A comparison of methods to assess cell mechanical properties.

The mechanical properties of cells influence their cellular and subcellular functions, including cell adhesion, migration, polarization, and differentiation, as well as organelle organization and trafficking inside the cytoplasm. Yet reported values of cell stiffness and viscosity vary substantially, which suggests differences in how the results of different methods are obtained or analyzed by different groups. To address this issue and illustrate the complementarity of certain approaches, here we present, analyze, and critically compare measurements obtained by means of some of the most widely used methods for cell mechanics: atomic force microscopy, magnetic twisting cytometry, particle-tracking microrheology, parallel-plate rheometry, cell monolayer rheology, and optical stretching. These measurements highlight how elastic and viscous moduli of MCF-7 breast cancer cells can vary 1,000-fold and 100-fold, respectively. We discuss the sources of these variations, including the level of applied mechanical stress, the rate of deformation, the geometry of the probe, the location probed in the cell, and the extracellular microenvironment.

Simultaneously defining cell phenotypes, cell cycle, and chromatin modifications at single-cell resolution.

Heterogeneity of cellular phenotypes in asynchronous cell populations placed in the same biochemical and biophysical environment may depend on cell cycle and chromatin modifications; however, no current method can measure these properties at single-cell resolution simultaneously and in situ. Here, we develop, test, and validate a new microscopy assay that rapidly quantifies global acetylation on histone H3 and measures a wide range of cell and nuclear properties, including cell and nuclear morphology descriptors, cell-cycle phase, and F-actin content of thousands of cells simultaneously, without cell detachment from their substrate, at single-cell resolution. These measurements show that isogenic, isotypic cells of identical DNA content and the same cell-cycle phase can still display large variations in H3 acetylation and that these variations predict specific phenotypic variations, in particular, nuclear size and actin cytoskeleton content, but not cell shape. The dependence of cell and nuclear properties on cell-cycle phase is assessed without artifact-prone cell synchronization. To further demonstrate its versatility, this assay is used to quantify the complex interplay among cell cycle, epigenetic modifications, and phenotypic variations following pharmacological treatments affecting DNA integrity, cell cycle, and inhibiting chromatin-modifying enzymes.

SMRT analysis of MTOC and nuclear positioning reveals the role of EB1 and LIC1 in single-cell polarization.

In several migratory cells, the microtubule-organizing center (MTOC) is repositioned between the leading edge and nucleus, creating a polarized morphology. Although our understanding of polarization has progressed as a result of various scratch-wound and cell migration studies, variations in culture conditions required for such assays have prevented a unified understanding of the intricacies of MTOC and nucleus positioning that result in cell polarization. Here, we employ a new SMRT (for sparse, monolayer, round, triangular) analysis that uses a universal coordinate system based on cell centroid to examine the pathways regulating MTOC and nuclear positions in cells plated in a variety of conditions. We find that MTOC and nucleus positioning are crucially and independently affected by cell shape and confluence; MTOC off-centering correlates with the polarization of single cells; acto-myosin contractility and microtubule dynamics are required for single-cell polarization; and end binding protein 1 and light intermediate chain 1, but not Par3 and light intermediate chain 2, are required for single-cell polarization and directional cell motility. Using various cellular geometries and conditions, we implement a systematic and reproducible approach to identify regulators of MTOC and nucleus positioning that depend on extracellular guidance cues.

Divergent roles of CD44 and carcinoembryonic antigen in colon cancer metastasis.

