Exosomal HPV oncogene and PD-L1 in cervical cancer: potential biomarkers for monitoring disease progression and treatment response

Objectives: Exosomes are nanosized extracellular vehicles released from cells as messengers for cell-cell communication. Growing evidence shows tumor-derived exosomes (TEXs) carry malignant materials from tumors contributing to metastasis and influencing tumor growth regulation. TEXs can be obtained from biofluids, thus, characterizing TEXs in serum and understanding the information they carry opens up the possibility of using TEXs as a valuable diagnostic and prognostic tool. While this potential has been demonstrated in other solid tumor cancers such as breast, lung, and liver, the role of exosomes in gynecologic malignancies remains largely unexplored. Breakthroughs in cervical cancer have revealed the importance of HPV in oncologic transformation of cervical cells and the roll of PD-1/PD-L1 immune checkpoint in evading regulatory pathways to allow for tumor cell growth. Exosomal PD-L1 has also been shown to contribute to tumor growth and correlate with disease progression in melanoma and head and neck cancers. This study aims to determine whether HPV and PD-L1 can be detected in cervical TEXs to further investigate what diagnostic and prognostic implications their presence may have. Methods: The cervical cancer cell lines C-33-A, Ca-Ski, and SiHa were used to demonstrate successful cervical cancer-derived exosome extraction and detect exosomal molecular and genetic material. For each cell line, PCR was used to confirm HPV presence in the cells and exosomes, and Western Blot was used to determine the presence of PD-L1. To collect exosomes, the cells were cultured in growth medium with exosome-depleted FBS for 72 hours. Then, differential ultracentrifugation was used to isolate the exosomes. The exosomes were collected and tested for presence of HPV transcript and PD-L1 protein using PCR and Western Blot, respectively. The presence of CD9, a universal surface marker of exosomes, was also tested on the exosomes as an internal control. Results: PCR confirmed that C-33-A is HPV negative while Ca-Ski and SiHa are HPV positive. Western Blot revealed that only Ca-Ski expresses PD-L1, while both Si-Ha and C-33-A are negative for PD-L1. Notably, the expression of HPV and PD-L1 in exosomes was consistent with the host cells. Ca-Ski-derived exosome expressed both HPV and PD-L1. SiHa-derived exosomes expressed HPV but not PD-L1, and C-33-A was negative for both HPV and PD-L1. CD9 expression was seen in all exosome samples from all 3 cell lines. Conclusions: As postulated, the presence of HPV and PD-L1 in cervical TEXs was correlated with their presence in the host cell, suggesting that the genetic and molecular information carried in the exosomes can be used to provide valuable information regarding primary and metastatic tumors. This has promising implications for the use of TEXs in diagnostics and monitoring treatment response in patients with cervical cancer. Future steps in realizing the potential of this discovery in cervical cancer diagnostics and management of the disease include demonstrating that HPV and PD-L1 are also present in exosomes isolated from serum of patients with cervical cancer.