PTM Based Tumor Microenvironment Monitoring: A Method for Tracking Changes in ECM Rigidity Using Tumor Spheroids

Abstract

Source: Jones, D. P., et al. Longitudinal Measurement of Extracellular Matrix Rigidity in 3D Tumor Models Using Particle-tracking Microrheology. J. Vis. Exp. (2014).

This protocol describes a technique for monitoring extracellular matrix rigidity changes by using particle-tracking microrheology (PTM). This is done by culturing 3D pancreatic tumor spheroids into ECM with embedded fluorescent probes for video particle-tracking and analysis.

Protocol

1. Preparing 3D Tumor Spheroids Embedded in ECM

1. Prepare a work station with needed materials inside a laminar flow hood.
2. Prepare a diluted mixture of carboxylate-modified 1 μm diameter fluorescent tracer probes by adding 2 parts stock probes (2% solids) to 25 parts sterile water.
3. Remove a bottle of 3.1 mg/ml bovine collagen from the refrigerator and place it on ice.
4. Aliquot 125 μl of collagen into an empty 2 ml vial.
5. Add 50 μl of diluted tracer probe solution to the vial containing the collagen and vortex briefly to distribute probes.
6. Add 235 μl of appropriate cell cultu....

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
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