After separating from a primary tumor, metastasizing cells enter the circulatory system and interact with host cells before lodging in secondary organs. Previous studies have implicated the surface glycoproteins CD44 and carcinoembryonic antigen (CEA) in adhesion, migration, and invasion, suggesting that they may influence metastatic progression. To elucidate the role of these multifunctional molecules while avoiding the potential drawbacks of ectopic expression or monoclonal antibody treatments, we silenced the expression of CD44 and/or CEA in LS174T colon carcinoma cells and analyzed their ability to metastasize in 2 independent mouse models. Quantitative PCR revealed that CD44 knockdown increased lung and liver metastasis >10-fold, while metastasis was decreased by >50% following CEA knockdown. These findings were corroborated by in vitro experiments assessing the metastatic potential of LS174T cells. Cell migration was decreased as a result of silencing CEA but was enhanced in CD44-knockdown cells. In addition, CD44 silencing promoted homotypic aggregation of LS147T cells, a phenotype that was reversed by additional CEA knockdown. Finally, CD44-knockdown cells exhibited greater mechanical compliance than control cells, a property that correlates with increased metastatic potential. Collectively, these data indicate that CEA, but not CD44, is a viable target for therapeutics aimed at curbing colon carcinoma metastasis.

A robust unsupervised machine-learning method to quantify the morphological heterogeneity of cells and nuclei.

Cell morphology encodes essential information on many underlying biological processes. It is commonly used by clinicians and researchers in the study, diagnosis, prognosis, and treatment of human diseases. Quantification of cell morphology has seen tremendous advances in recent years. However, effectively defining morphological shapes and evaluating the extent of morphological heterogeneity within cell populations remain challenging. Here we present a protocol and software for the analysis of cell and nuclear morphology from fluorescence or bright-field images using the VAMPIRE algorithm ( https://github.com/kukionfr/VAMPIRE_open ). This algorithm enables the profiling and classification of cells into shape modes based on equidistant points along cell and nuclear contours. Examining the distributions of cell morphologies across automatically identified shape modes provides an effective visualization scheme that relates cell shapes to cellular subtypes based on endogenous and exogenous cellular conditions. In addition, these shape mode distributions offer a direct and quantitative way to measure the extent of morphological heterogeneity within cell populations. This protocol is highly automated and fast, with the ability to quantify the morphologies from 2D projections of cells seeded both on 2D substrates or embedded within 3D microenvironments, such as hydrogels and tissues. The complete analysis pipeline can be completed within 60 minutes for a dataset of ~20,000 cells/2,400 images.

Integration of Fluorescence Detection and Image-Based Automated Counting Increases Speed, Sensitivity, and Robustness of Plaque Assays.

Plaque assays are used to measure the infectious titer of viral samples. These assays are multi-day and low-throughput and may be subject to analyst variability from biased or subjective manual plaque counting. Typically, on day 1, cells are adhered to plates overnight. On day 2, cells are infected with virus. After 3 additional days, plaques are fixed, stained with a horseradish peroxidase (HRP)-conjugated antibody and a HRP substrate, and counted by eye. Manual-based visual counting of plaques is time-consuming and laborious and may be subject to variability between analysts. Also, the assay must proceed for several days to allow the plaques to increase to sufficiently large sizes for manual identification. Here, we integrate fluorescent detection and automated plaque counting to increase the sensitivity and speed of the assay. First, we stain plaques with a fluorescent-labeled antibody. Second, we implement a plate-based cell imager to perform non-biased, non-subjective plaque counting. The integration of these two technologies decreases the assay length by 40%, from 5 days to 3 days, because plaque size, plaque signal to noise, and manual visualization are no longer limiting. This optimized plaque assay is sensitive, fast, and robust and expands the throughput and usage of this method for measuring plaque formation.

Analytical Strategies for Quantification of Adeno-Associated Virus Empty Capsids to Support Process Development.

Recombinant adeno-associated virus (rAAV)-mediated gene therapy is a fast-evolving field in the biotechnology industry. One of the major challenges in developing a purification process for AAV gene therapy is establishing an effective yet scalable method to remove empty capsids, or viral vectors lacking the therapeutic gene, from full capsids-viral product containing the therapeutic sequence. Several analytical methods that can quantify the empty-to-full capsid ratio have been reported in the literature. However, as samples can vary widely in viral titer, buffer matrix, and the relative level of empty capsids, understanding the specifications and limitations of different analytical methods is critical to providing appropriate support to facilitate process development. In this study, we developed a novel anion-exchange high-performance liquid chromatography assay to determine the empty-to-full capsid ratio of rAAV samples. The newly developed method demonstrated good comparability with both the transmission electron microscopy and analytical ultracentrifugation methods used in empty-to-full capsid ratio quantification, while providing much higher assay throughput and reducing the minimum sample concentration requirement to 2.7E11 viral genomes/mL.

