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Label Free Isolation and Molecular Analysis of Circulating Tumor Cells

  • Author(s): Dhar, Manjima
  • Advisor(s): Di Carlo, Dino
  • et al.
Abstract

Circulating Tumor Cells (CTCs) are important biomarkers for monitoring tumor dynamics and efficacy of cancer therapy. When these cells disseminate from tumors and enter the blood stream, they carry the tumor’s genetic and proteomic information. They are particularly attractive because they can be obtained in a minimally invasive manner. Using microfluidic devices, we can isolate these rare cells from the background of blood cells. Here we study clinically relevant use cases of these CTCs. We characterize two aspects of CTCs: i) the PD-L1 expression levels on CTCs from non-small cell lung cancer patients undergoing immunotherapy ii) the secretion of proteases as it relates to cancer metastasis. In the process of molecular profiling the CTCs, we use a novel label free Vortex isolation system. A modification to the original Vortex device enabled us to capture a wider size range of CTCs at a higher capture efficiency, uncovering further heterogeneity. This simple biophysical method opens doors for a range of downstream analysis.

After CTC isolation, we test the hypothesis that tumor cells secrete proteolytic enzymes to facilitate tissue invasion and spread. Given recent work suggesting CTCs can be produced by early stage tumors, MMP production by CTCs could provide a more precise biomarker of the level of metastatic activity for the disease beyond a simple CTC count. We developed a unique integrated microfluidic system able to perform three functions:

i) Isolate CTCs rapidly by size. ii) Exchange the fluid around CTCs to both remove contaminants, and introduce

a fluorogenic MMP-substrate. iii) Encapsulate purified CTCs into a small number of microdroplets to interrogate MMPs secreted at the single-CTC level.

Total analysis from blood input to secretion assay takes minutes and preserves viability, making this system compatible with studying live cells while they retain physiologic conditions. We found that isolated CTCs from metastatic prostate cancer patients, identified through surface markers and nucleus size, release active MMPs. Assaying protease activity of circulating tumor cells will ultimately be important in uncovering the biology of metastasis and serve as a key component of future phenotypic liquid biopsies, potentially enabling companion diagnostics for MMP inhibitor therapies currently in clinical trials.

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