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

Summary

Particle-tracking microrheology can be used to non-destructively quantify and spatially map changes in extracellular matrix mechanical properties in 3D tumor models.

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 time-dependent 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.

Introduction

It is clear from a growing body of evidence in the literature that cancer cells, as with non-malignant mammalian epithelial cells, are highly sensitive to the mechanical and biophysical properties of the surrounding extracellular matrix (ECM) and other microenvironment components^1-9. Elegant mechanistic studies have provided insights into the role of extracellular rigidity as a complex mechanosensitive signaling partner that regulates malignant growth behavior and morphogenesis^2,3,10,11. This work has been facilitated in particular by the development of 3D in vitro tumor models that restore biologically relevant tissue architecture ....

Protocol

1. Culturing Tumor Spheroids

1. Mix 10 ml of cell culture grade water with 0.1 g of agarose to obtain a 1% agarose solution.
2. Heat agarose solution to above 70 °C (roughly 14 sec in a standard microwave or by using a heating plate) before aliquoting 40 μl of agarose solution into a well on a 96-well plate.
3. Incubate plate at 37 °C for at least 1 hr while harvesting cells using standard techniques.
4. Use a hemacytometer to determine the concentration of cells in the suspension.
5. Dilute cell suspension to 1,000 cells/ml and add 100 µl of this dilution to the well containing the cured aga....

Results

To verify the validity of G*(ω) measurements at localized positions within a complex model tumor microenvironment, two initial validation experiments were conducted. First, we sought to validate our measurements against the "gold standard" of bulk oscillatory shear rheometry. We prepared identical samples of collagen matrix (without cells) at a concentration of 1.0 mg/ml collagen. These samples were probed with a bulk rheometer (TA Instruments AR-G2, using 40 mm parallel plate geometry) and by PTM (using the sam.......

Discussion

In this protocol we introduce a robust and widely applicable strategy for longitudinally tracking local changes in ECM rigidity in 3D tumor models. We envision that this methodology could be adopted by cancer biologists and biophysicists interested in mechanosensitive behavior implicated in matrix remodeling during tumor growth and invasion processes. Precise quantification of matrix degradation kinetics could be particularly valuable to those studying the activity of matrix metalloproteases, lysyl oxidase or other relev.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Bovine type 1 collagen	BD Biosciences, San Jose, CA	354231
PANC-1	American Type Cell Culture, Manassas, VA	CRL1469	or other appropriate cell type
Fluorescent Microspheres	Life Technologies, Carlsbad, CA	906906
Matrigel	BD Biosciences, Bedford, MA	354230
Agarose	Fisher Bioreagents, Waltham, MA	C12H18O9
NaOH	Fisher Bioreagents, Waltham, MA	NC0480985
96-well Imaging plates	Corning Inc., Corning, NY	3904
DMEM	Hyclone, Waltham, MA	SH30243.01	or appropriate cell culture media
Zeiss AxioObsever Microscope	Zeiss, Oberkochen, Germany		includes high-speed camera and imaging software
MATLAB software	The Mathworks, Natick, MA