target genes in MCF-7 cells and triggered a more invasive and proliferative phenotype concomitant with the acquisition of mesenchymal features. Neutralization of the N1ICD cargo in MDA-MB-231-derived SEVs significantly reduced their potential to enhance the aggressiveness of MCF-7 cells in vitro and in a xenograft model. Overall, our results indicate that a SEV-mediated non-classical pathway of Notch signal transduction in breast cancer models bypasses the need for classical ligand–receptor interactions, which may have important implications in cancer.

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Fig. 1: Characterization of small extracellular vesicles collected from MDA-MB-231 and MCF-7 cells in recollection medium.
Fig. 2: Notch components are present in proteins extracted from MDA-MB-231 and MCF-7 cells and associated small extracellular vesicles.
Fig. 3: Notch components packaged into MDA-MB-231 small extracellular vesicles trigger expression changes in Notch target genes and epithelial-to-mesenchymal transition marker genes in MCF-7 cells.
Fig. 4: MDA-MB-231 small extracellular vesicles induce a more aggressive phenotype in MCF-7 cells that is Notch signaling-dependent.
Fig. 5: N1ICD loaded into MDA-231-SEVs induces expression changes in Notch target genes and epithelial-to-mesenchymal transition marker genes in MCF-7 cells.
Fig. 6: N1ICD loaded into MDA-231-SEVs enhances cell motility and proliferation in recipient MCF-7 cells.
Fig. 7: N1ICD loaded into MDA-231-SEVs suppresses E-cadherin expression and increases Ki67 expression in MCF-7 tumors in vivo.
Fig. 8: Model of regulation of breast cancer cell progression by non-classical Notch signaling via small extracellular vesicles.

Data availability

Data are available upon request to the authors by email.


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We thank Dr. Kenneth McCreath for manuscript editing and helpful comments. The data presented herein were obtained using core facilities at IIS La Fe (Cell Culture Unit, Animal Housing Facility and Microscopy Unit) and the Cytometry Unit at Centro de Investigación Principe Felipe. This work was supported by grants from the Instituto de Salud Carlos III (grants: PI16/00107, PI19/00245, RD16/0011/0004, cofunded by the European Regional Development Fund (FEDER “una manera de hacer Europa”), Conselleria de Sanitat Universal i Salut Pública, fellowships ACIF/2018/259 and ACIF/2017/318 co-financed by the European Union through the Operational Program of FEDER of the Valencian Community 2014–2020.

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Conceptualization: HGK, NAG. Methodology: HGK, STG, MCC, MSN, MG. Investigation: HGK, NAG. Visualization: HGK, STG. Funding acquisition: PSS. Project administration: PS. Supervision: NAG. Writing—original draft: HGK, NAG. Writing—review & editing: NAG, PS, HGK, STG, RSS.

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Correspondence to Pilar Sepúlveda or Nahuel A. García.

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González-King, H., Tejedor, S., Ciria, M. et al. Non-classical Notch signaling by MDA-MB-231 breast cancer cell-derived small extracellular vesicles promotes malignancy in poorly invasive MCF-7 cells. Cancer Gene Ther 29, 1056–1069 (2022).

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