3D Traction Force Microscopy inside Compliant Confining Microchannels

Introduction: Cancer metastasis, responsible for the majority of cancer-related deaths, underscores the urgent need for sophisticated tools to unravel its complexities. The intricate process involves cells navigating through constrictive microenvironments to establish secondary tumors. Understanding how self-generated mechanical forces, alongside other contributing factors, play a role in cancer cell escape from primary tumor sites and their entry into confining spaces is crucial for comprehensive insights. Conventional in vitro models, predominantly constructed from Polydimethylsiloxane (PDMS), inadequately mirror the physiological conditions crucial for studying cancer cell migration. To bridge this gap, we introduce a pioneering approach utilizing polyacrylamide (PA)-based microfluidic devices with tunable physiological stiffness (8-40 kPa). This innovative platform, termed Hydrogel-Encapsulated Microchannel Array (HEMICA), not only enables the investigation of confined migration dynamics but also serves as the foundation for our novel 3D Traction Force Microscopy (3D-TFM) method.

Materials and

Methods: An array of parallel microchannels with a fixed channel length (L=200 µm) and height (H=10 µm) and widths of either 3, 6, 10, 15, 20, 50 µm (multichannel design) or only 3 µm (confining channel design) were fabricated via photolithography. After fabrication, the wafers were cut using a diamond cutter and used as molds for PA polymerization, which resulted in gels imprinted with desired features. Acrylamide (A) (40%) with N,N-Methylene bisacrylamide (B) (2%) were mixed with distilled water to final concentrations of 8% A/0.1% B, 8% A/0.2% B and 8% A/0.6% B corresponding to PA gels with an elastic modulus of 8, 15 and 21 kPa, respectively. For traction force microscopy experiments, 60 µL of the distilled water component was replaced with 200 nm fluorescent bead suspension (FluoSpheres, Invitrogen) resulting in 0.1% solids in the acrylamide mix. Polymerization was achieved using ammonium persulfate and TEMED initiators.
Beads (200 nm in diameter) embedded within microchannel walls and cells were imaged with a confocal microscope (Nikon) using a 40x water objective with a high numerical aperture and a 1024x1024 pixel resolution. z-stacks with a step size of 0.2 µm were acquired for all microchannels in the HEMICA device before cell seeding to record the positions of the fluorescent beads in the absence of cells. The z-stack images acquired at different time points were then registered and subjected to 3D traction stress analysis.

Results, Conclusions, and Discussions: We developed methods to optimize the study of cell traction in 3D confining channels at both high temporal and spatial resolution. This was achieved by minimizing the spherical aberration of embedded nanobeads and noise during z stack image acquisition via the use of a water immersion objective and the process of image deconvolution using a theoretical Point Spread Function (Fig. a) in addition to other denoising algorithms. This allowed for improved spatial resolution of displacement measurements crucial for force calculation.
Our investigation focused on the force exertion dynamics of highly migratory Triple Negative Breast cancer cells as they entered and migrated through confined spaces. Notably, during cell entry, using our 3D-TFM measurements we observed heightened displacements of the PA gel at the rear (Fig. b, d), indicating the critical role of rearward pushing forces for cell entry into confining spaces. Additionally, an increase in calcium flashes at the channel entrance, detected using GCaMP6s, a calcium biosensor, revealed potential enhancement of actomyosin-mediated reinforcement at the cell rear, facilitating entry. In marked contrast, displacements were uniformly distributed along the cell length when cell was entirely within the microchannel geometry (Fig. c, d). This is in contradiction of widely reported behavior on 2D surfaces where force exertion primarily occurs at edges. Confocal microscopy of Paxilin-GFP cells revealed adhesions distributed across the cell body in 3D confinement, correlating with uniformly distributed forces. Disruption of the actin cytoskeleton with Latrunculin-A reduced displacements, albeit incompletely, indicating a possible role of plasma membrane tension in addition to the adhesion (Fig. e).
In conclusion, our findings underscore the significance of studying cell traction in 3D confining channels, revealing unique force exertion dynamics distinct from those observed on 2D surfaces. The uniform distribution of forces across the cell body challenges traditional notions of force localization at adhesion edges, highlighting the importance of considering 3D microenvironmental cues. Additionally, our observations suggest potential roles for calcium signaling and plasma membrane tension in regulating cell traction dynamics in confined spaces. These insights advance our understanding of cell migration mechanics and offer new perspectives for designing targeted interventions against metastatic cancer progression.

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