Nanoparticle-based sorting of circulating tumor cells by epithelial antigen expression during disease progression in an animal model

doi:10.1016/j.nano.2015.04.017

Nanomedicine: Nanotechnology, Biology and Medicine

Volume 11, Issue 7, October 2015, Pages 1613-1620

https://doi.org/10.1016/j.nano.2015.04.017 Get rights and content

Abstract

Circulating tumor cells (CTCs) can be used as markers for the detection, characterization, and targeted therapeutic management of cancer. We recently developed a nanoparticle-mediated approach for capture and sorting of CTCs based on their specific epithelial phenotype. In the current study, we investigate the phenotypic transition of tumor cells in an animal model and show the correlation of this transition with tumor progression. VX2 tumor cells were injected into rabbits, and CTCs were evaluated during tumor progression and correlated with computerized tomography (CT) measurements of tumor volume. The results showed a dramatic increase of CTCs during the four weeks of tumor growth. Following resection, CTC levels dropped but then rebounded, likely due to lymph node metastases. Additionally, CTCs showed a marked loss of the epithelial cell adhesion molecule (EpCAM) relative to precursor cells. In conclusion, the device accurately traces disease progression and CTC phenotypic shift in an animal model.

From the Clinical Editor

The detection of circulating tumor cells (CTCs) has been used to predict disease prognosis. In this study, the authors developed a nanoparticle-mediated platform based on microfluidics to analyze the differential expressions of epithelial cell adhesion molecule (EpCAM) on CTCs in an animal model. It was found that the loss of EpCAM correlated with disease progression. Hence, the use of this platform may be further applied in other cancer models in the future.

Graphical abstract

A study is presented that tracks the surface expression phenotype of CTCs during tumor growth in an animal model of cancer. A microfluidic device sorts subpopulations of CTCs into discrete compartments based on EpCAM levels. Loss of EpCAM, and thus epithelial character, is observed in CTCs as tumor growth progresses.

Section snippets

Animal model

Experiments were performed using 6 New Zealand white rabbits weighing 2.5-3.0 kg. All animal studies were performed in accordance with the University Health Network/University of Toronto guidelines for the humane use of animals. Care, handling and maintenance of all animals used in this study were conducted in a humane manner, as per the animal care experimental protocol approved by the institutional Animal Care and Use Committee of University Health Network, University of Toronto. Male rabbits

Animal study and microfluidic chip design

The overall timeline of the study is shown in Figure 1, A. Following tumor induction, CT scans and CTC analysis were performed bi-weekly until the 4th week post-tumor induction. At this point, the primary tumor was resected and CT scan/CTC analysis continued until terminal surgery during the 6th week.

The microfluidic chip employed for CTC analysis demonstrates a number of key design features that allow for capture as a function of EpCAM expression. Cells are first tagged with magnetic

Discussion

It is well recognized that CTCs are precursors to metastasis and can serve as an integral component of tumor staging criteria. Furthermore, previous studies involving breast cancer patients have shown that CTC data can be the best indicator of disease state and can provide improvement to the staging process.²³ Because blood collection is simple and minimally invasive, CTCs can potentially be used as a real-time marker to monitor disease progression. They also possess the potential to guide

References (30)

P. Quirke et al.
The future of the TNM staging system in colorectal cancer: time for a debate?
Lancet Oncol
(2007)
N. Aceto et al.
Circulating tumor cell clusters are oligoclonal precursors of breast cancer metastasis
Cell
(2014)
F.C. Bidard et al.
Single circulating tumor cell detection and overall survival in nonmetastatic breast cancer
Ann Oncol
(2010)
H. Tsukamoto et al.
Irradiation-induced epithelial–mesenchymal transition (EMT) related to invasive potential in endometrial carcinoma cells
Gynecol Oncol
(2007)
F.C. Bidard et al.
Disseminated and circulating tumor cells in gastrointestinal oncology
Crit Rev Oncol Hematol
(2012)
M. Cristofanilli et al.
Circulating tumor cells in metastatic breast cancer: biologic staging beyond tumor burden
Clin Breast Cancer
(2007)
B. Baum et al.
Transitions between epithelial and mesenchymal states in develoment and disease
Semin Cell Dev Biol
(2008)
D. Wang et al.
Single cell analysis: the new frontier in ‘omics'
Trends Biotechnol
(2010)
N.N. Baxter et al.
Inadequacy of lymph node staging in gastric cancer patients: a population-based study
Ann Surg Oncol
(2005)
L. Schnipper
Clinical implications of tumor-cell heterogeneity
N Engl J Med
(1986)

M.R. Junttila et al.

