Specificity of TLS11a Aptamer Towards Hepatocellular Carcinoma as a Means of Detection and Targeted Drug Delivery
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Liver cancer is the sixth most commonly occurring cancer and second leading cause of cancer-related deaths worldwide with an estimated 75% of all liver malignancy cases being hepatocellular carcinoma (HCC), the most common and most lethal form of liver cancer . While the incidence rates for many cancers have slowly declined over the years, HCC incidence and death rates have been continuously rising due to late stage diagnoses and poor prognoses. This suggests that there is a lack of existing biomarkers and diagnostic equipment sensitive enough to identify this disease in early stages . More sophisticated methods are needed to detect HCC in early stages and specifically target this disease. Recently, an HCC-specific aptamer was created and shown to effectively discriminate mouse HCC from normal liver in vitro . In this thesis, the specificity of TLS11a towards HCC is addressed regarding human tissue. Specifically, immunohistochemical (IHC) staining of human tissue microarrays (TMA) was utilized to address what tissue types TLS11a can bind, if it can differentiate between normal liver and HCC tissue and if binding is correlated with tumor stage. Formalin fixed paraffin embedded (FFPE) TMAs were stained with fluorophore labeled TLS11a and imaged using epifluorescence microscopy. However, negative controls demonstrated high levels of autofluorescence (AF) which made aptamer-based fluorescence indistinguishable from background. Attempts to induce aptamer refolding, antigen retrieval and using alternate fluorophores were unsuccessful at increasing the signal to noise ratio. Colorimetric FFPE staining was also pursued; unfortunately, there was no indication of aptamer binding above background using these conditions either. Ultimately, this lack of specific binding between TLS11a and HCC tissue was attributed to disruption or loss of cell surface binding sites and epitopes during the formalin fixation and paraffin embedding process. Using immunofluorescence staining of fresh frozen (FF) TMAs, TLS11a was found to effectively distinguish between normal liver and HCC tissue (p<0.001) in human tissue with the mean integrated intensity increasing more than 10-fold on average in HCC tissues. TLS11a was also found to bind to other malignant tissue types including breast, lung, kidney and colon, among others, with little to no binding to normal tissues (p<0.0001). These results signify that TLS11a has the potential to be used for targeting multiple malignancies. Additional studies are needed to confirm these findings; however, these studies suggest TLS11a has the potential to be a tumor-specific marker, particularly with respect to HCC, and would be an excellent candidate for use in human studies for new HCC-targeted diagnostic and therapeutic applications.