Ian Y. Wong

Label-free tracking of adherent cell migration could enable important insights into biological processes such as tissue repair, inflammatory response, or cancer progression. Nevertheless, visualizing unlabeled animal cells using optical microscopy remains challenging due to low contrast as well as frequent changes in cell shape and number. A promising alternative uses electrical capacitance measurements, which are sensitive to cell adhesion to electrode surfaces. However, prior examples often utilized electrodes with areas larger than single cells, resulting in averaged readouts over multiple cells. Here, we demonstrate label-free, live-cell tracking using a capacitance sensor array with more than 1 million pixels on a 10 micron pitch across an area larger than 1 square centimeter. We show that single cell morphology can be clearly segmented, and then used to reconstruct migration and proliferation dynamics using optical flow. We further track the spreading of multicellular spheroids, revealing fast-moving peripheral regions led by a collective leader cell “front.” Finally, we demonstrate label-free imaging of millimeter-scale honeycomb-shaped tissues without the multi-image stitching often required for conventional microscopy. We utilize mutual capacitance measurements with electrically-programmable electrode spacing to reconstruct topographical features of these engineered tissues. Overall, CMOS capacitance imaging arrays enables label-free imaging spanning from single cells to large tissues, in a portable and scalable format for settings where optical microscopy may be difficult to access.

The epithelial-mesenchymal transition (EMT) alters cell-cell interactions to facilitate collective or individual migration during embryonic development, wound repair, or tumor invasion. Epithelial cells are typically cohesive and stationary while mesenchymal cells are individually dispersed and motile. Additional “partial” EMT states are thought to occur with distinct adhesive and migratory behaviors, but these functional phenotypes are poorly understood. Here, we show that cells treated with moderate TGF- β concentration exhibit collective migration that is fast and directionally persistent despite heterogeneity in epithelial, partial, and mesenchymal states. We find cells coordinate their motility by reorienting in similar directions after transient contacts, a distinct “collision guidance” mechanism that differs from epithelial arrest or mesenchymal repulsion. Moreover, partial EMT cells sustain collision guidance when interacting with epithelial or mesenchymal cells, which otherwise have increased tendency to repel. We corroborate these experimental observations with a computational model using self-propelled interacting particles that align their motion or repel upon contact. Finally, we show that partial EMT enables tissue monolayer fronts to overwhelm and displace monolayers of other cell types after collision. Overall, these results reveal that partial EMT promotes coherent and emergent behaviors that bridge from cell to tissue length scales, with potential implications for shaping epithelial tissue formation, regeneration, or disorganization.

Cell migration and morphology are fundamental to various biological processes, including tissue development, wound healing, and cancer progression. In this study, we investigated the migration and morphological characteristics of HeLa cells cultured on different extracellular matrix (ECM) components—laminin (Ln), collagen 1 (Col1), and collagen 3 (Col3)—using machine learning-based image analysis. Cells on Ln exhibited significantly higher motility, increased turning angles, and reduced directional persistence compared to those on Col1 and Col3. Morphologically, these cells showed larger areas and higher solidity, suggesting a distinct attachment pattern compared to those on collagens. In addition, principal component analysis (PCA) effectively classified cells based on motility parameters, with cells on Ln forming a clearly separate cluster in PCA space. Further biological assays demonstrated that cells on Ln exhibited increased cell-cell interactions and smaller but more numerous focal adhesions, indicating that Ln promotes enhanced migratory plasticity and intercellular interactions. These biological factors were identified as key contributors to the distinctions observed in PCA classification. This study highlights the differential effects of ECM components on cellular behavior and demonstrates the value of integrating machine learning-based quantitative analysis with biological interpretation to advance our understanding of cell-microenvironment interactions.

Coordinated cell rotation along a curved matrix interface can sculpt epithelial tissues into spherical morphologies. Subsequently, radially oriented invasion of multicellular strands or branches can occur by local remodelling of the confining matrix. These symmetry-breaking transitions emerge from the dynamic reciprocity between cells and matrix but remain poorly understood. Here we show that epithelial cell spheroids collectively transition from circumferential orbiting to radial invasion via bidirectional interactions with the surrounding matrix curvature. Initially, spheroids exhibit an ellipsoidal shape but become rounded as orbiting occurs. In turn, orbiting along sharper curvature results in locally stronger contractile tractions, which gradually align collagen fibres in the radial direction. Thus, the initially elongated morphology primes the matrix towards subsequent invasion of two to four strands that are roughly aligned with its major axis. We then show that orbiting can be arrested and invasion can be reversed using osmotic pressure. We also investigate coordinated orbiting in mosaic spheroids, showing that a small fraction of cells with weakened cell–cell adhesions can impede collective orbiting but still invade into the matrix. This work elucidates how symmetry breaking in tissue morphogenesis is governed by the interplay of collective migration and the local curvature of the cell–matrix interface, with relevance for embryonic development and tumour progression.

