Capillary Force Driven Single-Cell Spiking Apparatus for Studying Circulating Tumor Cells

2018 ◽  
Author(s):  
Jacob Amontree ◽  
Kangfu Chen ◽  
Jose Varillas ◽  
Z. Hugh Fan
Keyword(s):  
Single Cell ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Single Cells ◽  
Lymphoblastic Leukemia ◽  
Surface Membrane ◽  
Critical Element ◽  
Cell Capture ◽  
Biomedical Sciences ◽  
Low Concentrations

The characterization of single cells within heterogeneous populations has great impact on both biomedical sciences and cancer research. By investigating cellular compositions on a broad scale, pertinent outliers may be lost in the sample set. Alternatively, an investigation focused on the behavior of specific cells, such as circulating tumor cells (CTCs), will reveal genetic biomarkers or phenotypic characteristics associated with cancer and metastasis. On average, CTC concentration in peripheral blood is extremely low, as few as one to two per billion of healthy blood cells. Consequently, the critical element lacking in many methods of CTC detection is accurate cell capture efficiency at low concentrations. To simulate CTC isolation, researchers usually spike small amounts of tumor cells to healthy blood for separation. However, spiking tumor cells at extremely low concentrations is challenging in a standard laboratory setting. We report our study on an innovative apparatus and method designed for low-cost, precise, and replicable single-cell spiking (SCS). Our SCS method operates solely from capillary aspiration without the reliance on external laboratory equipment. To ensure that our method does not affect the viability of each cell, we investigated the effects of surface membrane tensions induced by aspiration. Finally, we performed affinity-based CTC isolation using human acute lymphoblastic leukemia cells (CCRF-CEM) spiked into healthy whole blood with the SCS technique. The results of the isolation experiments demonstrate the reliability of our method in generating low-concentration cell samples.

scholarly journals Application of single-cell sequencing technologies in pancreatic cancer

2021 ◽  
Author(s):  
Mastan Mannarapu ◽  
Begum Dariya ◽  
Obul Reddy Bandapalli
Keyword(s):  
Pancreatic Cancer ◽  
Single Cell ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Single Cells ◽  
Evolutionary Relationship ◽  
Intratumor Heterogeneity ◽  
Sequencing Technology ◽  
Single Cell Sequencing ◽  
Sequencing Technologies

AbstractPancreatic cancer (PC) is the third lethal disease for cancer-related mortalities globally. This is mainly because of the aggressive nature and heterogeneity of the disease that is diagnosed only in their advanced stages. Thus, it is challenging for researchers and clinicians to study the molecular mechanism involved in the development of this aggressive disease. The single-cell sequencing technology enables researchers to study each and every individual cell in a single tumor. It can be used to detect genome, transcriptome, and multi-omics of single cells. The current single-cell sequencing technology is now becoming an important tool for the biological analysis of cells, to find evolutionary relationship between multiple cells and unmask the heterogeneity present in the tumor cells. Moreover, its sensitivity nature is found progressive enabling to detect rare cancer cells, circulating tumor cells, metastatic cells, and analyze the intratumor heterogeneity. Furthermore, these single-cell sequencing technologies also promoted personalized treatment strategies and next-generation sequencing to predict the disease. In this review, we have focused on the applications of single-cell sequencing technology in identifying cancer-associated cells like cancer-associated fibroblast via detecting circulating tumor cells. We also included advanced technologies involved in single-cell sequencing and their advantages. The future research indeed brings the single-cell sequencing into the clinical arena and thus could be beneficial for diagnosis and therapy of PC patients.

scholarly journals Effect of Blood Collection Tube Type and Time to Processing on the Enumeration and High-Content Characterization of Circulating Tumor Cells Using the High-Definition Single-Cell Assay

2017 ◽  
Vol 142 (2) ◽  
pp. 198-207 ◽  
Author(s):  
Mariam Rodríguez-Lee ◽  
Anand Kolatkar ◽  
Madelyn McCormick ◽  
Angel D. Dago ◽  
Jude Kendall ◽  
...  
Keyword(s):  
Single Cell ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Single Cells ◽  
Blood Collection ◽  
High Definition ◽  
Cell Assay ◽  
Collection Tube ◽  
Blood Collection Tube

Context.— As circulating tumor cell (CTC) assays gain clinical relevance, it is essential to address preanalytic variability and to develop standard operating procedures for sample handling in order to successfully implement genomically informed, precision health care. Objective.— To evaluate the effects of blood collection tube (BCT) type and time-to-assay (TTA) on the enumeration and high-content characterization of CTCs by using the high-definition single-cell assay (HD-SCA). Design.— Blood samples of patients with early- and advanced-stage breast cancer were collected into cell-free DNA (CfDNA), EDTA, acid-citrate-dextrose solution, and heparin BCTs. Time-to-assay was evaluated at 24 and 72 hours, representing the fastest possible and more routine domestic shipping intervals, respectively. Results.— We detected the highest CTC levels and the lowest levels of negative events in CfDNA BCT at 24 hours. At 72 hours in this BCT, all CTC subpopulations were decreased with the larger effect observed in high-definition CTCs and cytokeratin-positive cells smaller than white blood cells. Overall cell retention was also optimal in CfDNA BCT at 24 hours. Whole-genome copy number variation profiles were generated from single cells isolated from all BCT types and TTAs. Cells from CfDNA BCT at 24-hour TTA exhibited the least noise. Conclusions.— Circulating tumor cells can be identified and characterized under a variety of collection, handling, and processing conditions, but the highest quality can be achieved with optimized conditions. We quantified performance differences of the HD-SCA for specific preanalytic variables that may be used as a guide to develop best practices for implementation into patient care and/or research biorepository processes.

