Reza M. Mohamadi

Beyond the Capture of Circulating Tumor Cells: Next-Generation Devices and Materials

Over the last decade, significant progress has been made towards the development of approaches that enable the capture of rare circulating tumor cells (CTCs) from the blood of cancer patients, a critical capability for noninvasive tumor profiling. These advances have leveraged new insights in materials chemistry and microfluidics and allowed the capture and enumeration of CTCs with unprecedented sensitivity. However, it has become increasingly clear that simply capturing and counting tumor cells launched into the bloodstream may not provide the information needed to advance our understanding of the biology of these rare cells, or to allow us to better exploit them in medicine. A variety of advances have now emerged demonstrating that more information can be extracted from CTCs with next-generation devices and materials featuring tailored physical and chemical properties. In this Minireview, the last ten years of work in this area will be discussed, with an emphasis on the groundbreaking work of the last five years, during which the focus has moved beyond the simple capture of CTCs and gravitated towards approaches that enable in-depth analysis.

Nanoparticle-Mediated Binning and Profiling of Heterogeneous Circulating Tumor Cell Subpopulations†

The analysis of circulating tumor cells (CTCs) is an important capability that may lead to new approaches for cancer management. CTC capture devices developed to date isolate a bulk population of CTCs and do not differentiate subpopulations that may have varying phenotypes with different levels of clinical relevance. Here, we present a new device for CTC spatial sorting and profiling that sequesters blood-borne tumor cells with different phenotypes into discrete spatial bins. Validation data are presented showing that cancer cell lines with varying surface expression generate different binning profiles within the device. Working with patient blood samples, we obtain profiles that elucidate the heterogeneity of CTC populations present in cancer patients and also report on the status of CTCs within the epithelial-to-mesenchymal transition (EMT).

Highly Specific Electrochemical Analysis of Cancer Cells using Multi‐Nanoparticle Labeling

Circulating tumor cells (CTCs) can be collected noninvasively and provide a wealth of information about tumor phenotype. For this reason, their specific and sensitive detection is of intense interest. Herein, we report a new, chip-based strategy for the automated analysis of cancer cells. The nanoparticle-based, multi-marker approach exploits the direct electrochemical oxidation of metal nanoparticles (MNPs) to report on the presence of specific surface markers. The electrochemical assay allows simultaneous detection of multiple different biomarkers on the surfaces of cancer cells, enabling discrimination between cancer cells and normal blood cells. Through multiplexing, it further enables differentiation among distinct cancer cell types. We showcase the technology by demonstrating the detection of cancer cells spiked into blood samples.

Tracking the dynamics of circulating tumour cell phenotypes using nanoparticle-mediated magnetic ranking

Profiling the heterogeneous phenotypes of rare circulating tumour cells (CTCs) in whole blood is critical to unravelling the complex and dynamic properties of these potential clinical markers. This task is challenging because these cells are present at parts per billion levels among normal blood cells. Here we report a new nanoparticle-enabled method for CTC characterization, called magnetic ranking cytometry, which profiles CTCs on the basis of their surface expression phenotype. We achieve this using a microfluidic chip that successfully processes whole blood samples. The approach classifies CTCs with single-cell resolution in accordance with their expression of phenotypic surface markers, which is read out using magnetic nanoparticles. We deploy this new technique to reveal the dynamic phenotypes of CTCs in unprocessed blood from mice as a function of tumour growth and aggressiveness. We also test magnetic ranking cytometry using blood samples collected from cancer patients.

Aptamer and Antisense-Mediated Two-Dimensional Isolation of Specific Cancer Cell Subpopulations

Cancer cells, and in particular those found circulating in blood, can have widely varying phenotypes and molecular profiles despite a common origin. New methods are needed that can deconvolute the heterogeneity of cancer cells and sort small numbers of cells to aid in the characterization of cancer cell subpopulations. Here, we describe a new molecular approach to capturing cancer cells that isolates subpopulations using two-dimensional sorting. Using aptamer-mediated capture and antisense-triggered release, the new strategy sorts cells according to levels of two different markers and thereby separates them into their corresponding subpopulations. Using a phenotypic assay, we demonstrate that the subpopulations isolated have markedly different properties. This system provides an important new tool for identifying circulating tumor cell subtypes.

In Situ Electrochemical ELISA for Specific Identification of Captured Cancer Cells

Circulating tumor cells (CTCs) are cancer cells disseminated from a tumor into the bloodstream. Their presence in patient blood samples has been associated with metastatic disease. Here, we report a simple system that enables the isolation and detection of these rare cancer cells. By developing a sensitive electrochemical ELISA method integrated within a microfluidic cell capture system, were we able to reliably detect very low levels of cancer cells in whole blood. Our results indicate that the new system provides the clinically relevant specificity and sensitivity needed for a convenient, point-of-need assay for cancer cell counting.

Interrogating Circulating Microsomes and Exosomes Using Metal Nanoparticles.

A chip-based approach for electrochemical characterization and detection of microsomes and exosomes based on direct electro-oxidation of metal nanoparticles (MNPs) that specifically recognize surface markers of these vesicles is reported. It is found that exosomes and microsomes derived from prostate cancer cells can be identified by their surface proteins EpCAM and PSMA, suggesting the potential of exosomes and microsomes for use as diagnostic biomarkers.

Sample-to-Answer Isolation and mRNA Profiling of Circulating Tumor Cells

The isolation and rapid molecular characterization of circulating tumor cells (CTCs) from a liquid biopsy could enable the convenient and effective characterization of the state and aggressiveness of cancerous tumors. Existing technologies enumerate CTCs using immunostaining; however, these approaches are slow, labor-intensive, and often fail to enable further genetic characterization of CTCs. Here, we report on an integrated circuit that combines the capture of CTCs with the profiling of their gene expression signatures. Specifically, we use a velocity valley chip to efficiently capture magnetic nanoparticle-bound CTCs, which are then directly analyzed for their gene expression profiles using nanostructured microelectrode biosensors. CTCs are captured with 97% efficiency from 2 mL of whole blood, yielding a 500-fold concentration within 1 h. We show efficient capture of as few as 2 cancer cells/(mL of blood) and demonstrate that the gene expression module accurately profiles the expression of prostate-specific genes in CTCs captured from whole blood. This advance provides the first sample-to-answer solution for gene-based testing of CTCs. The approach was successfully validated using samples collected from prostate cancer patients: both CTCs and prostate-specific antigen (PSA) mRNA sequences were detected in all cancer patient samples and not in the healthy controls.

Profiling Functional and Biochemical Phenotypes of Circulating Tumor Cells Using a Two-Dimensional Sorting Device

During cancer progression, tumors shed circulating tumor cells (CTCs) into the bloodstream. CTCs that originate from the same primary tumor can have heterogeneous phenotypes and, while some CTCs possess benign properties, others have high metastatic potential. Deconstructing the heterogeneity of CTCs is challenging and new methods are needed that can sort small numbers of cancer cells according to their phenotypic properties. Here we describe a new microfluidic approach that profiles, along two independent phenotypic axes, the behavior of heterogeneous cell subpopulations. Cancer cells are first profiled according to expression of a surface marker using a nanoparticle-enabled approach. Along the second dimension, these subsets are further separated into subpopulations corresponding to migration profiles generated in response to a chemotactic agent. We deploy this new technique and find a strong correlation between the surface expression and migration potential of CTCs present in blood from mice with xenografted tumors. This system provides an important new means to characterize functional diversity in circulating tumor cells.

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

UNLABELLED 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.