Microfluidic Apheresis to Isolate Circulating Tumor Clusters

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ABSTRACT Circulating tumor cells clusters (CTC-clusters), also called tumor emboli in 1970s, are known to play a major role in the metastatic process. For example, CTC-clusters have been inferred to be 50-fold as metastatic as one CTC. They also differ in many ways from single CTCs, including having distinct epigenetic markers. Due in part to their intact cell-cell junctions, the evidence also suggests they avoid anoikis, hence unlike single CTCs they are less apoptotic. However, CTC-clusters are even rarer than single CTCs, with in many cases an average of just one cluster-associated tumor cell in 10 mL of blood, meaning most samples of 10-20 mL do not contain a CTC-cluster. Isolating CTC-clusters from larger blood volumes is thus required to unlock the unique reservoir of biomarkers linked to this most metastatic population of tumor cells. Although there has been a number of approaches to isolate CTC-clusters, mostly using microfluidics, they primarily use a small amount of blood up to 10-20 mL and hence are not able to sort CTC-clusters reliably to impact clinical-decision making. To overcome the shortcomings of current approaches, we propose to develop core technology enabling a microfluidic CTC-cluster apheresis (CTApheresis) system that can interrogate 20-100% (i.e., 1-5 L) of the entire blood volume within an hour to isolate enough CTC-clusters in a majority of patients. We will reach our goal of CTApheresis by building an innovative microfluidic technology to gently and selectively sort CTC-clusters from undiluted blood in a high-throughput system. Here, we will create the “functional modular unit” for scalable CTApheresis. We will split the project in two distinct, integrated, aims. In aim 1, we will create a microfluidic chip to concentrate CTC-clusters within large volumes of whole blood. In aim 2, we will integrate this sorter into a functional modular unit sorter to process 120 mL of whole blood in 1 hour (scalable by nine-fold by parallelization to 1 L/h). Throughout and as a comprehensive safety study after aim 2 completes, we will run a battery of tests comparing processed and unprocessed blood, to examine if CTApheresis blood processed using the proposed microfluidic device will be safe to return back to the patient. Gentle sorting of CTC-clusters from liters of whole blood has the potential to unlock some of the mysteries of the metastatic process but also to enable ex-vivo culture of CTCs for “real time” functional drug testing for cancer patients. Routine sampling of the CTC-cluster genome, transcriptome and proteome could become a replacement for invasive surgical biopsies, and ultimately provide a powerful approach for early detection of cancer in high-risk patients.