Assessment of CD73 activity in breast cancer-derived small extracellular vesicles: application to monitoring of patients' responses to immunotherapy.

BACKGROUND

We previously discovered that small extracellular vesicles (sEV) isolated from melanoma cells produce immunosuppressive adenosine (ADO) via the ATP→ADP→AMP→ADO pathway and that CD73 is the 'gateway' ecto-nucleotidase used by melanoma sEV to generate ADO. Here we extend these findings to CD39(+)CD73(+) and CD39(+)CD73(-) sEV from breast cancer cells.

MATERIALS AND METHODS

sEV were isolated from supernatants of a triple-negative breast cancer cell line ± the genetic knockout of CD73. A newly developed high pressure liquid chromatography assay with fluorescence detection was used for assessment of N-etheno-AMP conversion to N-etheno-ADO by sEV. PSB12379 (selective CD73 inhibitor) and anti-CD73 antibodies were used to inhibit/neutralize CD73 activity in sEV.

RESULTS

Untreated sEV isolated from CD39(+)CD73(+) breast cancer cells readily metabolized N-etheno-AMP to N-etheno-ADO, and this activity was abolished by PSB12379. sEV from CD39(+)CD73(-) breast cancer cells were unable to metabolize N-etheno-AMP to N-etheno-ADO. Effects of three different anti-CD73 antibodies on CD73 activity in sEV were examined. Only one antibody, the direct binding pocket inhibitor of CD73, but not antibodies that allosterically inhibit recombinant CD73, attenuated conversion of N-etheno-AMP to N-etheno-ADO by cancer-derived sEV.

CONCLUSIONS

In breast cancer-derived sEV, as in melanoma-derived sEV, CD73 is the gateway enzyme regulating ADO formation from upstream AMP. The quantitation in sEV of N-etheno-AMP conversion to N-etheno-ADO ± neutralizing anti-CD73 antibodies provides a measure of the ability of these antibodies to suppress ADO production and could potentially serve as a personalized predictor of CD73 activity in patients with cancer.