Single-cell Gene Mutation Detection Of Rare Cells

There are a variety of rare cells circulating in the blood stream, such as circulating tumor cells(CTCs), circulating endothelial cells(CECs),benign circulating epithelial cells, as well as fetal nucleated red blood cells. These cells are extraordinarily rare but have important clinical value.For example, CTCs indicate the presence of the tumor in vivo and the risk of recurrence. In addition to the primary tumor sites, CTCs represent another source of tumor cells, which is called ‘liquid biopsy'. At present,many efforts have been devoted to count the total number of CTCs in the peripheral blood and conduct mutation analysis of CTCs. However, the heterogeneity of CTCs, especially the relationship between CTCs' genetic heterogeneity and functional heterogeneity have been ignored due to a lack of single-cell techniques. Therefore, we should develop a single-cell technology for genetic profiling of CTCs with single-cell resolution.This thesis relies on micromanipulation platform to retrieve single tumor cells captured by microfluidic devices, followed by single-cell whole genome amplification and detection of genetic mutations.Meanwhile, with the need of co-profiling of intracellular functional protein and genetic mutations at the single-cell level, we have developed protocols to retraive single-cell lysate for detection of genetic mutations.Finally, with the combination of techniques for microfluidics-based CTC capture and single-cell detection of genetic mutations, we measured clinical samples from lung cancer patients. The integrated microfluidic chip system that we developed is able to isolate CTCs from the patients' blood samples with high efficiency, followed by removal of red blood cells and white blood cells through negative selection, and thus leading to a CTC population with high purity for subsequent analysis.