Abstract 4117: Utilizing RNA aptamers for biomarker discovery in a novel cell culture system for hepatocellular carcinoma

Abstract

Abstract Introduction: Current serum biomarkers for hepatocellular carcinoma (HCC) lack the sensitivity and specificity to be effective screening tools. Aptamers are single-stranded RNA oligonucleotides that specifically bind to target proteins with high affinity and are generated by repeatedly screening complex RNA libraries for binding to protein targets. We have utilized this selection process to identify biomarkers from complex targets, such as secreted proteomes (secretomes). Our hypothesis is that aptamers that specifically bind the HCC secretome will identify serum protein biomarkers for HCC. Methods: We mirrored the microenvironments of HCC and normal liver tissue utilizing two co-culture systems: 1) human HCC cells (Huh-7) with normal hepatic stellate cells (HSCs) to model HCC livers and 2) normal human hepatocyte cells (THLE-2) with HSCs to model normal livers. Co-cultures were grown to confluence then switched to serum free media for collection of secreted cancer proteins (HCC secretome, HS) and normal secretome (NS). A nuclease-resistant RNA pool was then alternately incubated with NS (discarded for negative selection) and HS (recovered and amplified for positive selection). Results: Binding affinity for HS increased over 7 successive rounds, while binding affinity for NS decreased. The final RNA pool was cloned and sequenced yielding 3 candidate aptamers that were tested for binding to the secretomes and to human serum from patients with HCC and normal controls. In the samples tested we found that one candidate aptamer sequence (G-20) demonstrated increased binding to the sera of HCC pts (N=5) relative to normal sera (N=2) and to a non-binding control aptamer. The maximal percent fraction bound of the G-20 aptamer to cancer serum was 15% compared to only 7% in control. The maximal percent fraction bound of G-20 in normal serum was 8% compared to 6% in control. Conclusions: This co-culture system may better recapitulate the in vivo environment. Aptamers that specifically bind our cancer secretome over the normal secretome may bind to proteins that are specifically secreted by HCC tumors in vivo. The G-20 aptamer selectively binds to HCC patient sera. Future goals of this work are to utilize affinity purification techniques to identify the target of our G-20 aptamer and to continue testing our G-20 aptamer in patient sera to further validate its ability to differentiate between cancer and normal disease states. Citation Format: Ibtehaj A. Naqvi, Rebekah R. White, Cynthia A. Moylan, Anna Mae Diehl, Steve S. Choi. Utilizing RNA aptamers for biomarker discovery in a novel cell culture system for hepatocellular carcinoma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4117. doi:10.1158/1538-7445.AM2014-4117