Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology

Abstract

The mechanical microenvironment has been shown to act as a crucial regulator of tumor growth behavior and signaling, which is itself remodeled and modified as part of a set of complex, two-way mechanosensitive interactions. While the development of biologically-relevant 3D tumor models have facilitated mechanistic studies on the impact of matrix rheology on tumor growth, the inverse problem of mapping changes in the mechanical environment induced by tumors remains challenging. Here, we describe the implementation of particle-tracking microrheology (PTM) in conjunction with 3D models of pancreatic cancer as part of a robust and viable approach for longitudinally monitoring physical changes in the tumor microenvironment, in situ. The methodology described here integrates a system of preparing in vitro 3D models embedded in a model extracellular matrix (ECM) scaffold of Type I collagen with fluorescently labeled probes uniformly distributed for position-and timedependent microrheology measurements throughout the specimen. In vitro tumors are plated and probed in parallel conditions using multiwell imaging plates. Drawing on established methods, videos of tracer probe movements are transformed via the Generalized Stokes Einstein Relation (GSER) to report the complex frequency-dependent viscoelastic shear modulus, G*(ω). Because this approach is imaging-based, mechanical characterization is also mapped onto large transmitted-light spatial fields to simultaneously report qualitative changes in 3D tumor size and phenotype. Representative results showing contrasting mechanical response in sub-regions associated with localized invasion-induced matrix degradation as well as system calibration, validation data are presented. Undesirable outcomes from common experimental errors and troubleshooting of these issues are also presented. The 96-well 3D culture plating format implemented in this protocol is conducive to correlation of microrheology measurements with therapeutic screening assays or molecular imaging to gain new insights into impact of treatments or biochemical stimuli on the mechanical microenvironment. Video LinkThe video component of this article can be found at

ECM Composition and Rheology Regulate Growth, Motility, and Response to Photodynamic Therapy in 3D Models of Pancreatic Ductal Adenocarcinoma

“…Time-dependent changes in ECM compliance were obtained in normal and drug resistant 3D PDAC cell cultures using a methodology described previously (26). In this approach, the mobility of fluorescent tracer probes embedded in ECM surrounding 3D tumor nodules is analyzed using the Generalized Stokes Einstein Relation (GSER) to estimate local viscoelastic response, G*(ω) , of the material.…”

The Mechanics of Single Cell and Collective Migration of Tumor Cells

“…Particle-tracking microrheology of the cell cytoplasm, which was originally developed for use in cells plated on 2D substrates, has also been adapted for 3D systems [90][91][92]. Here, tracer beads are embedded within cells and tracked to determine various mechanical and viscoelastic properties [84] relatively noninvasively and over time [90].…”

In situ measurement of ECM rheology and microheterogeneity in embedded and overlaid 3D pancreatic tumor stroma co-cultures via passive particle tracking

“…Video data were analyzed using a methodology for PTM in multiwell 3D cultures described previously. 29 This analysis is based on open source particle-tracking routines from the laboratory of Maria Kilfoil. 32 These routines make it possible to track mean squared displacement (MSD) of°uorescent tracer probes throughout all frames of respective video data and then apply the GSER in order to extract frequency-dependent viscoelastic moduli.…”

“…28 PTM has been shown to be useful in measuring tumor level physical interactions, and can be easily collected over a grid in order to inform on the whole sample rheology. 29 This contrasts with certain active techniques such as optical tweezers which are typically used for studying single cell mechanics and making point measurements within a sample. 30 Leveraging the relative ease of PTM to acquire data over relatively large spatial scales (trading o® microscope¯eld of view and magni¯cation), this approach can also report intra sample microheterogeneity, obtained through statistical analysis of tracer probe trajectories.…”

“…30 Leveraging the relative ease of PTM to acquire data over relatively large spatial scales (trading o® microscope¯eld of view and magni¯cation), this approach can also report intra sample microheterogeneity, obtained through statistical analysis of tracer probe trajectories. 24 Here, building on previous studies establishing the utility of particle tracking measurements for nondestructive longitudinal characterization of ECM remodeling in 3D tumor models, 29,31 we speci¯cally evaluate the capability of this approach to measure the impact 3D PDAC co-culture composition (with and without broblasts) and geometry on the sti®ness and microstructure of collagen I ECM. By embedding polystyrene microspheres in the ECM hydrogel at the time of culture preparation, short video sequences can be acquired nondestructively while cultures are removed from incubation for brief durations.…”