Sharif Ahmed

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.

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.

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.

Multifunctional quantum dot DNA hydrogels

Biotemplated nanomaterials offer versatile functionality for multimodal imaging, biosensing, and drug delivery. There remains an unmet need for traceable and biocompatible nanomaterials that can be synthesized in a precisely controllable manner. Here, we report self-assembled quantum dot DNA hydrogels that exhibit both size and spectral tunability. We successfully incorporate DNA-templated quantum dots with high quantum yield, long-term photostability, and low cytotoxicity into a hydrogel network in a single step. By leveraging DNA-guided interactions, we introduce multifunctionality for a variety of applications, including enzyme-responsive drug delivery and cell-specific targeting. We report that quantum dot DNA hydrogels can be used for delivery of doxorubicin, an anticancer drug, to increase potency 9-fold against cancer cells. This approach also demonstrated high biocompatibility, trackability, and in vivo therapeutic efficacy in mice bearing xenografted breast cancer tumors. This work paves the way for the development of new tunable biotemplated nanomaterials with multiple synergistic functionalities for biomedical applications.The development of nanomaterials for imaging and drug delivery has been of great interest to the field. Here, the authors synthesized multifunctional enzyme-responsive hydrogels with self-assembling quantum dots for nucleic acid and drug delivery as well as having imaging capability.

Isolation of Phenotypically Distinct Cancer Cells Using Nanoparticle-Mediated Sorting

Isolating subpopulations of heterogeneous cancer cells is an important capability for the meaningful characterization of circulating tumor cells at different stages of tumor progression and during the epithelial-to-mesenchymal transition. Here, we present a microfluidic device that can separate phenotypically distinct subpopulations of cancer cells. Magnetic nanoparticles coated with antibodies against the epithelial cell adhesion molecule (EpCAM) are used to separate breast cancer cells in the microfluidic platform. Cells are sorted into different zones on the basis of the levels of EpCAM expression, which enables the detection of cells that are losing epithelial character and becoming more mesenchymal. The phenotypic properties of the isolated cells with low and high EpCAM are then assessed using matrix-coated surfaces for collagen uptake analysis, and an NAD(P)H assay that assesses metabolic activity. We show that low-EpCAM expressing cells have higher collagen uptake and higher folate-induced NAD(P)H responses compared to those of high-EpCAM expressing cells. In addition, we tested SKBR3 cancer cells undergoing chemically induced hypoxia. The induced cells have reduced expression of EpCAM, and we find that these cells have higher collagen uptake and NAD(P)H metabolism relative to noninduced cells. This work demonstrates that nanoparticle-mediated binning facilitates the isolation of functionally distinct cell subpopulations and allows surface marker expression to be associated with invasiveness, including collagen uptake and metabolic activity.