Heterogeneity in circulating tumor cells : the relevance of the stem-cell subset

The release of circulating tumor cells (CTCs) into vasculature is an early event in the metastatic process. The analysis of CTCs in patients has recently received widespread attention because of its clinical implications, particularly for precision medicine. Accumulated evidence documents a large heterogeneity in CTCs across patients. Currently, the most accepted view is that tumor cells with an intermediate phenotype between epithelial and mesenchymal have the highest plasticity. Indeed, the existence of a meta-stable or partial epithelial⁻mesenchymal transition (EMT) cell state, with both epithelial and mesenchymal features, can be easily reconciled with the concept of a highly plastic stem-like state. A close connection between EMT and cancer stem cells (CSC) traits, with enhanced metastatic competence and drug resistance, has also been described. Accordingly, a subset of CTCs consisting of CSC, present a stemness profile, are able to survive chemotherapy, and generate metastases after xenotransplantation in immunodeficient mice. In the present review, we discuss the current evidence connecting CTCs, EMT, and stemness. An improved understanding of the CTC/EMT/CSC connections may uncover novel therapeutic targets, irrespective of the tumor type, since most cancers seem to harbor a pool of CSCs, and disclose important mechanisms underlying tumorigenicity

The Selection Strategy for Circulating Tumor Cells (CTCs) Isolation and Enumeration: Technical Features, Methods, and Clinical Applications

The key aim of the proposed chapter is to provide readers a brief description for the most important parts of the field of circulating tumor cells (CTCs): the core techniques, including negative and positive selection‐based CTC isolation, and the differences between them. Most importantly, we will also review the clinical applications and important findings in clinical trials. The evidence‐based review will not only help clinicians use CTCs to predict recurrence and foresee the disease‐related outcomes but also to inspire the researchers in this field to conduct further investigations

Isolation and Analysis of Circulating Tumor Cells in Genitourinary Cancers

While accessible by a relatively noninvasive blood draw, circulating tumor cells (CTCs) remain difficult to study because of their rarity and their presence amongst the billions of surrounding normal blood cells. Of particular promise and utility to the in-depth study of CTCs are those technologies making use of microfluidics and nanomaterials, such as the graphene oxide (GO) Chip. The GO Chip has been applied to a 41-patient metastatic castrate resistant prostate cancer (mCRPC) cohort. CTCs were enumerated from whole blood for all patients (range: 3-166 CTCs/mL, median: 20 CTCs/mL). Clusters of CTCs, defined as two or more directly adjacent CTCs, were observed in 26/41 patients, and ranged in size from 2-8 CTCs/cluster. Within the CTC population, the percentage of CTCs present as clusters ranged from 0-54.8%. Additionally, a parallel device was run for 36 patients to ultimately obtain RNA to use in RT-qPCR to assess levels of 96 genes of interest. Enumeration and RNA expression data were compared with clinical outcomes including overall survival, radioclinical progression, and PSA progression. An eight-gene score was determined to be highly prognostic of overall survival (AUC: 0.88), with the genes comprising the score suggesting the importance of a dedifferentiated expression phenotype in poor prognosis. Follow-up work in prostate cancer investigated the role of HER2 and EGFR in prostate cancer metastasis. Analysis of tissue microarrays showed HER2 expression in prostate cancer and bone metastases. Primary and secondary prostate sphere formation was dependent on high EGFR expression as determined by FACS, but not on HER2 expression. EGFR was also implicated in survival in transit as shown by the presence of EGFR+ CTCs isolated by the GO Chip in 9/10 mCRPC patients assays, with an average of 35.5% of CTCs showing EGFR expression. Dual inhibition of HER2 and EGFR in mouse xenograft models prevented tumor growth. HER2 and EGFR as well as ADAM15 and CD31 were studied in bladder cancer CTCs as well. In a preliminary study primarily for optimization, antibodies were chosen for higher sensitivity capture as well as to stain bladder CTCs for the markers of interest. Ultimately, CTCs were isolated from five metastatic bladder cancer patients (range: 5-499 CTCs/mL), and a combination of staining antibodies that showed low background in the healthy control was chosen. EGFR+ and CD31+ CTCs were observed, while HER+ and ADAM15+ CTCs were not, and clusters of CTCs were isolated from some patients. To address drawbacks in the current technology, two strategies were attempted to enable cell release. A layer-by-layer (LbL) substrate enclosed in a microfluidic chamber featured different disadvantages based on film composition, but a thermosensitive polymer substrate enable release when cooled below its lower critical solution temperature of 12-13°C. The polymer-GO composite showed between 84.9 and 95.2% capture efficiency of EpCAM expressing cell lines and released over 91% of cells captured from whole blood. Using this device, CTCs were captured from 2/3 pancreatic cancer patients and 8/10 breast cancer patients. FISH for HER2 was performed on CTCs isolated from one breast cancer patient. With high performing technologies to separate them from the noise of other cells in the blood, CTCs can provide information about disease spread in genitourinary cancers. The future incorporation of CTC-related information into clinical decision making has the potential to better inform treatment selection and disease prognosis.PHDChemical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/140934/1/mollykoz_1.pd

