MICROFLUIDIC CLUSTER ASSAY FOR PATIENTDERIVED CIRCULATING TUMOR CELLS WITH RELEVANCE IN ANTI-CANCER TREATMENT
Bee Luan Khoo1, Gianluca Grenci2, Tengyang Jing3, Ying Bena Lim3, Soo Chin Lee4, Jean Paul Thiery5, Jongyoon Han6, Chwee Teck Lim5
1Singapore MIT Alliance for Research and Technology, Singapore; 2Mechanobiology Institute; 3SMART - Singapore MIT Alliance for Research and Technology.; 4Cancer Science Institute of Singapore; 5National University of Singapore; 6Massachusetts Institute of Technology
We developed a novel microfluidic assay for primary circulating tumor cell (CTC) clusters, incorporating conditions to mimic the in vivo tumor microenvironment for label-free and high sensitivity expansion of CTCs from clinically relevant blood volumes. CTC clusters were successfully generated from >50% of clinical blood samples (n=73) in a short period of two weeks, displaying an efficiency superior to other reported assays (<20%).
CTCs are rare cells originating from either primary or secondary tumors. The ability to enrich CTCs from blood (liquid biopsy) is attractive as it is not invasive, and can be rapidly done on a regular basis. Current CTC enrichment techniques are limited in sensitivity, and present an underestimate of total CTCs (100s to 1000s per ml blood). Current attempts for in vitro expansion of CTCs still require pre-enrichment procedures as well growth factors, which limits clinical utility as they are selective for only some CTC subpopulations.
We report a unique methodology relying solely on the combinatorial use of hypoxia and specialized microwells to concentrate patient-derived blood cells and CTCs for mimicking the in vivo tumor environment. Flat-based cylindrical microwells did not achieve cluster formation. Blood cells provided ‘goodies’ (e.g. growth factors), in place of growth supplements. Incorporation of microfluidic parameters allowed screening for a wider drug range. Clusters might comprise single CTCs or microemboli.
This one-step protocol does not require pre-enrichment, thus enhancing cell recovery and maintenance of viability. Our clinical studies demonstrated the potential correlation of CTC cluster formation with patient survival. This robust liquid biopsy technique can be incorporated into a non-invasive and inexpensive clinical assessment for drug screening. Studies are ongoing to compare the IC50 values of CTCs from various time points throughout the therapy regime, which may reflect the onset of drug resistance and prompt changes in treatment strategy.