scholarly journals Label-free enrichment of rare unconventional circulating neoplastic cells using a microfluidic dielectrophoretic sorting device

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Jose Montoya Mira ◽  
Ajay A. Sapre ◽  
Brett S. Walker ◽  
Jesus Bueno Alvarez ◽  
Kyle T. Gustafson ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Mononuclear Cells ◽  
Digital Pcr ◽  
Clinical Samples ◽  
Label Free ◽  
Neoplastic Cells ◽  
Peripheral Blood Mononuclear ◽  
Potential Methods ◽  
Circulating Neoplastic Cells ◽  
Free Enrichment

AbstractCellular circulating biomarkers from the primary tumor such as circulating tumor cells (CTCs) and circulating hybrid cells (CHCs) have been described to harbor tumor-like phenotype and genotype. CHCs are present in higher numbers than CTCs supporting their translational potential. Methods for isolation of CHCs do not exist and are restricted to low-throughput, time consuming, and biased methodologies. We report the development of a label-free dielectrophoretic microfluidic platform facilitating enrichment of CHCs in a high-throughput and rapid fashion by depleting healthy peripheral blood mononuclear cells (PBMCs). We demonstrated up to 96.5% depletion of PBMCs resulting in 18.6-fold enrichment of cancer cells. In PBMCs from pancreatic adenocarcinoma patients, the platform enriched neoplastic cells identified by their KRAS mutant status using droplet digital PCR with one hour of processing. Enrichment was achieved in 75% of the clinical samples analyzed, establishing this approach as a promising way to non-invasively analyze tumor cells from patients.

scholarly journals Label-free enrichment of rare unconventional circulating neoplastic cells using a microfluidic dielectrophoretic sorting device

2021 ◽  
Author(s):  
Jose Montoya Mira ◽  
Ajay Sapre ◽  
Brett Walker ◽  
Jesus Bueno Alvarez ◽  
Kyle Gustafson ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Tumor Cells ◽  
Mononuclear Cells ◽  
Digital Pcr ◽  
Clinical Samples ◽  
Label Free ◽  
Circulating Biomarkers ◽  
Neoplastic Cells ◽  
Peripheral Blood Mononuclear ◽  
Potential Methods

Abstract Technologies that facilitate analyses of circulating biomarkers from blood for cancer detection are powerful tools for improving patient outcomes. Circulating biomarkers derived directly from the primary tumor have been identified including circulating tumor cells (CTCs) and circulating hybrid cells (CHCs), described to harbor phenotypes of both tumor cells and leukocytes. CHCs are present in higher numbers than CTCs which support their translational potential. Methods for isolation of CHCs do not exist and are restricted to low-throughput, time consuming, and biased methodologies. Here, we report development of a label-free dielectrophoretic (DEP) microfluidic platform facilitating enrichment of CHCs in a high-throughput and rapid fashion. Unlike other methods, depletion of healthy peripheral blood mononuclear cells (PBMCs) drives enrichment of CHCs. We analyzed DEP differential response of PBMCs and cancer cells and demonstrated up to 96.5% depletion of PBMCs resulting in 18.6-fold enrichment of cancer cells. In PBMCs from pancreatic adenocarcinoma patients, the platform enriched for neoplastic cells identified by their KRAS mutant status using droplet digital PCR from only 2 mL of whole blood with one hour of processing. Enrichment was achieved in 75% of the clinical samples analyzed, establishing this approach as a promising way to non-invasively analyze tumor cells from patients.

scholarly journals Dielectrophoretic and Electrical Impedance Differentiation of Cancerous Cells Based on Biophysical Phenotype

Biosensors ◽  
2021 ◽  
Vol 11 (10) ◽  
pp. 401
Author(s):  
Ina Turcan ◽  
Iuliana Caras ◽  
Thomas Gabriel Schreiner ◽  
Catalin Tucureanu ◽  
Aurora Salageanu ◽  
...  
Keyword(s):  
Impedance Spectroscopy ◽  
Tumor Cells ◽  
Cancer Cell ◽  
Primary Tumor ◽  
Electrical Impedance ◽  
Mononuclear Cells ◽  
Equivalent Circuit Model ◽  
Electrical Impedance Spectroscopy ◽  
Label Free ◽  
Peripheral Blood Mononuclear