Swelling the micelle core surrounding single-walled carbon nanotubes with water-immiscible organic solvents.

Solvatochromic shifts in the absorbance and fluorescence spectra are observed when surfactant-stabilized aqueous single-walled carbon nanotube (SWNT) suspensions are mixed with immiscible organic solvents. When aqueous surfactant-suspended SWNTs are mixed with o-dichlorobenzene, the spectra closely match the peaks for SWNTs dispersed in only pure o-dichlorobenzene. These spectral changes suggest that the hydrophobic region of the micelle surrounding SWNTs swells with the organic solvent when mixed. The solvatochromic shifts of the aqueous SWNT suspensions are reversible once the solvent evaporates. However, some surfactant-solvent systems show permanent changes to the fluorescence emission intensity after exposure to the organic solvent. The intensity of some large diameter SWNT (n, m) types increase by more than 175%. These differences are attributed to surfactant reorganization, which can improve nanotube coverage, resulting in decreased exposure to quenching mechanisms from the aqueous phase.

Improving the effectiveness of interfacial trapping in removing single-walled carbon nanotube bundles.

Single-walled carbon nanotube (SWNT) bundles are selectively removed from an aqueous dispersion containing individually suspended carbon nanotubes coated with gum Arabic via interfacial trapping. The suspensions are characterized with absorbance, fluorescence, and Raman spectroscopy as well as atomic force microscopy (AFM) and rheology. The resulting aqueous suspensions have better dispersion quality after interfacial trapping and can be further improved by altering the processing conditions. A two-step extraction process offers a simple and fast approach to preparing high-quality dispersions of individual SWNTs comparable to ultracentrifugation. Partitioning of SWNTs to the liquid-liquid interface is described by free energy changes. SWNT bundles prefer to reside at the interface over individually suspended SWNTs because of greater free energy changes.

Isomeric control of the mechanical properties of supramolecular filament hydrogels.

Supramolecular filament hydrogels are an emerging class of biomaterials that hold great promise for regenerative medicine, tissue engineering, and drug delivery. However, fine-tuning of their bulk mechanical properties at the molecular level without altering their network structures remains a significant challenge. Here we report an isomeric strategy to construct amphiphilic peptides through the conjugation of isomeric hydrocarbons to influence the local viscoelastic properties of their resulting supramolecular hydrogels. In this case, the packing requirements of the chosen isomeric hydrocarbons within the supramolecular filaments are dictated by their atomic arrangements at the molecular and intermolecular levels. Atomistic molecular dynamics simulations suggest that this design strategy can subtly alter the molecular packing at the interface between the peptide domain and the hydrophobic core of the supramolecular assemblies, without changing both the filament width and morphology. Our results from wide-angle X-ray scattering and molecular simulations further confirm that alterations to the intermolecular packing at the interface impact the strength and degree of hydrogen bonding within the peptide domains. This subtle difference in the isomeric hydrocarbon design and their consequent packing difference led to variations in the persistence length of the individual supramolecular filaments. Microrheological analysis reveals that this difference in filament stiffness enables the fine-tuning of the mechanical properties of the hydrogel at the macroscopic scale. We believe that this isomeric platform provides an innovative method to tune the local viscoelastic properties of supramolecular polymeric hydrogels without necessarily altering their network structures.

Evolution of cellular morpho-phenotypes in cancer metastasis.