Influence of tumor micro-environment heterogeneity on therapeutic response

Nature

(2013)

J.P. Thiery

Epithelial–mesenchymal transitions in tumour progression

Nat Rev Cancer

(2002)

E. Heitzer et al.

Complex tumor genomes inferred from single circulating tumor cells by array-CGH and next-generation sequencing

Cancer Res

(2013)

C. Zong et al.

Genome-wide detection of single-nucleotide and copy-number variations of a single human cell

Science

(2012)

S.J. Cohen et al.

Relationship of circulating tumor cells to tumor response, progression-free survival, and overall survival in patients with metastatic colorectal cancer

J Clin Oncol

(2008)

Cited by (23)

Microfluidic chip for graduated magnetic separation of circulating tumor cells by their epithelial cell adhesion molecule expression and magnetic nanoparticle binding
2021, Journal of Chromatography A
Citation Excerpt :
The device was tested using cultured MCF-7 (high EpCAM) and MDA-NB-231 (low EpCAM) cells, and it showed some degree of selective capture. Kelley and co-workers have developed the Velocity Valley Chip (or VV Chip) [69–72] and the related magnetic ranking cytometry (MagRC) device [73–75]. The VV Chip uses a series of thin channels containing arrays of small X-shaped obstacles.
The enumeration of circulating tumor cells (CTCs) in the peripheral bloodstream of metastatic cancer patients has contributed to improvements in prognosis and therapeutics. There have been numerous approaches to capture and counting of CTCs. However, CTCs have potential information beyond simple enumeration and hold promise as a liquid biopsy for cancer and a pathway for personalized cancer therapy by detecting the subset of CTCs having the highest metastatic potential. There is evidence that epithelial cell adhesion molecule (EpCAM) expression level distinguishes these highly metastatic CTCs. The few previous approaches to selective CTC capture according to EpCAM expression level are reviewed. A new two-stage microfluidic device for separation, enrichment and release of CTCs into subpopulations sorted by EpCAM expression level is presented here. It relies upon immunospecific magnetic nanoparticle labeling of CTCs followed by their field- and flow-based separation in the first stage and capture as discrete subpopulations in the second stage. To fine tune the separation, the magnetic field profile across the first stage microfluidic channel may be modified by bonding small Vanadium Permendur strips to its outer walls. Mathematical modeling of magnetic fields and fluid flows supports the soundness of the design.
CTC analysis: an update on technological progress
2019, Translational Research
Citation Excerpt :
The captured MUNPs are fluorescently illuminated by an imaging system and the cells can be recovered by turning the magnet off for further study. A microfluidic device with a gradient of EpCAM capture areas from high to low developed by Muhanna et al may help determine differentiation of CTCs as tumor progresses.93 The VERSA platform presents an all-in-one handheld chip with the ability to do size-based CTC isolation and further separation of fluids for mRNA and DNA analysis.94
There is a growing need for a more accurate, real-time assessment of tumor status and the probability of metastasis, relapse, or response to treatment. Conventional means of assessment include imaging and tissue biopsies that can be highly invasive, may not provide complete information of the disease's heterogeneity, and not ideal for repeat analysis. Therefore, a less-invasive means of acquiring similar information at greater time points is necessary. Liquid biopsies are samples of a patients’ peripheral blood and hold potential of addressing these criteria. Ongoing research has revealed that a tumor can release circulating cells, genetic materials (DNA or RNA), and exosomes into circulation. These potential biomarkers can be captured in a liquid biopsy and analyzed to determine disease status. To achieve these goals, numerous technologies have been developed. In this review, we discuss both prominent and newly developed technologies for circulating tumor cell capture and analysis and their clinical impact.
AlN and AlP doped graphene quantum dots as novel drug delivery systems for 5-fluorouracil drug: Theoretical studies
2018, Journal of Fluorine Chemistry
Citation Excerpt :
There are several advantages to the use of nanocarriers as DDSs. Firstly, the small size of nanocarriers allow them to diffuse more quickly than larger particles [19]. Secondly, the nanocarriers have larger surface-to-volume ratio, which provided a larger contact surface between the drug and body in the same drug concentration.
With the help of density functional theory (DFT) calculations, it was attempted to investigate the interaction of 5-fluorouracil (FU) drug and undoped/doped graphene quantum dots (GQDs). To this aim, hexa-peri-hexabenzocoronene as well as its BN, BP, AlN and AlP doped (C₃₆X₃Y₃H₁₈ where XB, Al and YN, P) forms were selected as model GQDs. Results showed that doping of GQDs with AlN and AlP could significantly enhance the tendency of GQDs for adsorption of FU drug. Moreover, Gibbs free energy values indicated that the adsorption of FU drug on the GQDs surfaces was spontaneously occurred. It was found that AlN-FU and AlP-FU complexes had high chemical reactivity, which was crucial for binding of the drug onto the target site. Also, the nature of intermolecular interactions was analyzed based on quantum theory of atoms in molecules (QTAIM) and noncovalent interaction (NCI) analyses. Consequently, the results demonstrated that the AlN and AlP doped GQDs could be used as potential carriers for delivery of FU drug in the nanomedicine domain.
