Characterizing deformability and surface friction of cancer cells.

Metastasis requires the penetration of cancer cells through tight spaces, which is mediated by the physical properties of the cells as well as their interactions with the confined environment. Various microfluidic approaches have been devised to mimic traversal in vitro by measuring the time required for cells to pass through a constriction. Although a cell's passage time is expected to depend on its deformability, measurements from existing approaches are confounded by a cell's size and its frictional properties with the channel wall. Here, we introduce a device that enables the precise measurement of (i) the size of a single cell, given by its buoyant mass, (ii) the velocity of the cell entering a constricted microchannel (entry velocity), and (iii) the velocity of the cell as it transits through the constriction (transit velocity). Changing the deformability of the cell by perturbing its cytoskeleton primarily alters the entry velocity, whereas changing the surface friction by immobilizing positive charges on the constriction's walls primarily alters the transit velocity, indicating that these parameters can give insight into the factors affecting the passage of each cell. When accounting for cell buoyant mass, we find that cells possessing higher metastatic potential exhibit faster entry velocities than cells with lower metastatic potential. We additionally find that some cell types with higher metastatic potential exhibit greater than expected changes in transit velocities, suggesting that not only the increased deformability but reduced friction may be a factor in enabling invasive cancer cells to efficiently squeeze through tight spaces.

Characterizing Cellular Biophysical Responses to Stress by Relating Density, Deformability, and Size.

Cellular physical properties are important indicators of specific cell states. Although changes in individual biophysical parameters, such as cell size, density, and deformability, during cellular processes have been investigated in great detail, relatively little is known about how they are related. Here, we use a suspended microchannel resonator (SMR) to measure single-cell density, volume, and passage time through a narrow constriction of populations of cells subjected to a variety of environmental stresses. Osmotic stress significantly affects density and volume, as previously shown. In contrast to density and volume, the effect of an osmotic challenge on passage time is relatively small. Deformability, as determined by comparing passage times for cells with similar volume, exhibits a strong dependence on osmolarity, indicating that passage time alone does not always provide a meaningful proxy for deformability. Finally, we find that protein synthesis inhibition, cell-cycle arrest, protein kinase inhibition, and cytoskeletal disruption result in unexpected relationships among deformability, density, and volume. Taken together, our results suggest that by measuring multiple biophysical parameters, one can detect unique characteristics that more specifically reflect cellular behaviors.

Correlation of Optical and Automated Patch Clamp Electrophysiology for Identification of NaV1.7 Inhibitors.

The voltage-gated sodium channel Nav1.7 is a genetically validated target for pain; pharmacological blockers are promising as a new class of nonaddictive therapeutics. The search for Nav1.7 subtype selective inhibitors requires a reliable, scalable, and sensitive assay. Previously, we developed an all-optical electrophysiology (Optopatch) Spiking HEK platform to study activity-dependent modulation of Nav1.7 in a format compatible with high-throughput screening. In this study, we benchmarked the Optopatch Spiking HEK assay with an existing validated automated electrophysiology assay on the IonWorks Barracuda (IWB) platform. In a pilot screen of 3520 compounds, which included compound plates from a random library as well as compound plates enriched for Nav1.7 inhibitors, the Optopatch Spiking HEK assay identified 174 hits, of which 143 were confirmed by IWB. The Optopatch Spiking HEK assay maintained the high reliability afforded by traditional fluorescent assays and further demonstrated comparable sensitivity to IWB measurements. We speculate that the Optopatch assay could provide an affordable high-throughput screening platform to identify novel Nav1.7 subtype selective inhibitors with diverse mechanisms of action, if coupled with a multiwell parallel optogenetic recording instrument.

Microfluidic platform for characterizing TCR-pMHC interactions.

The physical characteristics of the T cell receptor (TCR)-peptide-major histocompatibility complex (pMHC) interaction are known to play a central role in determining T cell function in the initial stages of the adaptive immune response. State-of-the-art assays can probe the kinetics of this interaction with single-molecular-bond resolution, but this precision typically comes at the cost of low throughput, since the complexity of these measurements largely precludes "scaling up." Here, we explore the feasibility of detecting specific TCR-pMHC interactions by flowing T cells past immobilized pMHC and measuring the reduction in cell speed due to the mechanical force of the receptor-ligand interaction. To test this new fluidic measurement modality, we fabricated a microfluidic device in which pMHC-coated beads are immobilized in hydrodynamic traps along the length of a serpentine channel. As T cells flow past the immobilized beads, their change in speed is tracked via microscopy. We validated this approach using two model systems: primary CD8+ T cells from an OT-1 TCR transgenic mouse with beads conjugated with H-2Kb:SIINFEKL, and Jurkat T cells with beads conjugated with anti-CD3 and anti-CD28 antibodies.

Amino Acids Rather than Glucose Account for the Majority of Cell Mass in Proliferating Mammalian Cells.