Genomic heterogeneity of circulating tumor cells in castration-resistant prostate cancer (CRPC) revealed by single-cell sequencing.

2013 ◽  
Vol 31 (15_suppl) ◽  
pp. 5030-5030
Author(s):  
Allison Welsh ◽  
Daniel Costin Danila ◽  
Aseem Anand ◽  
Jude Kendall ◽  
Charles L. Sawyers ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Dna Sequencing ◽  
Single Cell ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Single Cells ◽  
Dna Amplification ◽  
Copy Number Variations ◽  
Castration Resistant Prostate Cancer ◽  
Blood Draw

5030 Background: Circulating tumor cells (CTC) provide an opportunity to sample multiple metastatic tumor sites through a single blood draw – a ”fluid biopsy.” NextGen DNA sequencing provides the means to obtain detailed genetic information from captured cells prior to and during treatment. Here we demonstrate the use of DNA sequencing to interrogate genome-wide copy number variations (CNV) at the single-cell level in CTC isolated from pts with CRPC. Methods: Pre- and post-treatment blood samples were obtained from pts treated at MSKCC. EpCAM+ events were collected singly and in groups by cytometric flow sorting and were subjected to DNA amplification and Illumina NextGeneration sequencing. Parallel samples were assayed using the Veridex CellSearch method to ensure the presence of malignant cells. Results: Samples with up to 50 EpCAM+ events analyzed in bulk displayed CNV patterns expected from published CRPC data. Subsequent single cell analyses showed that the method could reliably detect common genomic markers in CRPC, including AR amplification, PTEN and RB1 loss, and the TMPRSS-ERG fusion. Individual genomic CNV profiles obtained from 125 single cells isolated from 15 patients were then analyzed. Using unsupervised clustering, cells from each pt showed a closely related lineage structure, consistent with an evolution from a common ancestor. The degree of genomic heterogeneity within CTC from an individual pt was highly variable, with R2 correlation coefficients ranging from >0.92 (nearly homogeneous) to <0.75 (mixed populations). Two pts harbored separate subpopulations with both amplified AR and non-amplified AR cells and another displayed mixtures of genetic markers that changed over the course of treatment. Conclusions: The observed variation in complexity of CTC populations in CRPC pts underscores the importance of being able to sample and analyze multiple cells from an individual pt on multiple occasions and with real time analytics. Doing so is essential to understand and identify mechanisms of resistance so that they can be targeted effectively. Supported by STARR Cancer Consortium, NCI SPORE in Prostate Cancer; Department of Defense; Prostate Cancer Foundation.

scholarly journals 56P Single circulating tumor cells RNA profiling by label-free enrichment and active single cell selection on an integrated fluidic system

2016 ◽  
Vol 27 ◽  
pp. ix15-ix16
Author(s):  
Y.F. Lee ◽  
N. Ramalingam ◽  
L. Szpankowski ◽  
A. Leyrat ◽  
N.D. Angeles ◽  
...  
Keyword(s):  
Single Cell ◽  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Cell Selection ◽  
Label Free ◽  
Rna Profiling ◽  
Free Enrichment

scholarly journals A small molecule screen identified N-Acetyl-L-cysteine for promoting ex vivo growth of single circulating tumor cells

2020 ◽  
Author(s):  
Teng Teng ◽  
Mohamed Kamal ◽  
Oihana Iriondo ◽  
Yonatan Amzaleg ◽  
Chunqiao Luo ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Small Molecule ◽  
Ex Vivo ◽  
Single Cells ◽  
Small Subset ◽  
Blood Draw ◽  
Small Molecule Screen ◽  
Functional Analyses ◽  
Single Ctcs

AbstractCirculating tumor cells (CTCs) can be isolated via a minimally invasive blood draw and are considered a “liquid biopsy” of their originating solid tumors. CTCs contain a small subset of metastatic precursors that can form metastases in secondary organs, and provide a resource to identify mechanisms underlying metastasis-initiating properties. Despite technological advancements that allow for highly sensitive approaches of detection and isolation, CTCs are very rare and often present as single cells, posing an extreme challenge for ex vivo expansion after isolation. Here, using previously established patient-derived CTC lines, we performed a small molecule drug screening to identify compounds that can improve ex vivo culture efficiency for single CTCs. We found that N-acetylcysteine (NAC) and other antioxidants can promote ex vivo expansion of single CTCs, by reducing oxidative and other stress particularly at the initial stage of single cell expansion. RNA-seq analysis of growing clones and non-growing clones confirmed the effect by NAC, but also indicate that NAC-induced decrease in oxidative stress is insufficient for promoting proliferation of a subset of cells with heterogeneous quiescent and senescent features. Despite the challenge in expanding all CTCs, NAC treatment lead to establishment of single CTC clones that have similar tumorigenic features, which will facilitate future functional analyses.

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