Epithelial and mesenchymal circulating tumor cell isolation and discrimination using dual-immunopatterned device with newly-developed anti-63B6 and anti-EpCAM
2018, Sensors and Actuators, B: Chemical
Citation Excerpt :
Although further validation for this newly developed antibody is required, these results may help the CTC researchers to develop potential competitors of anti-EpCAM antibody. Second, unlike MCF-7 cells, MDA-MB-231 cells show a very low level of EpCAM expression; [10,40,41] thus, anti-EpCAM antibody is not quite suitable for them. In the test using heterogeneous CTC model samples, anti-EpCAM antibody-immobilized layer showed much lower capture efficiency against MDA-MB-231.
We report a dual-immunopatterned microfluidic device for capturing both epithelial and mesenchymal circulating tumor cells (CTCs) simultaneously in a single device using the two antibodies of anti-epithelial cell adhesion molecule (EpCAM) antibody and newly developed anti-63B6 antibody. In addition to the conventional epithelial antibody of anti-EpCAM, our in-house produced anti-63B6 antibody targets mesenchymal stem cell-like cancer cells and intermediate cancer cells, thus overcoming limitation of the conventional single anti-EpCAM based CTC isolations. These two antibodies are immobilized to each top and bottom layer of the present device and the combination of two layers forms a circular chamber with the center inlet. The mutual complementary cell isolation through competing reaction in different velocity zones between mesenchymal and epithelial antibodies enables the precise screening and profiling of CTCs depending on their positivity and degree of epithelial/mesenchymal surface antigen expression, which may differ in compliance with disease status.
From the experiments using epithelial and mesenchymal-like cancer cells, the present device captures 94.47% of cancer cells, which is substantially higher than the EpCAM-only-based method in terms of heterogeneous cancer cell isolation including mesenchymal–like cancer cells. Patient blood samples were employed to assess the clinical application of the present device for examining the patient status based on their CTC heterogeneity. The CTCs captured by both antibodies exhibit considerably varied expression profiles of epithelial protein including cytokeratin and EpCAM. The present device facilitates the isolation of heterogeneous subtypes of CTCs. These undiscovered heterogeneities would be helpful for precise analysis of CTCs to find their underlying meaning in cancer progression.
Recent insights into the development of nanotechnology to detect circulating tumor cells
2016, TrAC - Trends in Analytical Chemistry
Citation Excerpt :
This approach is readily amenable to translation on HbCTC-chips that are currently used for clinical applications, and can also be translated to a range of different device surfaces, from silicon and glass to plastic or paper, to generate low-cost detection devices. A nanoparticle-mediated approach to capture and sort CTCs based on their specific epithelial phenotype was developed by Muhanna et al. [48]. They investigated the phenotypic transition of tumor cells in an animal model and showed the correlation of this transition with tumor progression.
Circulating tumor cells (CTCs) produced from primary tumors act as seeds for metastasis, leading to the majority of cancer-related deaths. Currently, in cancer research, these cells have attracted much attention in studying the process of metastasis. Various studies in the past decade have enlightened the role of CTCs as potential biomarkers in cancer diagnosis and prognosis. As a result, the analysis of CTCs could act as a substitute for characterizing the nature of primary tumors and provide unique insights into the metastatic process. The detection of CTCs in the blood samples of a cancer patient is technically challenging because of the extremely low abundance of CTCs among a large number of other blood cells. Therefore, novel methods for the detection of CTCs are highly recommended. In this feature article, we discuss the recent progress in nanotechnology for the detection of CTCs along with perspectives on future opportunities.
Facile, rapid and efficient isolation of circulating tumor cells using aptamer-targeted magnetic nanoparticles integrated with a microfluidic device
2022, RSC Advances

View all citing articles on Scopus

: This research was sponsored by the Canadian Institutes of Health Research (CIHR), the Ontario Research Fund (ORF), the Canadian Cancer Society Research Institute (CCSRI), CMC Microsystems and the Natural Sciences and Engineering Research Council (NSERC).

¹: Equally contributing authors.

View full text

Feature ArticleNanoparticle-based sorting of circulating tumor cells by epithelial antigen expression during disease progression in an animal model

Abstract

From the Clinical Editor

Graphical abstract

Section snippets

Animal model

Animal study and microfluidic chip design

Discussion

Lancet Oncol

Cell

Ann Oncol

Gynecol Oncol

Crit Rev Oncol Hematol

Clin Breast Cancer

Semin Cell Dev Biol

Trends Biotechnol

Inadequacy of lymph node staging in gastric cancer patients: a population-based study

Ann Surg Oncol

Clinical implications of tumor-cell heterogeneity

N Engl J Med