Intratumoral heterogeneity greatly complicates the study of molecular mechanisms driving cancer progression and our ability to predict patient outcomes. Here we have developed an automated high-throughput cell-imaging platform (htCIP) that allows us to extract high-content information about individual cells, including cell morphology, molecular content and local cell density at single-cell resolution. We further develop a comprehensive visually-aided morpho-phenotyping recognition (VAMPIRE) tool to analyze irregular cellular and nuclear shapes in both 2D and 3D microenvironments. VAMPIRE analysis of ~39,000 cells from 13 previously sequenced patient-derived pancreatic cancer samples indicate that metastasized cells present significantly lower heterogeneity than primary tumor cells. We found the same morphological signature for metastasis for a cohort of 10 breast cancer cell lines. We further decipher the relative contributions to heterogeneity from cell cycle, cell-cell contact, cell stochasticity and heritable morphological variations.

Functional interplay between the cell cycle and cell phenotypes.

Cell cycle distribution of adherent cells is typically assessed using flow cytometry, which precludes the measurements of many cell properties and their cycle phase in the same environment. Here we develop and validate a microscopy system to quantitatively analyze the cell-cycle phase of thousands of adherent cells and their associated cell properties simultaneously. This assay demonstrates that population-averaged cell phenotypes can be written as a linear combination of cell-cycle fractions and phase-dependent phenotypes. By perturbing the cell cycle through inhibition of cell-cycle regulators or changing nuclear morphology by depletion of structural proteins, our results reveal that cell cycle regulators and structural proteins can significantly interfere with each other's prima facie functions. This study introduces a high-throughput method to simultaneously measure the cell cycle and phenotypes at single-cell resolution, which reveals a complex functional interplay between the cell cycle and cell phenotypes.

A physical sciences network characterization of non-tumorigenic and metastatic cells.

To investigate the transition from non-cancerous to metastatic from a physical sciences perspective, the Physical Sciences-Oncology Centers (PS-OC) Network performed molecular and biophysical comparative studies of the non-tumorigenic MCF-10A and metastatic MDA-MB-231 breast epithelial cell lines, commonly used as models of cancer metastasis. Experiments were performed in 20 laboratories from 12 PS-OCs. Each laboratory was supplied with identical aliquots and common reagents and culture protocols. Analyses of these measurements revealed dramatic differences in their mechanics, migration, adhesion, oxygen response, and proteomic profiles. Model-based multi-omics approaches identified key differences between these cells' regulatory networks involved in morphology and survival. These results provide a multifaceted description of cellular parameters of two widely used cell lines and demonstrate the value of the PS-OC Network approach for integration of diverse experimental observations to elucidate the phenotypes associated with cancer metastasis.

High-throughput ballistic injection nanorheology to measure cell mechanics.

High-throughput ballistic injection nanorheology is a method for the quantitative study of cell mechanics. Cell mechanics are measured by ballistic injection of submicron particles into the cytoplasm of living cells and tracking the spontaneous displacement of the particles at high spatial resolution. The trajectories of the cytoplasm-embedded particles are transformed into mean-squared displacements, which are subsequently transformed into frequency-dependent viscoelastic moduli and time-dependent creep compliance of the cytoplasm. This method allows for the study of a wide range of cellular conditions, including cells inside a 3D matrix, cell subjected to shear flows and biochemical stimuli, and cells in a live animal. Ballistic injection lasts <1 min and is followed by overnight incubation. Multiple particle tracking for one cell lasts <1 min. Forty cells can be examined in <1 h.

Coating individual single-walled carbon nanotubes with nylon 6,10 through emulsion polymerization.

Solvent microenvironments are formed around individual single-walled carbon nanotubes (SWNTs) by mixing SWNT suspensions with water-immiscible organic solvents. These microenvironments are used to encapsulate the SWNTs with the monomer sebacoyl chloride. Hexamethylene diamine is then injected into the aqueous phase so the formation of nylon 6,10 is restricted to the interface between the microenvironment and water. This emulsion polymerization process results in uniform coatings of nylon 6,10 around individual SWNTs. The nylon-coated SWNTs remain dispersed in the aqueous phase and are highly luminescent at pH values ranging from 3 to 12. This emulsion polymerization method provides a general approach to coat nanotubes with various polymers.