Background: Circulating tumor cells (CTCs) could provide significant insights into cancer metastatic events and potentially even inform clinical decisions. However, the scarcity of CTCs, compounded by the abundance of circulating white blood cells, poses a major technical challenge in the isolation, yield and downstream molecular analysis. Moreover, next-generation sequencing (NGS) of CTCs has been challenged by low CTC purity and nucleic acid yields commonly derived from existing technologies. Here, we present a label-free microfluidic approach that utilizes curvilinear microchannel geometry for high purity CTC enrichment from 7.5 ml whole blood.
Methods: We showed high purity isolation of CTCs using a label-free inertial focusing microfluidics platform, ClearCell® FX. The lung cancer cell line, NCI-H1975, with known EGFR, CDKN2A and TP53 mutations, were spiked into 7.5ml healthy donor peripheral blood samples at varying concentrations (50, 100, 200 cells). DNA was extracted using the QIAamp Micro kit. The Thunderbolts Cancer Panel droplet digital PCR NGS library preparation method was used to enrich 230 loci in 50 known cancer genes from the isolated CTC gDNA. The libraries were sequenced on a MiSeq system.
Results: The CTCs were sufficiently enriched with an average recovery of 45.8% and purities ranging from 1% - 70%. All libraries were successfully generated and sequenced with ≥1400× mean coverage, even with as little as 2 ng of starting material. Known EGFR, CDKN2A and TP53 mutations in H1975 were successfully detected in all the samples (n = 9), with a significant variant frequency that correlates with CTC purity levels.
Conclusion: We have demonstrated a highly accurate and sensitive workflow utilizing a combination of inertial focusing and digital droplet microfluidics to detect low numbers of rare CTCs and interrogate their molecular profiles from as little as 10 cells/mL frequency. This workflow has the potential for further development and adoption as a liquid biopsy approach for non-invasive genomic profiling of metastatic cancers.