The Elucidation of Mutation-Specific Glycoprotein and Exosomal Non-small Cell Lung Cancer Biomarkers

Abstract

Lung cancer is the leading cause of cancer-related death in the world. This high rate of mortality is often a result of late-stage diagnosis, which occurs in the majority of lung cancer cases. The high false positive rate associated with the current screening technique leads to unnecessary and invasive follow-up procedures, thus newer diagnostics must be developed. Here, we evaluated the potential role of exosomes as "biomarker vesicles" in non-small cell lung cancer (NSCLC). We employed quantitative proteomics to evaluate both N-linked glycoprotein expression patterns, and exosomal protein cargo abundance differences between an immortalized bronchial epithelial cell line and two NSCLC cell lines each harboring distinct, non-overlapping mutations in cell signaling molecules: Kirsten rat sarcoma viral oncogene homolog (KRAS) and epidermal growth factor receptor (EGFR). The inherent benefits of this strategy included: (i) the identification of several glycoproteins that were overexpressed in both NSCLC cells, as well as glycoproteins uniquely overexpressed in each profiled NSCLC cell line, and (ii) characterization of NSCLC exosomes to determine proteins enriched in NSCLC exosomes. These results allowed us to generate a preliminary panel of overexpressed glycoproteins that could be further validated as enriched NSCLC exosomal protein cargo, and identify several potential biomarkers of NSCLC to be examined in future clinical studies. We also developed a novel strategy to unbiasedly characterize the exosome proteome by pairing quantitative proteomics and multivariate cluster analysis. Using the breast cancer cell line SKBR3B as a proof-of-concept, we were able to calculate the probability of an identified protein being exosomal in origin. With this method, we were able to determine proteins enriched in the exosome fraction in an unbiased manner, and identify potential residual contaminant proteins that were identified in the final analyte. In summary, our study provides an in-depth characterization of NSCLC exosomes and the identification of several exosomal proteins that could serve as candidate markers of NSCLC In addition, we described a methodology to classify a protein as a "true" constituent of the exosome proteome. Taken together, these studies provide a foundation for the development of a platform that utilizes exosomes in a diagnostic setting.