Microfluidic continuum sorting of sub-populations of tumor cells via surface antibody expression levels.

The extent of inter- and intra-tumor cell heterogeneity observed in patient tumors appears to be directly associated with patient prognosis. Moreover, studies indicate that targeting distinct subpopulations of tumor cells may be more relevant to successfully managing cancer metastasis. The ability to distinguish and characterize unique tumor cell subpopulations within a given sample is thus exigent. Existing platforms separate cells binarily, based on some threshold level of phenotypic characteristics without consideration of the continuum levels of biomarker expression and the associated implications. Herein we describe how specific tumor cell groups have been immunomagnetically enriched according to a continuum of EpCAM surface marker expression levels. Even among a relatively homogenous group of cells such as the PANC-1 cell line, cells could be separated according to their EpCAM levels into low, moderate and high expression. To physiologically assess each subpopulation, a wound healing assay was performed which revealed distinct invasive potentials among each subset. Furthermore, the clinical relevance of the approach was demonstrated by isolating pancreatic cancer CTCs from the same patient sample based on their EpCAM levels. We demonstrate a robust method of isolating CTCs according to their varying protein levels, which enables extensive studies on tumor cell heterogeneity. Interestingly, 5 of 6 samples had CTCs that could be recovered at all three levels of EpCAM expression though the majority of CTCs were recovered as low expression events. Preliminary studies that compare tumor cell subpopulations in this continuum manner can potentially increase our understanding of the dynamic nature of cell heterogeneity and how it relates to patient outcomes. Ultimately further investigation may yield therapeutic targets against virulent cell subpopulations.

Ultra-Specific Isolation of Circulating Tumor Cells Enables Rare-Cell RNA Profiling.

The clinical potential of circulating tumor cells (CTCs) in managing cancer metastasis is significant. However, low CTC isolation purities from patient blood have hindered sensitive molecular assays of these rare cells. Described herein is the ultra-pure isolation of CTCs from patient blood samples and how this platform has enabled highly specific molecular (mRNA and miRNA) profiling of patient CTCs.

Opportunities and Challenges for Pancreatic Circulating Tumor Cells.

Sensitive and reproducible platforms have been developed for detection, isolation, and enrichment of circulating tumor cells (CTCs)-rare cells that enter the blood from solid tumors, including those of the breast, prostate gland, lung, pancreas, and colon. These might be used as biomarkers in diagnosis or determination of prognosis. CTCs are no longer simply detected and quantified; they are now used in ex vivo studies of anticancer agents and early detection. We review what we have recently learned about CTCs from pancreatic tumors, describing advances in their isolation and analysis and challenges to their clinical utility. We summarize technologies used to isolate CTCs from blood samples of patients with pancreatic cancer, including immunoaffinity and label-free physical attribute-based capture. We explain methods of CTC analysis and how findings from these studies might be used to detect cancer at earlier stages, monitor disease progression, and determine prognosis. We review studies that have expanded CTCs for testing of anticancer agents and how these approaches might be used to personalize treatment. Advances in the detection, isolation, and analysis of CTCs have increased our understanding of the dissemination and progression of pancreatic cancer. However, standardization of methodologies and prospective studies are needed for this emerging technology to have a significant effect on clinical care.