Abstract LB-038: Development and characterization of cancer cell line exosomes as reference standards in cancer research

Exosomes are subcellular particles 50-200 nm in size released from cells through a fusion of multicellular bodies with the plasma membrane. Exosomes are currently being evaluated as potential diagnostic tools in a number of diseases, including cancer. Exosomes are stable carriers of cell-free cargo in the form of DNA, RNA, and protein, thereby making them an attractive candidate for early detection of cancer via liquid biopsy. Tumor-derived exosomes have been linked to stimulation of tumor cell growth, angiogenesis, metastasis, and suppression of the immune system. However, isolating a consistent population of exosomes can be challenging and there is an unmet need for highly characterized exosomes for use as reference standards for research and diagnostic applications. We have developed a novel method employing tangential flow filtration for the isolation of large quantities of highly purified, ready to use exosomes from cell culture supernatant. Exosomes from a variety of cancer cell lines representing the most prevalent cancer types, including prostate (LNCaP), colorectal (HCT116), breast (MDA-MB-231, MCF-7), lung (A549), cervical (HeLa), and glioblastoma (U87-MG) were isolated and characterized in this study. We employed a stringent quality control approach in order to define and characterize these exosomes. Our method generated a high yield (>1×1010 exosomes/mL) and average protein equivalent of 2 mg/mL. Our data demonstrated expression of a number of different characteristic exosomal proteins including tetraspanin, which was confirmed through western blot and PCR. Isolated exosomes had a median size of 102 nm through nanoparticle tracking analysis. Western blot data revealed cell line specific protein marker expression of CD63, CD81, flotillin 1, Alix and TSG101 protein. We assessed the functionality of these purified exosomes through cellular uptake, migration assay, and anchorage independent growth assay in this study. We also evaluated exosomal RNA from these purified exosomes for the presence of common mutations associated with cancer through PCR. Here we report that exosomes isolated by our method not only had high exosome yield and quality, but also retained relevant functionality and thereby make ideal reference standards/controls in the field of cancer research and diagnostics development. Citation Format: Siddhartha Paul, Nanda Mahashetty, Joy Wells, Dezhong Yin, Fang Tian. Development and characterization of cancer cell line exosomes as reference standards in cancer research [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr LB-038.