Here, we reported a study on the detection and electrical characterization of both cancer cell line and primary tumor cells. Dielectrophoresis (DEP) and electrical impedance spectroscopy (EIS) were jointly employed to enable the rapid and label-free differentiation of various cancer cells from normal ones. The primary tumor cells that were collected from two colorectal cancer patients and cancer cell lines (SW-403, Jurkat, and THP-1), and healthy peripheral blood mononuclear cells (PBMCs) were trapped first at the level of interdigitated microelectrodes with the help of dielectrophoresis. Correlation of the cells dielectric characteristics that was obtained via electrical impedance spectroscopy (EIS) allowed evident differentiation of the various types of cell. The differentiations were assigned to a “dielectric phenotype” based on their crossover frequencies. Finally, Randles equivalent circuit model was employed for highlighting the differences with regard to a series group of charge transport resistance and constant phase element for cancerous and normal cells.

scholarly journals Integrative analysis and machine learning based characterization of single circulating tumor cells

2019 ◽  
Author(s):  
Arvind Iyer ◽  
Krishan Gupta ◽  
Shreya Sharma ◽  
Kishore Hari ◽  
Yi Fang Lee ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Circulating Tumor Cells ◽  
Expression Profiles ◽  
Gene Expression Pattern ◽  
Integrative Analysis ◽  
Label Free ◽  
Peripheral Blood Mononuclear ◽  
Cell Expression ◽  
Free Enrichment

ABSTRACTWe collated publicly available single-cell expression profiles of circulating tumor cells (CTCs) and showed that CTCs across cancers lie on a near-perfect continuum of epithelial to mesenchymal (EMT) transition. Integrative analysis of CTC transcriptomes also highlighted the inverse gene expression pattern between PD-L1 and MHC, which is implicated in cancer immunotherapy. We used the CTCs expression profiles in tandem with publicly available peripheral blood mononuclear cell (PBMC) transcriptomes to train a classifier that accurately recognizes CTCs of diverse phenotype. Further, we used this classifier to validate circulating breast tumor cells captured using a newly developed microfluidic systems for label-free enrichment of CTCs.

Carfilzomib can induce tumor cell death through selective inhibition of the chymotrypsin-like activity of the proteasome

Blood ◽  
2009 ◽  
Vol 114 (16) ◽  
pp. 3439-3447 ◽  
Author(s):  
Francesco Parlati ◽  
Susan J. Lee ◽  
Monette Aujay ◽  
Erika Suzuki ◽  
Konstantin Levitsky ◽  
...  
Keyword(s):  
Tumor Cells ◽  
Mononuclear Cells ◽  
Proteasome Inhibitors ◽  
Selective Inhibition ◽  
Tumor Cell Death ◽  
Clinical Safety ◽  
Peripheral Blood Mononuclear ◽  
Non Hodgkin Lymphoma ◽  
Proteasome Subunits ◽  
The Impact

Abstract Carfilzomib is a proteasome inhibitor in clinical development that primarily targets the chymotrypsin-like (CT-L) subunits in both the constitutive proteasome (c20S) and the immunoproteasome (i20S). To investigate the impact of inhibiting the CT-L activity with carfilzomib, we set out to quantitate the levels of CT-L subunits β5 from the c20S and LMP7 from the i20S in normal and malignant hematopoietic cells. We found that the i20S is a major form of the proteasome expressed in cells of hematopoietic origin, including multiple myeloma (MM) CD138+ tumor cells. Although specific inhibition of either LMP7 or β5 alone was insufficient to produce an antitumor response, inhibition of all proteasome subunits was cytotoxic to both hematologic tumor cells and peripheral blood mononuclear cells. However, selective inhibition of both β5 and LMP7 was sufficient to induce an antitumor effect in MM, non-Hodgkin lymphoma, and leukemia cells while minimizing the toxicity toward nontransformed cells. In MM tumor cells, CT-L inhibition alone was sufficient to induce proapoptotic sequelae, including proteasome substrate accumulation, Noxa and caspase 3/7 induction, and phospho-eIF2α suppression. These data support a hypothesis that hematologic tumor cells are uniquely sensitive to CT-L inhibition and provide a mechanistic understanding of the clinical safety profile and antitumor activity of proteasome inhibitors.

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 Human Autologous In Vitro Models of Glioma Immunogene Therapy Using B7-2, GM-CSF, and IL12

2002 ◽  
Vol 29 (3) ◽  
pp. 267-275 ◽  
Author(s):  
Ian F. Parney ◽  
Maxine A. Farr-Jones ◽  
Kevin Kane ◽  
Lung-Ji Chang ◽  
Kenneth C. Petruk
Keyword(s):  
Tumor Cells ◽  
Peripheral Blood Mononuclear Cells ◽  
Peripheral Blood ◽  
Mononuclear Cells ◽  
Wild Type ◽  
Peripheral Blood Mononuclear ◽  
Gm Csf ◽  
Blood Mononuclear Cells ◽  
Immunogene Therapy ◽  
Tumor Cytotoxicity

