The Predictive Value of Circulating Exosomal PD-L1 in Cervical Cancer Immunotherapy

Abstract

Programmed death ligand 1 (PD-L1) expression was wildly used as a predictor of immune Check-Point Inhibitors (ICIs) efficiency. However, emerging results showed that PD-L1 was of great heterogeneity in sampling time and site. Recently, some studies found that exosomal PD-L1(ExoPD-L1) was related to ICIs response. In this study, we aimed to explore the predictive value of ExoPD-L1 in ICIs treatment of cervical cancer (CC) for the first time. A total of 40 primarily diagnosed CC patients who accepted radical radiotherapy (RT) from March 2021 to October 2022 were included. The consecutive tumor sample were collected before and during RT. Another 37 advanced CC patients who accepted ICIs combination therapy from June 2020 to October 2022 were enrolled in this study. Blood samples were collected from each participant before and during treatment. Exosomes were derived by differential centrifugation, which was further identified by Western blot (WB) (CD9/TSG101/Calnexin), transmission electron microscope analysis and nanoparticle tracking analysis. ExoPD-L1 detection was conducted by enzyme-linked immuno-sorbent assay (ELISA). The knockout of PD-L1 was conducted via CRISPR/Cas9 assay and the overexpress of PD-L1 was conducted by lentiviral transfection. CD8+ T cells were extracted from murine spleen by CD8+ T Cell Isolation Kit. Immune cells and cytokines markers were detected by multicolor flow cytometry. The consecutive detection of PD-L1 showed a dynamic change during RT. Compared with the level before RT, PD-L1 expression elevated in most patients (87.5%, 35/40) after RT. And the responders (n = 18) had elevated ExoPD-L1 level at the first two circles in the ICIs combination therapy (P<0.001). Whereas the level of pre-treatment ExoPD-L1 couldn't stratified clinical responders and non-responders (P = 0.181). The median follow-up time was 14.13 months. The mPFS in increased group vs. decreased group: not reach vs.11.02 months (P = 0.025, HR: 0.218, 0.052-0.913). Continuous blood sampling of mice models also found that effective therapeutic intervention could increase ExoPD-L1 in the early stage. The combination of exosome inhibitor GW4869 and anti-PD-1 further inhibited tumor growth. Mice were injected with external ExoPD-L1OE and ExoPD-L1KO. The results showed that ExoPD-L1OE suppressed body immunity and promoted tumor growth. The results of flow cytometry showed that ExoPD-L1OE inhibited CD8+ T cells from releasing interferon-and granzyme B. And ExoPD-L1OE also suppressed the CD8+ T cells proliferation in murine spleen. The coculture of CD8+ T cells and exosomes in vitro also confirmed the above conclusion. Compared with unstable and impressionable tumoral PD-L1, ExoPD-L1 seems to be better predictor for the efficacy of immunotherapy in CC, which was with easy accessibility and continuation. Exosome PD-L1 played an immunosuppressive role by inhibiting the proliferation and functional factor release of CD8+ T cell.