Profiling circulating tumour cells (CTCs) in cancer patients' blood samples is critical to understand the complex and dynamic nature of metastasis. This task is challenged by the fact that CTCs are not only extremely rare in circulation but also highly heterogeneous in their molecular programs and cellular functions. Here we report a combinational approach for the simultaneous biochemical and functional phenotyping of patient-derived CTCs, using an integrated inertial ferrohydrodynamic cell separation (i 2 FCS) method and a single-cell microfluidic migration assay. This combinatorial approach offers unique capability to profile CTCs on the basis of their surface expression and migratory characteristics. We achieve this using the i 2 FCS method that successfully processes whole blood samples in a tumor cell marker and size agnostic manner. The i 2 FCS method enables an ultrahigh blood sample processing throughput of up to 2 × 10 5 cells s −1 with a blood sample flow rate of 60 mL h −1 . Its short processing time (10 minutes for a 10 mL sample), together with a close-to-complete CTC recovery (99.70% recovery rate) and a low WBC contamination (4.07-log depletion rate by removing 99.992% of leukocytes), results in adequate and functional CTCs for subsequent studies in the single-cell migration device. For the first time, we employ this new approach to query CTCs with single-cell resolution in accordance with their expression of phenotypic surface markers and migration properties, revealing the dynamic phenotypes and the existence of a high-motility subpopulation of CTCs in blood samples from metastatic lung cancer patients. This method could be adopted to study the biological and clinical value of invasive CTC phenotypes.
more »
« less
Fundamentals of integrated ferrohydrodynamic cell separation in circulating tumor cell isolation
Methods to separate circulating tumor cells (CTCs) from blood samples were intensively researched in order to understand the metastatic process and develop corresponding clinical assays. However current methods faced challenges that stemmed from CTCs' heterogeneity in their biological markers and physical morphologies. To this end, we developed integrated ferrohydrodynamic cell separation (iFCS), a scheme that separated CTCs independent of their surface antigen expression and physical characteristics. iFCS integrated both diamagnetophoresis of CTCs and magnetophoresis of blood cells together via a magnetic liquid medium, ferrofluid, whose magnetization could be tuned by adjusting its magnetic volume concentration. In this paper, we presented the fundamental theory of iFCS and its specific application in CTC separation. Governing equations of iFCS were developed to guide its optimization process. Three critical parameters that affected iFCS's cell separation performance were determined and validated theoretically and experimentally. These parameters included the sample flow rate, the volumetric concentration of magnetic materials in the ferrofluid, and the gradient of the magnetic flux density. We determined these optimized parameters in an iFCS device that led to a high recovery CTC separation in both spiked and clinical samples.
more »
« less
- Award ID(s):
- 1648035
- NSF-PAR ID:
- 10312458
- Date Published:
- Journal Name:
- Lab on a Chip
- Volume:
- 21
- Issue:
- 9
- ISSN:
- 1473-0197
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
More Like this
-
-
In this report, the relative efficiency of cellulose nanocrystals (CNCs) and nanofibers (CNFs) to capture circulating tumor cells (CTCs) from the blood sample of head and neck cancer (HNC) patients was evaluated. Detection and enumeration of CTCs are critical for monitoring cancer progression. Both types of nanostructured cellulose were chemically modified with Epithelial Cell Adhesion Molecule (EpCAM) antibody and iron oxide nanoparticles. The EpCAM antibody facilitated the engagement of CTCs, promoting entrapment within the cellulose cage structure. Iron oxide nanoparticles, on the other hand, rendered the cages activatable via the use of a magnet for the capture and separation of entrapped CTCs. The efficiency of the network structures is shown in head and neck cancer (HNC) patients' blood samples. It was observed that the degree of chemical functionalization of hydroxyl groups located within the CNCs or CNFs with anti-EpCAM determined the efficiency of the system's interaction with CTCs. Further, our result indicated that inflexible scaffolds of nanocrystals interacted more efficiently with CTCs than that of the fibrous CNF scaffolds. Network structures derived from CNCs demonstrated comparable CTC capturing efficiency to commercial standard, OncoDiscover®. The output of the work will provide the chemical design principles of cellulosic materials intended for constructing affordable platforms for monitoring cancer progression in 'real time'.more » « less