Background:Cancer immunogene therapy is based on vaccination with radiated, autologous tumor cells transduced with immunostimulatory genes. To help determine an optimal glioma immunogene therapy strategy, we stimulated lymphocytes with autologous human glioma cells transduced with B7-2 (CD86), granulocyte-macrophage colony-stimulating factor (GM-CSF), and/or interleukin-12 (IL12).Methods:A human glioma-derived cell culture (Ed147.BT) was transduced with B7-2, GM-CSF, and/or IL12 using retroviral vectors. Autologous peripheral blood mononuclear cells (PBMC) were co-cultured with irradiated gene-transduced tumor alone or a combination of radiated wild type and gene-transduced cells. Peripheral blood mononuclear cells proliferation was determined by serial cell counts. Peripheral blood mononuclear cells phenotype was assessed by flow cytometry for CD4, CD8, and CD16. Anti-tumor cytotoxicity was determined by chromium-51 (51Cr) release assay.Results:Peripheral blood mononuclear cells cell numbers all decreased during primary stimulation but tumor cells expressing B7-2 or GM-CSF consistently caused secondary proliferation. Tumors expressing B7-2 and GM-CSF or B7-2, GM-CSF, and IL12 consistently increased PBMC CD8+ (cytotoxic T) and CD16+ (natural killer) percentages. Interestingly, anti-tumor cytotoxicity only exceeded that of PBMC stimulated with wild type tumor alone when peripheral blood mononuclear cells were stimulated with both wild type tumor and B7-2/GM-CSF- (but not IL12) transduced cells.Conclusion:PBMC proliferation and phenotype is altered as expected by exposure to immunostimulatory gene-transduced tumor. However, transduced tumor cells alone do not stimulate greater anti-tumor cytotoxicity than wild type tumor. Only B7-2/GM-CSF-transduced cells combined with wild type produced increased cytotoxicity. This may reflect selection of tumor subclones with limited antigenic spectra during retrovirus-mediated gene transfer.

scholarly journals FACS-Based Proteomics Enables Profiling of Proteins in Rare Cell Populations

2020 ◽  
Vol 21 (18) ◽  
pp. 6557
Author(s):  
Evelyne Maes ◽  
Nathalie Cools ◽  
Hanny Willems ◽  
Geert Baggerman
Keyword(s):  
Proteomic Analysis ◽  
Mononuclear Cells ◽  
Protein Extraction ◽  
Cell Types ◽  
Clinical Samples ◽  
Cell Populations ◽  
Peripheral Blood Mononuclear ◽  
Limited Protein ◽  
Instrument Sensitivity ◽  
Acquisition Method

Understanding disease pathology often does not require an overall proteomic analysis of clinical samples but rather the analysis of different, often rare, subpopulations of cells in a heterogeneous mixture of cell types. For the isolation of pre-specified cellular subtypes, fluorescence activated cell sorting (FACS) is commonly used for its ability to isolate the required cell populations with high purity, even of scarce cell types. The proteomic analysis of a limited number of FACS-sorted cells, however, is very challenging as both sample preparation inefficiencies and limits in terms of instrument sensitivity are present. In this study, we used CD14+CD15+ immune cells sorted out of peripheral blood mononuclear cells isolated from whole blood to improve and evaluate FACS-based proteomics. To optimize both the protein extraction protocol and the mass spectrometry (MS) data acquisition method, PBMCs as well as commercialized HeLa digest were used. To reflect the limited number of sorted cells in some clinical samples, different numbers of sorted cells (1000, 5000, 10,000, or 50,000) were used. This allowed comparing protein profiles across samples with limited protein material and provided further insights in the benefits and limitations of using a very limited numbers of cells.

Non-inertial lift induced migration for label-free sorting of cells in a co-flowing aqueous two-phase system

The Analyst ◽  
2019 ◽  
Vol 144 (8) ◽  
pp. 2574-2583 ◽  
Author(s):  
S. Hazra ◽  
K. S. Jayaprakash ◽  
K. Pandian ◽  
A. Raj ◽  
S. K. Mitra ◽  
...  
Keyword(s):  
Cancer Cells ◽  
Mononuclear Cells ◽  
Phase System ◽  
Label Free ◽  
Two Phase ◽  
Peripheral Blood Mononuclear ◽  
Aqueous Two Phase System ◽  
System P ◽  
Blood Mononuclear Cells ◽  
Microfluidic Technique

We present a novel label-free passive microfluidic technique for isolation of cancer cells (EpCAM+ and CD45−) from peripheral blood mononuclear cells (PBMCs) (CD45+ and EpCAM−) in aqueous two-phase system (ATPS).

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