Shuhuan Hu

Multiparametric Biomechanical and Biochemical Phenotypic Profiling of Single Cancer Cells Using an Elasticity Microcytometer

Deep phenotyping of single cancer cells is of critical importance in the era of precision medicine to advance understanding of relationships between gene mutation and cell phenotype and to elucidate the biological nature of tumor heterogeneity. Existing microfluidic single-cell phenotyping tools, however, are limited to phenotypic measurements of 1-2 selected morphological and physiological features of single cells. Herein a microfluidic elasticity microcytometer is reported for multiparametric biomechanical and biochemical phenotypic profiling of free-floating, live single cancer cells for quantitative, simultaneous characterizations of cell size, cell deformability/stiffness, and surface receptors. The elasticity microcytometer is implemented for measurements and comparisons of four human cell lines with distinct metastatic potentials and derived from different human tissues. An analytical model is developed from first principles for the first time to convert cell deformation and adhesion information of single cancer cells encapsulated inside the elasticity microcytometer to cell deformability/stiffness and surface protein expression. Together, the elasticity microcytometer holds great promise for comprehensive molecular, cellular, and biomechanical phenotypic profiling of live cancer cells at the single cell level, critical for studying intratumor cellular and molecular heterogeneity using low-abundance, clinically relevant human cancer cells.

A microfluidic device for isolation and characterization of transendothelial migrating cancer cells

Transendothelial migration of cancer cells is a critical stage in cancer, including breast cancer, as the migrating cells are generally believed to be highly metastatic. However, it is still challenging for many existing platforms to achieve a fully covering endothelium and to ensure transendothelial migration capability of the extracted cancer cells for analyses with high specificity. Here, we report a microfluidic device containing multiple independent cell collection microchambers underneath an embedded endothelium such that the transendothelial-migrated cells can be selectively collected from only the microchambers with full coverage of an endothelial layer. In this work, we first optimize the pore size of a microfabricated supporting membrane for the endothelium formation. We quantify transendothelial migration rates of a malignant human breast cell type (MDA-MB-231) under different shear stress levels. We investigate characteristics of the migrating cells including morphology, cytoskeletal structures, and migration (speed and persistence). Further implementation of this endothelium-embedded microfluidic device can provide important insights into migration and intracellular characteristics related to cancer metastasis and strategies for effective cancer therapy.

Protein–Substrate Adhesion in Microcontact Printing Regulates Cell Behavior

Microcontact printing (μCP) is widely used to create patterns of biomolecules essential for studies of cell mechanics, migration, and tissue engineering. However, different types of μCPs may create micropatterns with varied protein-substrate adhesion, which may change cell behaviors and pose uncertainty in result interpretation. Here, we characterize two μCP methods for coating extracellular matrix (ECM) proteins (stamp-off and covalent bond) and demonstrate for the first time the important role of protein-substrate adhesion in determining cell behavior. We found that, as compared to cells with weaker traction force (e.g., endothelial cells), cells with strong traction force (e.g., vascular smooth muscle cells) may delaminate the ECM patterns, which reduced cell viability as a result. Importantly, such ECM delamination was observed on patterns by stamp-off but not on the patterns by covalent bonds. Further comparisons of the displacement of the ECM patterns between the normal VSMCs and the force-reduced VSMCs suggested that the cell traction force plays an essential role in this ECM delamination. Together, our results indicated that μCPs with insufficient adhesion may lead to ECM delamination and cause cell death, providing new insight for micropatterning in cell-biomaterial interaction on biointerfaces.

Microfluidic biosensing of viscoelastic properties of normal and cancerous human breast cells

Biomechanical properties have been revealed as potential biomarkers for distinguishing cancer cells from normal cells. In this work, we report a novel technique using a confining microchannel embedded with microelectrodes for biomechanical phenotyping for floating human cells, including one normal breast cell line (MCF-10A) and two breast cancer cell lines (MCF-7 and MDA-MB-231). The floating cells move under a defined pressure profile along the microchannel, in which the cells deform dynamically under compression by the channel sidewalls. We adopt Hertz and Tatara models to convert the deformed cell shapes to cell diameters and transient stress-strain ratios. By further considering the cell viscoelasticity as a Standard Linear Solid (SLS) model, we compute for whole-cell viscosity, and instantaneous and relaxed moduli. Our results show that the selected cell types have significant different viscoelastic properties. Applications of the electrode-embedded confining microchannel can achieve high-throughput, continuous-flow deep phenotyping of rare cells by functionalizing channel side walls with antibodies for both biomechanical and biochemical biomarkers for more comprehensive and promising cell characterization.