Cells must duplicate their mass in order to proliferate. Glucose and glutamine are the major nutrients consumed by proliferating mammalian cells, but the extent to which these and other nutrients contribute to cell mass is unknown. We quantified the fraction of cell mass derived from different nutrients and found that the majority of carbon mass in cells is derived from other amino acids, which are consumed at much lower rates than glucose and glutamine. While glucose carbon has diverse fates, glutamine contributes most to protein, suggesting that glutamine's ability to replenish tricarboxylic acid cycle intermediates (anaplerosis) is primarily used for amino acid biosynthesis. These findings demonstrate that rates of nutrient consumption are indirectly associated with mass accumulation and suggest that high rates of glucose and glutamine consumption support rapid cell proliferation beyond providing carbon for biosynthesis.

Biophysical changes reduce energetic demand in growth factor-deprived lymphocytes.

Cytokine regulation of lymphocyte growth and proliferation is essential for matching nutrient consumption with cell state. Here, we examine how cellular biophysical changes that occur immediately after growth factor depletion promote adaptation to reduced nutrient uptake. After growth factor withdrawal, nutrient uptake decreases, leading to apoptosis. Bcl-xL expression prevents cell death, with autophagy facilitating long-term cell survival. However, autophagy induction is slow relative to the reduction of nutrient uptake, suggesting that cells must engage additional adaptive mechanisms to respond initially to growth factor depletion. We describe an acute biophysical response to growth factor withdrawal, characterized by a simultaneous decrease in cell volume and increase in cell density, which occurs before autophagy initiation and is observed in both FL5.12 Bcl-xL cells depleted of IL-3 and primary CD8(+) T cells depleted of IL-2 that are differentiating toward memory cells. The response reduces cell surface area to minimize energy expenditure while conserving biomass, suggesting that the biophysical properties of cells can be regulated to promote survival under conditions of nutrient stress.

Deformability of Tumor Cells versus Blood Cells.

The potential for circulating tumor cells (CTCs) to elucidate the process of cancer metastasis and inform clinical decision-making has made their isolation of great importance. However, CTCs are rare in the blood, and universal properties with which to identify them remain elusive. As technological advancements have made single-cell deformability measurements increasingly routine, the assessment of physical distinctions between tumor cells and blood cells may provide insight into the feasibility of deformability-based methods for identifying CTCs in patient blood. To this end, we present an initial study assessing deformability differences between tumor cells and blood cells, indicated by the length of time required for them to pass through a microfluidic constriction. Here, we demonstrate that deformability changes in tumor cells that have undergone phenotypic shifts are small compared to differences between tumor cell lines and blood cells. Additionally, in a syngeneic mouse tumor model, cells that are able to exit a tumor and enter circulation are not required to be more deformable than the cells that were first injected into the mouse. However, a limited study of metastatic prostate cancer patients provides evidence that some CTCs may be more mechanically similar to blood cells than to typical tumor cell lines.

Measuring single cell mass, volume, and density with dual suspended microchannel resonators.

Cell size, measured as either volume or mass, is a fundamental indicator of cell state. Far more tightly regulated than size is density, the ratio between mass and volume, which can be used to distinguish between cell populations even when volume and mass appear to remain constant. Here we expand upon a previous method for measuring cell density involving a suspended microchannel resonator (SMR). We introduce a new device, the dual SMR, as a high-precision instrument for measuring single-cell mass, volume, and density using two resonators connected by a serpentine fluidic channel. The dual SMR designs considered herein demonstrate the critical role of channel geometry in ensuring proper mixing and damping of pressure fluctuations in microfluidic systems designed for precision measurement. We use the dual SMR to compare the physical properties of two well-known cancer cell lines: human lung cancer cell H1650 and mouse lymphoblastic leukemia cell line L1210.

Intracellular water exchange for measuring the dry mass, water mass and changes in chemical composition of living cells.

We present a method for direct non-optical quantification of dry mass, dry density and water mass of single living cells in suspension. Dry mass and dry density are obtained simultaneously by measuring a cell's buoyant mass sequentially in an H2O-based fluid and a D2O-based fluid. Rapid exchange of intracellular H2O for D2O renders the cell's water content neutrally buoyant in both measurements, and thus the paired measurements yield the mass and density of the cell's dry material alone. Utilizing this same property of rapid water exchange, we also demonstrate the quantification of intracellular water mass. In a population of E. coli, we paired these measurements to estimate the percent dry weight by mass and volume. We then focused on cellular dry density - the average density of all cellular biomolecules, weighted by their relative abundances. Given that densities vary across biomolecule types (RNA, DNA, protein), we investigated whether we could detect changes in biomolecular composition in bacteria, fungi, and mammalian cells. In E. coli, and S. cerevisiae, dry density increases from stationary to exponential phase, consistent with previously known increases in the RNA/protein ratio from up-regulated ribosome production. For mammalian cells, changes in growth conditions cause substantial shifts in dry density, suggesting concurrent changes in the protein, nucleic acid and lipid content of the cell.