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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.

No two tumors are the same, and even within a single patient, the intratumor heterogeneity that exists contributes to the overall complexity in disease treatment and management. Yet, tumors are constantly shedding fragments into circulation with genetic material that help to fingerprint the tumor of origin. Whether in the form of circulating tumor cells (CTCs), circulating DNA, or extracellular vesicles, these tumor fragments hold critical genetic information with the potential to predict disease onset, if and how a patient will respond to different therapeutic approaches, and whether tumors are developing resistance to treatment, for instance. Formally known as “liquid” or blood biopsies, tests involving these tumor fragments allow for routine monitoring and are less invasive than other traditional approaches such as tissue biopsies. As such, liquid biopsies and the study of tumor-derived genetic material can potentially lead to significant advances in personalized medicine where each patient is treated based on the unique tumor biology of the patient. Circulating tumor cell–based biopsies hold particular appeal among researchers because availability of intact cell enables interrogation of tumor-specific cellular processes and mechanisms. One major area of CTC-based research efforts is to establish techniques for robust molecular and functional characterization of these cells. Another area of emphasis is in developing EpCAM-independent approaches to CTC isolation with heightened sensitivity. Greater understanding of the role of cellular heterogeneity and the varied roles of tumor cells in disease will offer insight into the mechanisms of how cancer develops resistance to therapies, and how subsets of cells are able to spread to distant organs. In addition to these areas of research emphasis, one key topic has been in the development of CTC integrated cultures and expansion of cells outside the native CTC environment. While many challenges are faced to successfully expand CTCs, efforts are ongoing as successful culture of CTCs has potential for discovery of new therapeutics and precision cancer management. Tumor cell dissemination is an inextricable process in the metastasis cascade. The fraction of cells that are suitably adapted to seed new metastases demonstrates unique biomolecular processes that evade anticancer microenvironmental cues. Among these processes is mechanotransduction during which mechanosensitive ion channels can be activated, and upon activation, they help form tumor neovasculature, promote transendothelial migration, and increase cell motility. As such, these ion channels play a role in metastasis and tumor cell dissemination. However, the same ion channels can be involved in antimetastatic pathways, which convolutes them being targeted in antimetastatic cancer therapies. Before these channels can be accurately targeted, the desired levels of activation will need to be determined so that their apoptotic effects can be leveraged, while preventing the stimulation of prometastatic pathways. The implicit role of tumor-derived extracellular vesicles (TEVs) in tumor progression has been supported by a number of studies. It is believed that TEVs essentially modulate the tumor microenvironment and coordinate paths of least resistance during metastasis via cellular crosstalk. Modulation of the tumor microenvironment comes as a result of TEVs promoting cell invasion and other epigenetic modulations, which can ultimately lead to genotypic and phenotypic modification of target cells. Tumor-derived extracellular vesicles can be isolated from various body fluids, which adds to their appeal as a potential diagnostic tool. Strides have been made toward the feasibility of precision medicine, resulting from more powerful computational platforms for data acquisition and analysis, which can be leveraged in molecular profiling of liquid biopsy samples. Because sample material typically occurs with low frequency, analytical tools that enable high analytical sensitivity and specificity are needed for liquid biopsy molecular assays.

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.

Graphene Nanocutting Through Nanopatterned Vacancy Defects Rhonda Jack1 2, Dipanjan Sen1 3, and Markus J. Buehler1 ∗ 1Laboratory for Atomistic and Molecular Mechanics (LAMM), Department of Civil and Environmental Engineering, Massachusetts Institute of Technology, 77 Mass. Ave., Room 1-235A&B, Cambridge, MA, 02139, USA 2Department of Chemical Engineering, Hampton University, Hampton VA, USA 3Department of Materials Science and Engineering, Massachusetts Institute of Technology, 77 Mass. Ave., Cambridge, MA, 02139, USA

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.