Revealing elasticity of largely deformed cells flowing along confining microchannels

Deformability is a hallmark of malignant tumor cells. Characterizing cancer cell deformation can reveal how cancer cell metastasizes through tiny gaps in tissues. However, many previous reports only focus on the cancer cell behaviors under small deformation regimes, which may not be representative for the behaviors under large deformations as in the in vivo metastatic processes. Here, we investigate a wide range of cell elasticity using our recently developed confining microchannel arrays. We develop a relation between the elastic modulus and cell shape under different deformation levels based on a modified contact theory and the hyperelastic Tatara theory. We demonstrate good agreements between the model prediction and experimental results. Strikingly, we discover a clear ‘modulus jump’ of largely deformed cells compared to that of small deformed cells, offering further biomechanical properties of the cells. Likely, such a modulus jump can be considered as a label-free marker reflecting the elasticity of intracellular components including the nucleus during cell translocation in capillaries and tissue constrictions. In essence, we perform cell classification based on the distinct micromechanical properties of four cell lines, i.e. one normal cell line (MCF-10A) and three cancer cell lines (MCF-7, MDA-MB-231 and PC3) and achieved reasonable efficiencies (efficiency >65%). Finally, we study the correlation between large-deformational elasticity and translocation rates of the floating cells in the microchannels. Together, our results demonstrate the quantitative analysis of the biomechanical properties of single floating cells, which provide an additional label-free physical biomarker toward more effective cancer diagnosis.

Characterization of Cytoskeletal Pore Size Using Quantum Dots

Cytoskeletal pore size is a key factor in determining the intracellular molecular transportation, yet it is technically challenging to investigate correlations between the cytoskeletal pore size and cytoplasmic diffusion. Here, we report the application of quantum dots (QD) to measure the cytoskeletal pore size. QDs are first transfected into a cell, leading to different sizes of QD-encapsulating vesicles. The distribution of QD vesicles in cells reflects the cytoskeletal pore size. Further, we can predict the cytosolic viscosity from the measured pore size using the poroelasticity scaling theory. This predicted cytosolic viscosity is verified by measuring Brownian motions of the QDs in cells and transforming the motion characteristics into cytosolic viscosity via the Stocks–Einstein relation. This strategy also provides a practical method for quantifying the cytoskeletal pore sizes. Together, this research demonstrates the efficacy of applying QDs in further studies of intracellular physical properties that play key roles in intracellular molecular transports.

Cell behaviors mediated by the scale of extracellular matrix micro-islands

Extracellular matrix (ECM) plays a critical role in regulating cell behaviors such as proliferation, spreading, migration and differentiation. Here we investigate ECM micro-patterns as a factor of cell-ECM interactions. Different sizes of circular ECM islands were fabricated using the microcontact printing (μCP) technique as an array of cell attachment sites spreading over the substrates. We investigate the scale of ECM islands on their effects on cell morphology and the focal adhesions (FAs) characteristics. Further, we observe morphological changes and cytoskeleton formation during the cell spreading stage of cells on different ECM patterns, revealing that the size and gap distance between the patterns influences the cell spreading as well as the dynamic cytoskeleton architecture significantly.

Large field-of-view super-resolution imaging of endo-cellular structures through micro-beads array

This work presents a super-resolution cell imaging method that uses an array of polystyrene (PS) micro-beads lenses embedded in an elastomer thin film on top of a glass substrate. The micro-beads lens array collects reflected near-field light waves from an objects surface and amplifies the waves to allow the formation of enlarged virtual images of the object. We have shown that object images could be magnified up to at least 5 times of the original scale while having a field-of-view of 6 μm (for a single lens), i.e., visible light images of 200 nm features could be captured using a microscope objective lens of 100x (with overall magnification factor of 1000×). Furthermore, using the micro-beads lens array, NIH/3T3 cell internal structures have been successfully imaged under bright-field conditions, showing a much higher resolution image of the internal structures. In summary, we have shown that micro-beads lens array could resolve sub-diffraction-limit features with an overall field-of-view of at least 5000 μm2. Hence, micro-beads lens array shows promising applications in the rapid imaging of large quantity of biomolecules and internal cell structures with wide field-of-view capability.