-
null (Ed.)We demonstrate a label free and high-throughput microbubble-based acoustic microstreaming technique to isolate rare circulating cells such as circulating cancer associated fibroblasts (cCAFs) in addition to circulating tumor cells (CTCs) and immune cells ( i.e. leukocytes) from clinically diagnosed patients with a capture efficiency of 94% while preserving cell functional integrity within 8 minutes. The microfluidic device is self-pumping and was optimized to increase flow rate and achieve near perfect capturing of rare cells enabled by having a trapping capacity above the acoustic vortex saturation concentration threshold. Our approach enables rapid isolation of CTCs, cCAFs and their associated clusters from blood samples of cancer patients at different stages. By examining the combined role of cCAFs and CTCs in early cancer onset and metastasis progression, the device accurately diagnoses both cancer and the metastatic propensity of breast cancer patients. This was confirmed by flow cytometry where we observed that metastatic breast cancer blood samples had significantly higher percentage of exhausted CD8 + T cells expressing programmed cell death protein 1 (PD1), higher number of CD4 + T regulatory cells and T helper cells. We show for the first time that our lateral cavity acoustic transducers (LCATs)-based approach can thus be developed into a metastatic propensity assay for clinical usage by elucidating cancer immunological responses and the complex relationships between CTCs and its companion tumor microenvironment.more » « less
-
more » « less
Abstract Circulating tumor cells (CTCs) have been proven to have significant prognostic, diagnostic, and clinical values in early‐stage cancer detection and treatment. The efficient separation of CTCs from peripheral blood can ensure intact and viable CTCs and can, thus, give proper genetic characterization and drug innovation. In this study, continuous and high‐throughput separation of MDA‐231 CTCs from overlapping sized white blood cells (WBCs) is achieved by modifying inertial cell focusing with dielectrophoresis (DEP) in a single‐stage microfluidic platform by numeric simulation. The DEP is enabled by embedding interdigitated electrodes with alternating field control on a serpentine microchannel to avoid creating two‐stage separation. Rather than using the electrokinetic migration of cells which slows down the throughput, the system leverages the inertial microfluidic flow to achieve high‐speed continuous separation. The cell migration and cell positioning characteristics are quantified through coupled physics analyses to evaluate the effects of the applied voltages and Reynolds numbers (
Re ) on the separation performance. The results indicate that the introduction of DEP successfully migrates WBCs away from CTCs and that separation of MDA‐231 CTCs from similar sized WBCs at a highRe of 100 can be achieved with a low voltage of magnitude 4 ×106 V/m. Additionally, the viability of MDA‐231 CTCs is expected to be sustained after separation due to the short‐term DEP exposure. The developed technique could be exploited to design active microchips for high‐throughput separation of mixed cell beads despite their significant size overlap, using DEP‐modified inertial focusing controlled simply by adjusting the applied external field. -
The number of Circulating Tumor Cells (CTCs) in blood indicates the tumor response to chemotherapeutic agents and disease progression. In early cancer diagnosis and treatment monitoring routine, detection and enumeration of CTCs in clinical blood samples have significant applications. In this paper, we design a Deep Convolutional Neural Network (DCNN) with automatically learned features for image-based CTC detection. We also present an effective training methodology which finds the most representative training samples to define the classification boundary between positive and negative samples. In the experiment, we compare the performance of auto-learned feature from DCNN and hand-crafted features, in which the DCNN outperforms hand-crafted feature. We also prove that the proposed training methodology is effective in improving the performance of DCNN classifiers.more » « less
- Free Publicly Accessible Full Text
-
Accepted Manuscript1.0
- Journal Article:
- https://doi.org/10.1039/D1LC00119A