Carbon nanotubes micro-arrays: characterization and application in biosensing of free proteins and label-free capture of breast cancer cells

Circulating tumor cells (CTCs) are cells released into the bloodstream from primary tumors and are suspected to be one of the main causes behind metastatic spreading of cancer. The ability to capture and analyze circulating tumor cells in clinical samples is of great interest in prevailing patient prognosis and clinical management of cancer. Carbon nanotubes, individual rolled-up graphene sheets, have emerged as exciting materials for probing the biomolecular interactions. With diameter of about 1 nm, they can attach themselves to cell surface receptors through specific antibodies and hold a great potential for diagnostic cellular profiling. Carbon nanotubes can be either semiconducting or metallic, and the electronic properties of either type rivals the best known materials. Small size of nanotubes and the ability to functionalize their surface using 1-Pyrenebutanoic Acid, Succinimidyl Ester (PASE), enables a versatile probe for developing a platform for capture and analysis of cancer biomarkers and circulating tumor cells. Although nanotubes have previously been used to electrically detect a variety of molecules and proteins, here for the first time we demonstrate the label free capture of spiked breast cancer cells using ultra-thin carbon nanotube film micro-array devices in a drop of buffy coat and blood. A new statistical approach of using Dynamic Time Warping (DTW) was used to classify the electrical signatures with 90% sensitivity and 90% specificity in blood. These results suggest such label free devices could potentially be useful for clinical capture and further analysis of circulating tumor cells. This thesis will go in-depth the properties of carbon nanotubes, device fabrication and characterization methodologies, functionalization protocols, and experiments in buffy coats and in blood. Combination of nano and biological materials, functionalization protocols and advanced statistical classifiers can potentially enable clinical translation of such devices in the future

Study of Circulating Tumor Cells using Microfluidic Technology: From Isolation to Analysis

An intimidating aspect of cancer is its ability to spread out to distant organs causing 90% of cancer-associated deaths. This metastatic progression is driven by circulating tumor cells (CTCs) shed from the primary tumor into bloodstream of carcinoma patients. As a result, CTCs hold great promise as a potential biomarker in areas of cancer diagnosis, monitoring, and evaluation of therapeutic efficacy for personalized medicine, which can serve as surrogate for invasive tissue biopsy. However, theses cells are extremely rare with a frequency of only 1-10 cells surrounded by billions of normal blood cells in 1mL of blood. This thesis delineates the shortcomings of existing CTC isolation methods followed by development and implementation of new microfluidic-based platforms to improve the sensitivity, specificity, and throughput for CTC enrichment. First, an affinity-based CTC isolation chip is introduced incorporating functional graphene oxide for high-density tumor specific antibody presentation. The two-dimensional surface-capture approach shows an overall CTC capture efficiency of >82.3% for flow rates up to 3mL/hr, while maintaining high viability (>90%) from low shear stress generated during sample processing. The extremely low blood cell contamination rate in the order of 100 cells/mL enables subsequent downstream analysis of CTCs. The clinical validity of the chip is demonstrated in a cohort of 47 metastatic breast cancer patients. Second, a size based CTC isolation chip is presented utilizing the inertial force effects to isolate CTCs by differentially focusing. Channel design parameters including the height, width, and radius of curvature and flow conditions are investigated to observe their effect on particle/cell focusing and streak migration. Optimal flow regimes to achieve maximum separation of 10/20 μm particles, representing leukocytes and CTCs respectively, in various channel configurations are identified. Based on these results, a cascaded spiral chip is designed for label-free CTC isolation achieving 87.76% recovery rate with 97.91% leukocyte depletion. Finally, a catheter based in-vivo CTC isolation system is implemented for large blood volume CTC screening. The system includes a dual lumen catheter to connect the patient blood veins, a peristaltic pump for continuous blood sampling, heparin injector to prevent blood clogging and clotting, and a CTC capture module.PHDElectrical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/138501/1/tztaebo_1.pd