Animal cells direct their migration in response to diverse microenvironmental cues during developmental, physiological, and pathological processes. Emerging biomaterial and microfluidic technologies enable exquisite control over these external signals to manipulate cell migration, facilitating new fundamental insights and biomedical applications. Here, we highlight recent advances in cell migration assays, with particular emphasis on collective and individual epithelial invasion. First, we consider cell migration in confined geometries, either through micropatterned topographies or microchannels. Next, we address cell invasion in 3D matrix, including both natural and synthetic hydrogels. We then discuss microfluidic devices for applying flow and gradients. Finally, we consider emerging technologies and the potential for bench to bedside translation in cancer biology.

A one megapixel CMOS capacitance sensor with 10 micron pixels tracks adherent migrating cells and tissues across one square centimeter.

Animal cells can sense and "remember" the stiffness of their extracellular environment, resulting in sustained changes in form and function. Such "mechanical memory" has been previously explored using individual cells and attributed to epigenetic changes and transcriptional activity. However, it is unclear whether such memory is retained across collective cells. Here, we report that collective cells sustain mechanical memory through self-organized actin-CK18 networks spanning multiple cell lengths, even under dramatically changing mechanical environments, such as those encountered during cancer metastasis. As a case study, we modeled ovarian cancer metastasis and found that cells initially cultured on different stiffness retained distinct migratory phenotypes throughout the environmental transitions of the metastasis model. Notably, soft-primed cells, in particular, demonstrated stronger cell-cell adhesions than stiff-primed cells. Upon aggregation into multicellular spheroids, mimicking malignant spheroids found in patient ascites, the soft-primed spheroids exclusively developed a dense cage-like supracellular actin-CK18 structure at their peripheral surfaces. Furthermore, these soft-primed spheroids exhibited impeded collective invasion, instead becoming confined by the long-lasting cytoskeletal cage. Inhibition of gap junctions attenuated the formation of cytoskeletal cages, indicating that dynamic intercellular communication via gap junctions is essential for maintaining collective mechanical memory. This work demonstrates a collective mechanism of mechanical memory that is not solely dependent on epigenetic and transcriptional activation, advancing our understanding of the elevated metastatic potential of tumor cell clusters originating in stiffened matrices.

During a variety of physiological and pathological processes, such as development, wound healing, and tumor progression, epithelial cells collectively invade into their surroundings. Vimentin intermediate filaments (VIFs) are often observed to play a role in the epithelial cells located at the margins of 2D cultures. However, their role in 3D collective cell behavior remains underexplored. Here, we investigate how induced vimentin expression affects 3D multicellular architecture and mechanics in luminal breast cancer cells (MCF-7) that ordinarily express keratin intermediate filaments only. We find that vimentin expression significantly alters 3D cell cluster morphology, inducing protrusions and increasing boundary fluctuations. Furthermore, cells in vimentin-expressing clusters show enhanced, more stochastic migration. In addition, these clusters exert stronger and localized traction forces on the surrounding matrix, indicating increased cell-matrix interactions. Transcriptomic analysis corroborates these biophysical findings, revealing upregulated gene expression for cell migration and matrix adhesion, and downregulated cell-cell adhesion genes. Our results demonstrate that VIFs are critical in modulating 3D multicellular collective morphology and dynamics, promoting invasive-like behavior by enhancing cell migration and cell-matrix interactions. These results provide fundamental insights into understanding tissue morphogenesis and disease progression.

Vimentin is a cytoskeletal intermediate filament protein that governs the form and function of mesenchymal cells, although the mechanistic details have been poorly understood. Here we highlight recent findings that reveal the diverse role of vimentin in dynamically organizing intracellular architecture and enhancing mechanical resilience. The exceptional deformability of vimentin can now be understood from its high-resolution three-dimensional structure resolved using cryo-electron microscopy. Vimentin also organizes the motion and positioning of numerous organelles, including mitochondria and the nucleus. Furthermore, it synergizes with the actin cytoskeleton to protect cells from extreme mechanical deformations. Finally, vimentin expression in epithelial–mesenchymal transitions has a functional role in tumour invasion analogous to embryonic development and wound healing. These recent developments emphasize the importance of understanding the multifaceted roles of vimentin intermediate filaments in human health and disease.