The concept of studying tumor-derived circulating tumor cells (CTCs) from a simple blood-draw of a patient, has acquired significant attention in recent years. This relatively non-invasive approach opens many avenues in understanding and monitoring quite a number of cancers where correlations have since been made with CTCs and patient survival as well as patient response to therapeutics. Additionally, studies geared towards identifying genetic markers as well as gene profiling of CTCs are ongoing in efforts to develop early screening markers as well as gain a more holistic understanding of the various forms of the disease. Moreover, the concept of personalized medicine in treating several types of cancer is deemed indispensable due to the highly heterogeneous nature of the disease. Extensive studies involving genetic material of CTCs are expected to considerably develop effective therapeutics that are tailored to meet each patient's need. However, several challenges need to be overcome in order to harness the wealth of information that can be gained from CTC studies, such as the rarity of such cells among other blood cells, where CTCs occur as rarely as 1 to 1 million among other mononuclear blood cells. In this vein we discuss the development of an integrated, continuous microfluidic device designed to isolate and label CTCs disseminated from cancer tumors. The 3-part device initially exploits the size disparity of the majority of CTCs with other blood cells and couples this with the use of size-based inertial forces to presort CTCs from whole blood at 1.2mL/min. The CTC-rich fluid stream is then mixed passively, on-chip, with EpCAM coated micro-beads which allows the cells and beads to mix at the micron length scale. This is followed by a brief period of incubation which allows cells to be sufficiently labeled, where the majority of cells have experienced at least two-thirds magnetic bead coverage using this mixing-incubation approach. Thereafter on-chip magnetic sorting of CTCs from any remaining blood cells is achieved by application of an external magnet along the magnetic sorter of the integrated device. Cells with as little as one-third bead coverage experience magnetic deflection to the CTC collection stream. To identify CTCs, cells were stained positively for DAPI and CK-19, and CD45 was used to distinguish leukocytes. We demonstrate that the isolated CTCs are not only viable but that the range of WBC decontamination is the lowest yet reported in literature. Based on PANC-1 cell-line experiments with the device, an 80% recovery rate of cancer cells from other blood cells was consistently achieved. Testing of metastatic patient samples with the device has yielded high counts of CTCs, coupled with high purity rates. We believe that the use of this system in characterizing CTCs and studying cancer will contribute to understanding this aggressive disease in a non-invasive, efficient manner. Citation Format: Rhonda M. Jack, Meggie MG Grafton, Robert A. Cieslak, Denise C. Jue, Cathy Griffith, Danika Rodrigues, Sunitha Nagrath, Diane Simeone. Continuous isolation, labeling and collection of viable CTCs using an integrated microfluidic device. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3477. doi:10.1158/1538-7445.AM2014-3477

Pancreatic cancer has one of the highest mortality rates of all cancers. The mortality is associated with both resistance to therapeutics and late stage detection. Often by the time symptoms are presented the disease has advanced to an incurable state. Due to the aggressive nature of pancreatic cancer and the difficulty in obtaining biopsies, it has been challenging to examine the behavior and characteristics of pancreatic tumors over time. Recently, circulating tumor cells (CTCs) have been isolated from peripheral blood of cancer patients and correlated with survival and therapeutic response. CTCs can also be analyzed to provide insight into tumor genetics and metastatic capabilities. Since CTCs are present in early cancers, they have the potential to be used as an early screening marker of metastatic disease. Especially for pancreatic cancer, isolating CTCs would perhaps allow for detection prior to advanced disease states and aid in determining appropriate therapeutic treatments. We demonstrate an integrated microfluidic device to detect CTCs in pancreatic cancer patients. The three-part device which integrates an inertial pre-sorter, a passive mixer and a magnetic sorter can process 1ml of blood in less than 20min to achieve nearly 100% pure cancer cells at the outlet. High-throughput pre-sorting of CTCs from the diluted blood is achieved using the inertial sorter and further specificity and isolation of the CTCs is achieved through magnetic labeling and re-sorting. First diluted whole blood is run through the inertial spiral sorter at nearly 2ml/min. The curvature of the spiral focuses and aligns cancer cells to the inner channel wall, thus making it possible to enrich the cancer cells. The outlet of the spiral is designed to collect the enriched CTC stream, which are then passed through a passive mixer with EpCAM-labeled superparamagnetic beads. Only the cancer cells will be labeled with the magnetic beads as leukocytes and erythrocytes do not express the epithelial markers that are targeted during the mixing process. The mixed and labeled sample is sorted by a continuous flow magnetic sorter, where the application of an external magnetic field deflects the magnetically labeled cancer cells to a single collection outlet. Once isolated, the highly pure CTC population can be counted, genetically profiled, immunostained, or cultured for further analysis. Cells were stained using CK7/8, N-cadherin, CD45, and DAPI to assess epithelial and mesenchymal characteristics. Isolating pancreatic CTCs with both high sensitivity and high specificity can enable earlier cancer detection and downstream molecular analysis can identify key targets for pancreatic cancer treatment. Thereupon, the development of effective CTC isolation tools will improve patient prognosis and therapeutic treatment plans. Citation Format: Meggie MG Grafton, Rhonda M. Jack, Robert Cieslak, Danika Rodriguez, Somya Sharma, Diane M. Simeone, Sunitha Nagrath. An integrated microfluidic device for circulating tumor cell detection in pancreatic cancer patients. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1457. doi:10.1158/1538-7445.AM2013-1457