1. Field of the Invention
The present invention relates generally to the fields of genetics, protein biochemistry, and oncology. More particularly, it concerns the use of exosomal genomic DNA and proteins in genetic analysis and treatment.
2. Description of Related Art
Pancreaticoduodenectomy (Whipple procedure) can be curative for PDAC patients if tumors are detected early with clear surgical margins. Due to the late diagnosis of pancreatic cancer, only around 15% of patients present with surgically resectable tumors (Conlon et al., 1996). Studies comparing stage of disease with outcome following surgery suggest that death rates for PDAC would be reduced if the disease were diagnosed at an earlier stage (Bilimoria et al., 2007).
In addition to direct cell-to-cell contact via soluble factors, such as cytokines and chemokines, there is emerging evidence that exosomes play a pivotal role in intercellular communication (Kahlert and Kalluri, 2013). Exosomes are small, membrane-bound vesicles with a size of 40-150 nm (Pan et al., 1985; Trams et al., 1981). They are secreted by many different cell types, such as cancer cells, mesenchymal cells, thrombocytes (Kahlert and Kalluri, 2013; Heijnen et al., 1999; Raposo et al., 1996), immune cells (Thery et al., 2009), platelets (Janowska-Wieczorek et al., 2005), and endothelial cells (Hergenreider et al., 2012). The first step in exosomes biogenesis involves the inward budding from the limiting membrane of late endosomes (Trajkovic et al., 2008). During this process, exosomes are packed with RNA molecules and proteins from the parental cell (Trams et al., 1981; Trajkovic et al., 2008). After the release into the extracellular space, tumor-derived exosomes can transfer proteins and RNAs with oncogenic activity to recipient cells (Kacharzewska et al., 2012; Grange et al., 2011; Peinado et al., 2012). Because exosomes are very stable under different conditions, they can protect their biological cargo against degradation and denaturation in the extracellular environment (Taylor and Gercel-Taylor, 2008). Genomic DNA in circulation is mainly contained in exosomes (Kahlert et al., 2014). Exosomes from astrocytes and glioblastoma cells carry mitochondrial DNA (Guescini et al., 2010). Furthermore, it has been shown that exosomes from glioblastoma cell lines contain small amounts of single-stranded DNA as well as high levels of transposable elements (Balaj et al., 2011).
Exosomes are found in all body fluids of cancer patients, such as serum, saliva, cerebrospinal fluid, bone marrow aspirates, eye exudate/tears, and ascites (Peinado et al., 2012; Lau et al., 2013; Choi et al., 2011). As such, exosomes are promising diagnostic and predictive biomarkers in cancer. However, genetic profiling studies on circulating DNA from cancer patients are confounded by the fact that the isolated DNA represents all cells of the body, thus making mutation and genetic defects challenging (Murtaza et al., 2013; Yong, 2014; Kirk, 2013; Corwley et al., 2013).
Several exosomes markers have been proposed and include members of the tetraspanin family (CD9, CD63, CD81), members of the endosomal sorting complexes required for transport (ESCRT; TSG101, Alix), and heat shock proteins (Hsp60, Hsp70, Hsp90) (Taylor and Gercel-Taylor, 2011). Epithelial tumor cells secrete exosomes carrying the epithelial cell adhesion molecule (EpCAM) (Taylor and Gercel-Taylor, 2008; Silva et al., 2012; Runz et al., 2007). Melanoma-derived exosomes contain the tumor-associated antigen Mart-1 and tyrosinase-related protein-2 (TYRP2) (Peinado et al., 2012; Mears et al., 2004; Andre et al., 2002). Exosomes from gastric cancer, breast cancer, and pancreatic cancer carry members of the human epidermal growth factor receptor (HER) family (Adamczyk et al., 2011; Baran et al., 2010; Ciravolo et al., 2012). However, none of these markers are specific to cancer-derived exosomes and specific isolation of exosomes from the serum of cancer patients remains a challenge due to the lack of specific markers that can be used to identify and distinguish cancer exosomes from exosomes produced by other cells. A marker for cancer-derived exosomes will significantly increase the sensitivity of detection for low frequency mutations in circulation. Thus, a procedure to specifically detect and isolate cancer cell-derived exosomes in circulation is needed.