D. DePeralta1, W. Michaud1, M. Hammond1, G. Boland1 1Massachusetts General Hospital,Surgical Oncology/Surgery,Boston, MA, USA
Introduction: Circulating microvesicles, exosomes, have been described since 1981 [1], but their specific role in cancer biology remains unclear. They are small membrane vesicles (40-100nm) that contain microRNAs (miRNAs), messenger RNAs (mRNAs), DNA, and proteins. Given the variety of nucleic acids contained by exosomes, there is interest in their use as biomarkers of cancer. Studies show that circulating serum exosomes contain RNA signatures representative of the parental tumor [2, 3]. Applications of circulating exosomes to tumor diagnosis have been described for various cancers [4-6]. The work herein is focused on utilizing exosomes as markers of response to therapy, as well as to identify potential mediators active in this process. Our pilot set of serum samples have been collected at both pre-treatment and on-treatment timepoints. Our hypothesis is that there may be exosomal signatures that correspond with treatment response or resistance to current therapies.
Methods: We have collected serial tumor and blood samples from patients with metastatic melanoma enrolled on clinical trials with various therapies since these trials opened. Tissue and blood are collected using protocols approved by the institutional review board (IRB). Exosome isolation has been performed by both serial centrifugation and exoRNeasy serum/plasma kits (Qiagen Inc.). Assesment of exosome size and concentration is done using Nanosight analyses. Western blots have been performed for CD63/flotillin, markers associated with exosomes. We have also isolated exosomes from normal healthy donors to serve as a control.
Results: We can demonstrate a purified exosomal population (Fig. 1A) and an increased exosomal concentration in patients with metastatic melanoma as compared to normal controls (Fig. 1B). In all samples assessed (n=94) exosome concentration correlates with tumor burden, decreasing after successful treatment (surgical or systemic) and increasing at the time of clinical progression (representative patient shown in Fig. 1C). We can now model these findings in an in vitro system, showing that non-cancer cells have a much lower concentration of exosomal proteins in cell culture as compared to those derived from melanoma cell lines (data not shown).
Conclusion: Circulating microvesicles are enriched in melanoma patients as compared to normal healthy controls, a finding replicable in an in vitro system. In patient samples, the concentration correlates with tumor burden, suggesting potential utility as a circulating biomarker of disease. Efforts at profiling the mRNA/miRNA content of these exosomes is ongoing and may shed light on the mechanisms of response or resistance to current therapies.
