Saliva Diagnostics

Abstract

Cancer remains one of the leading causes of death, and early detection of this disease is crucial for increasing survival rates. Although cancer can be diagnosed following tissue biopsy, the biopsy procedure is invasive; liquid biopsy provides an alternative that is more comfortable for the patient. While blood, urine, and cerebral spinal fluid can all be used as a source of liquid biopsy, saliva is an ideal source of body fluid that is readily available and easily collected in the most noninvasive manner. Characterization of salivary constituents in the disease setting provides critical data for understanding pathophysiology and the evaluation of diagnostic potential. The aim of saliva diagnostics is therefore to develop a rapid and noninvasive detection of oral and systemic diseases that could be used together with compact analysis systems in the clinic to facilitate point-of-care diagnostics.

Keywords: circulating biomarkers; saliva exosomics; saliva liquid biopsy; salivaomics; salivary exosomes.

Figure 1

Structure and contents of typical salivary exosomes. The exosome is surrounded by a phospholipid bilayer, carrying many cell type–specific cargos. Abbreviations: ctDNA, circulating tumor DNA; mRNA, messenger RNA; miRNA, micro RNA; piRNA, piwi-interacting RNA; snoRNA, small nucleolar RNA.

Figure 2

Nanostructure of salivary exosomes observed under atomic force microscopy (AFM) and field emission scanning electron microscopy (FESEM). (a) AFM phase image of salivary exosomes exhibits a trilobular substructure. Surface contrast is presumably attributed to variable constitutive elements on exosomal membrane (e.g., protein and lipid). (b) FESEM reveals round-shaped salivary exosomes with intervesicular connections. (c) Electron microscopy with anti-CD63 antibody-conjugated gold beads identifies dense tetraspanin molecules on the exosome surface. Figure adapted with permission from Reference ; copyright 2010 American Chemical Society.

Figure 3

Exosomes and multivesicular bodies (MVBs) seen in saliva of oral cancer patients. (a) Salivary exosomes from healthy donors appear as homogeneous circular structures. (b) Salivary exosomes from oral cancer patients show irregular morphology with varying sizes and vesicle aggregation (arrow). (c) Elongated intervesicular filaments and exosome-like vesicles in MVBs are observed in cancer saliva. (d) At higher resolution, membrane ruptures are observed in cancer salivary MVBs (arrows). (e) Schematic of MVB endosomal membrane rupture and exosome release from oral cancer cell. Figure adapted with permission from Reference ; copyright 2011 American Chemical Society.

Figure 4

Schematic of the EFIRM assay. (a) Surface preparation: The gold electrodes are precoated with pyrrole and DNA capture probe is immobilized onto the pyrrole-coated electrodes. Electrophoresis and target hybridization: The surface is incubated with the target ctDNA-containing saliva sample and a cyclic square wave electric field is applied at 30 cycles of +200 mV for 1 s and −300 mV for 9 s during hybridization. Detector probe hybridization: A complementary biotinylated single-stranded oligonucleotide detector probe hybridizes with the ctDNA target. Electrochemical detection: HRP-conjugated streptavidin and 3,3′,5,5′-tetramethylbenzidine substrate generate electrical current, which is detected by an electric sensor. (b) Steric effect: The negative potential makes a closed hairpin structure of DNA capture probe stretch and form an open structure required for highly efficient intermolecular hybridization. Abbreviations: ctDNA, circulating tumor DNA; EFIRM, electric field–induced release and measurement; HRP, horseradish peroxidase.

Figure 5

Saliva-based liquid biopsy for non-small cell lung cancer. ctDNA and tumor-derived exosomes enter the circulation and reach the salivary glands. ctDNA and exosomes are uptaken by salivary gland acinar cells via endocytosis or membrane fusion. Central to saliva liquid biopsy techniques is the capture and analysis of ctDNA, which includes EGFR exon 19 deletion and L858R mutation. Combining salivary ctDNA and exosome analyses can provide more comprehensive panels of molecular markers for precision medicine application in a minimally invasive manner. Abbreviations: ctDNA, circulating tumor DNA; EGFR, epidermal growth factor receptor.