Update on Circulating Tumor Cells in Genitourinary Tumors with Focus on Prostate Cancer

Current developments in the treatment of genitourinary tumors underline the unmet clinical need for biomarkers to improve decision-making in a challenging clinical setting. The detection of circulating tumor cells (CTCs) has become one of the most exciting and important new approaches to identifying biomarkers at different stages of disease in a non-invasive way. Potential applications of CTCs include monitoring treatment efficacy and early detection of progression, selecting tailored therapies, as well as saving treatment costs. However, despite the promising implementation of CTCs in a clinical scenario, the isolation and characterization of these cells for molecular studies remain expensive with contemporary platforms, and significant technical challenges still need to be overcome. This updated, critical review focuses on the state of CTCs in patients with genitourinary tumor with focus on prostate cancer, discussing technical issues, main clinical results and hypothesizing potential future perspectives in clinical scenarios

Investigating the Clinical Utility of Circulating Tumor Cells Via Nanomaterial Based Microfluidic Platforms

To realize personalized treatment for cancer patients, it is crucial to identify and monitor the molecular drivers of tumors. Currently, the molecular analysis of tumors is mostly performed on tissue biopsies. However, due to the invasiveness of the procedure, biopsies typically cannot be obtained repeatedly during the course of treatment and thus cannot reveal the dynamic evolution of tumors on both the genetic and epigenetic levels. There is a pressing need to monitor tumor evolution and to predict the treatment response to guide the clinical decision-making in the practice of personalized therapy. Circulating tumor cells (CTCs) shed from the primary tumor, travel through the blood, and have the potential to cause metastases. As CTCs can be frequently sampled from peripheral blood, CTC isolation and analysis hold great potential as a biomarker in real-time monitoring of tumor status. This work highlights the clinical utility of CTCs for providing prognostic and predictive information for specific treatments in cancer patients. First, dynamic changes of PD-L1(+) CTCs during radio(chemo)therapy were investigated in NSCLC. The real-time monitoring of PD-L1 expression in tumor microenvironment is crucial in guiding the therapeutic management of anti-PD-1/PD-L1 immunotherapy. CTCs were isolated using a nanomaterial based microfluidic device, the GO chip. PD-L1 (+) CTCs were detected in 25 out of 36 (69%) samples from 12 NSCLC patients undergoing radiation or radiochemotherapy. After the initiation of radiation, the proportion of PD-L1 (+) CTCs in total CTCs increased significantly (median 4% vs 24%, P=0.018). Furthermore, patients who were PD-L1 positive (5% of CTCs stained with PD-L1) at baseline had shorter PFS, suggesting the prognostic value of PD-L1 (+) CTCs (6.7 months vs 14.75 months, P = 0.017) Secondly, CTC number and the molecular features of CTCs were monitored at different time points during the course of treatment for locally advanced pancreatic patients. The reduction of CTC numbers after chemotherapy correlated with shorter progressed free survival (PFS), indicating that changes of CTC numbers may be an early indicator for treatment failure (6.5 months vs 13.5 months, P value= 0.002). Furthermore, in the mRNA profiling of CTCs, the expression levels of three genes that have been shown to play a role in drug resistance, BAX, CHK1 and EZH2, are associated with poor prognosis, which could act as makers to predict and monitor the treatment response. Thirdly, this work presents two technical advances of CTC technologies. A highly sensitive microfluidic device to capture and release circulating tumor cells from whole blood of cancer patients is developed. Graphene oxide is embeded into a thermoresponsive polymer film to serve as the first step of an antibody functionalization chemistry. As the temperature decreases to around 5 °C, the polymer film dissolves and detaches from the device and captured cells are released. Over 90% capture efficiency and release efficiency have been achieved. Released CTCs were viable and structurally intact, enabling subsequent analysis such as standard clinical cytopathological and genetic testing. Finally, to develop a high throughput CTC isolation technology, a herringbone mixer is incorporated into the previously developed GO chip and optimized the structure of the herringbone mixer and the channel geometry to maximize the throughput while achieving high capture efficiency (> 80%) and cell viability (> 90%). The time required to process a 1-mL blood sample is reduced to 10 minutes, 6 times faster than in the previous design.PHDChemical EngineeringUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttps://deepblue.lib.umich.edu/bitstream/2027.42/138765/1/wyangela_1.pd