Non-coding RNAs such as microRNAs (miRNAs) have been identified as potential biomarkers for cancer detection because their profiles reflect developmental lineage and differentiation state of many tumor types. Since miRNAs are more stable than other nucleic acids such as mRNA in blood or other body fluids because they could be protected by bounding to endogeneous RNase proteins or contained within cell secreted nanoparticles, i.e. exosomes, using stable miRNAs as viable biomarkers in body fluids provide an excellent means to achieve noninvasive assays for early cancer detection. Although miRNAs have been quantitatively measured in human sera or plasma using quantitative reverse transcription polymerase chain reaction (qRT-PCR) with pre-concentration techniques such as ultra-centrifugation or ExoQuickTM exosome precipitation kit (SBI Inc.), this approach is tedious, expensive and time consuming. For clinic use, it would be highly valuable if a simple, fast and low-cost method can be developed as a routine pre-screening tool such that more in-depth but also more invasive and expensive mammography or magnetic resonance imaging (MRI) detection methods as well as disease treatment can be conducted as follow-up in early stage of cancer. We recently developed a simple tethered Cationic Lipoplex Nanoparticle (tCLN) biochip with pre-loaded molecular beacons (MBs) in the lipoplex nanoparticles as probes to capture and detect targeted miRNAs in human plasma and cell culture medium without any need of pre- or post-sample treatment. The negatively charged miRNAs, whether being bounded to endogeneous RNase proteins or contained within exosomes, can be easily captured by and fused with positively charged liposomes, allowing detection by MBs. Since both targeted miRNAs and MB probes are all confined within nanometer sized liposome particles (detection volume 1012 times smaller than that in PCR), this method provides very high detection sensitivity without the need of amplification as in PCR. Similar MB probes are also able to capture and identify mRNA in the same sample. This tCLN biochip has been successfully demonstrated in comparing exosomes isolated from cell culture media of a lung cancer cell line, A549 and a normal epithelial cell line using miR-21 locked nucleic acid (LNA) MB and TTF-1 mRNA MB as biomarkers. Preliminary results using lung cancer patient blood samples also demonstrate promising results. Lung cancer is the leading cause of cancer deaths worldwide with a disappointing 15% overall 5-year survival rate. A patient-friendly early detection and surveillance method would substantially reduce the mortality in this serious disease. The same technique may also be applied to many other cancers. We have assembled a strong interdisciplinary team to further develop the tCLN biochip method and to evaluate its feasibility in lung cancer patients. Our primary objectives are (1) to optimize the tCLN biochip and to evaluate its performance for capture and detection of 5-6 targeted miRNAs and mRNAs in lung cancer patient plasma, and (2) to conduct pilot scale test using well defined human plasma samples from early lung cancer patients and smokers.

Public Health Relevance

Non-coding RNAs such as microRNAs (miRNAs) have been identified as potential biomarkers for cancer detection because their profiles reflect developmental lineage and differentiation state of many tumor types. Lung cancer is the leading cause of cancer deaths worldwide with a disappointing 15% overall 5-year survival rate. Thus, lung cancer carries a significant socioeconomic burden. Poor survival rates are likely due to late presentation of disease, lack of methods for early detection, and molecular heterogeneity. We propose a simple tethered Cationic Lipoplex Nanoparticle (tCLN) biochip with pre-loaded molecular beacons (MBs) in the lipoplex nanoparticles as probes to capture and detect targeted noncoding RNAs in human plasma samples, which may aid in the early detection and surveillance of lung cancer.

Agency
National Institute of Health (NIH)
Type
Exploratory/Developmental Grants (R21)
Project #
5R21CA179403-02
Application #
8735903
Study Section
Cancer Biomarkers Study Section (CBSS)
Program Officer
Krueger, Karl E
Project Start
Project End
Budget Start
Budget End
Support Year
2
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Ohio State University
Department
Engineering (All Types)
Type
Biomed Engr/Col Engr/Engr Sta
DUNS #
City
Columbus
State
OH
Country
United States
Zip Code
43210