Abstract LB-064: Proteomic analysis of exosomes purified from HPV+ and HPV- oral squamous cell carcinoma cell lines
Authors: Wu Vivian F. , Wang Zhanxiang 
Published: July 2016

Abstract
Background: Exosomes are a subset of extracellular vesicles, considered packets of information derivative of a mother cell carrying a molecular signature that is used for intercellular communication. This type of communication is particularly important in the maintenance of homeostasis in viral infection. It is possible that this molecular signature can be exploited as a predictive biomarker of disease progression, treatment response, metastatic potential. To date, there have been no published studies evaluating exosomes in HPV+ OPSCC. We hypothesize HPV+/- OPSCC have a unique molecular signature mirrored in exosomal protein and that application of these proteins could facilitate disease progression and alter metastatic potential.
Methods: Fadu and UMSCC-47 cell lines were cultured as models of HPV negative and HPV positive HNSCC, respectively. Standard differential ultracentrifugation with a DTT/sucrose wash step were used to extract and purify exosomes from culture medium. Western blot was applied to detect marker gene expression. Particle size distribution and concentration were processed by NanoSight NS300. Electron microscopy was used to show the morphology of exosomes. Samples were then prepared for MS evaluation and proteomic data obtained using an UHPLC system on-line to a Q-Exactive mass spectrometer equipped with a nano-electrospray ion source. Raw data will be analyzed using the MaxQuant platform to obtain peptide /protein identification. Functional annotation and comparisons to other published data will be accomplished using the David bioinformatics resource, Ingenuity IPA software and the ProteinCenter software suite.
Results: Western blot results showed the enrichment of exosome marker Alix, Tsg101 and CD9 in sucrose gradient fractions with the density between 1.10 g/cm3 and 1.19 g/cm3. The sizes of most particles are around 100nm. Exosomes displayed a typical “cup-like” morphology under electron microscopy. These data indicate the success in obtaining the highly purified exosomes from cell culture medium. Also, we show that expression levels of exosome marker, Alix, are higher in Fadu exosomes than that in UMSCC-47 exosomes. β-catenin, a marker of epithelial-mesenchymal transition (EMT), as well as a key factor in canonical WNT pathway, is highly expressed in exosomes from both tested cell lines, but shows lower expression levels in Fadu cell lysate. Proteomic analysis is pending.
Conclusion: Successful extraction of purified exosomes from HPV+ and HPV- HNSCC cell lines was performed and early data suggests that protein expression levels are different between tumor cell types, as well as between exosomes and their host cell lysates. These differences indicate a dynamic intra- and inter-cellular regulation mechanism in cancer progression. This study is the first to query protein content in the exosomes extracted from HPV+ vs. HPV- cell lines. These vesicles can provide key information to help target the tumor cells from which they emerge. An identifiable target would help broaden our now limited arsenal of treatments for head and neck cancer patients.
Citation Format: Vivian F. Wu, Zhanxiang Wang. Proteomic analysis of exosomes purified from HPV+ and HPV- oral squamous cell carcinoma cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr LB-064.