High quality CTC isolation using microbubbles for downstream molecular analysis
Principal Investigator: Yu Tsueng Liu
Abstract: ABSTRACT Early cancer detection and intervention are crucial for long-term survival of either primary or recurrent tumors. Circulating tumor cells (CTCs) could be isolated for the diagnosis, prognosis, and treatment planning of cancer. Genetic profiling and expression analysis of CTC could provide additional invaluable information for accurate diagnosis and prognosis. Moreover, in vitro culture of CTC can be applied for functional analysis of cancer cells, such as drug sensitivity test. To achieve this goal, a transformative technology must be able to unmistakably isolate a few cancer cells using cost-effective and non-invasive procedures. Under the previous R21 IMAT award, we developed a novel technology for rare tumor cell isolation. Lipid shell-perfluorocarbon buoyant microbubbles (MBs) coated with anti-EpCAM antibody immediately attached to tumor cells in unfractionated blood, and isolated the cells after a quick centrifugation step (Shi et al., PLoS One, 2013). A short video clip to demonstrate this technology is included in this application. In conclusion, MB technique offers unique advantages over the existing immuno-enrichment technologies: (a) Short processing time can avoid RNA and protein degradation;(b) Scalability (isolation of cells from large volume sample) can help obtain large numbers of CTCs;(c) Specificity (minimal carryover of leukocytes) can improve sensitivity and specificity of molecular analysis;(d) Flexibility (cells are collected in a very concentrated volume of 5-10 [unreadable]l and could be used for cell growth, immunostaining and/or molecular analysis. The R33 phase of this proposal is focused to further develop MB isolation method to achieve high purity isolation of CTCs from metastatic brain cancers for enumeration and molecular and functional analyses. We set the following specific aims of the study: (1) Optimize MB method to achieve high speed, high purity harvesting of tumor cells spiked in 7-20ml blood sample;(2) Applications of MB isolated CTCs for PCR analysis and culturing;(3) Validate the MB method for sensitivity and specificity of CTC enumeration from blood of metastatic brain tumor patients using CellSearch system as a benchmark. Simple but robust CTC isolation technologies that result in high quality CTC sample for downstream analysis will have transformative impact on clinical management of malignancies and early diagnostics of asymptomatic cancers.
Funding Period: 2013-09-18 - 2016-08-31
more information: NIH RePORT
- Binding and isolation of tumor cells in biological media with perfluorocarbon microbubblesGuixin Shi
Solid Tumor Therapeutics Program, Moores UCSD Cancer Center, UC San Diego, La Jolla, CA 92093, USA
Methods 64:102-7. 2013..In conclusion, MB-assisted cell isolation is a promising method for rapid enrichment of cells and biomarkers from biological samples...
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