Identification of markers for tumor- and immune-derived extracellular vesicles (EVs) in preclinical models.
Authors: Johnson Dove-Anna , Pleet Michelle , Welsh Joshua Aden , Cook Sean , Savage Jason , Mirza Nasiri Nooshin , Camphausen Kevin A. , Aldape Kenneth D. , Gulley James L. , Mock Beverly A , Berzofsky Jay A. , Jacobson Steve , Jones Jennifer C 
Published: June 2022

3046 Background: Extracellular Vesicles (EV) are of broad interest as carriers of molecular signatures of tumor progression and cancer treatment response. EVs, which contain nucleic acids, lipids, and proteins, are released from cells for waste excretion and communication. Numerous proteins and markers are expressed within and on the surface of EVs, but classification markers for murine EV subsets are lacking. To identify tumor and dendritic cell- derived EV markers for preclinical models of breast cancer, we investigated surface marker repertoires of EVs produced by the murine breast cancer and dendritic cell lines, 4T1 and DC2.4. Methods: Cells were cultured in serum free media for 2 days. EVs were harvested and isolated by ultrafiltration followed by size exclusion chromatography. EV particle size and concentration were estimated by nanoparticle tracking analysis and microBCA. To identify highly expressed EV markers, a mouse EV multiplex flow cytometry assay was performed using detection antibodies, CD9, CD63, and CD81, with sets of >35 barcoded capture beads, representing more than 100 specific capture: detection combinations. EV marker expression was analyzed using the FCMPASS/MPAPASS software (nano.ccr.cancer.gov). > 250 beads were assessed for each capture- and detection- antibody combination for each EV type and dilution tested; mean fluorescent intensity was determined; and pairwise comparisons between test and control sample sets were evaluated by t-tests. Results: Breast cancer (4T1)-derived EVs but not dendritic cell (DC2.4)-derived EVs were strongly detected with CD326 (EpCAM) and CD49b (integrin alpha5, VLA-2) capture beads, using each of the three tetraspanin antibodies. Both types of EVs were detected with anti-CD9 and anti-CD81 when captured by anti-CD44 and anti-CD49e (integrin beta1, VLA-5) beads. DC2.4 EVs were distinctively identified by CD11b capture. CD63 capture and detection antibodies robustly recognized EVs from 4T1 but provided minimal recognition of DC2.4 EVs. Mouse serum EVs from non-tumor bearing mice, showed minimal or no detectable CD326 or CD11b. Conclusions: Multiparametric MPAPASS-processed EV repertoire analysis of EVs from murine breast cancer and dendritic cell lines identified CD9, CD81, CD44, and CD49e as common epitopes among both types of evaluated EVs. CD326, CD49b, and CD63 distinguished 4T1 from DC2.4 EVs, and CD11b distinctively identified the DC2.4 EVs. The absence of detected CD326+ and CD11b+ in the serum of non-tumor bearing mice indicates the potential of these two markers for detection of specific tumor and antigen presenting cell EV subsets in serum from mice bearing CD326+ tumors such as 4T1. These results establish a foundation for further tests of detection and tracking of tumor-specific CD326+ EVs as "liquid biopsies" in blood samples as correlates to tumor progression and/or response to treatment.