We describe a method for quantifying the contractile forces that tumor spheroids collectively exert on highly nonlinear three-dimensional collagen networks. While three-dimensional traction force microscopy for single cells in a nonlinear matrix is computationally complex due to the variable cell shape, here we exploit the spherical symmetry of tumor spheroids to derive a scale-invariant relationship between spheroid contractility and the surrounding matrix deformations. This relationship allows us to directly translate the magnitude of matrix deformations to the total contractility of arbitrarily sized spheroids. We show that our method is accurate up to strains of 50% and remains valid even for irregularly shaped tissue samples when considering only the deformations in the far field. Finally, we demonstrate that collective forces of tumor spheroids reflect the contractility of individual cells for up to 1h after seeding, while collective forces on longer timescales are guided by mechanical feedback from the extracellular matrix.

LuB25-spheroid.zip

This zip-file contains a series of brightfield images through the equatorial plane of a spheroid created from 4,000 primary mesenchymal Luminal-B breast cancer cells. The spheroid is embedded in a three-dimensional 1.2mg/ml collagen gel, together with micron-sized fiducial markers. Images were taken at an interval of 10min, spanning a period of 24h in total. The pixel-size of the image series is 1.02µm.

Triduc1-spheroid.zip

This zip-file contains a series of brightfield images through the equatorial plane of a spheroid created from 4,000 primary mesenchymal triple-negative breast cancer cells. The spheroid is embedded in a three-dimensional 1.2mg/ml collagen gel, together with micron-sized fiducial markers. Images were taken at an interval of 10min, spanning a period of 24h in total. The pixel-size of the image series is 1.02µm.

LuB25-tumoroids.zip

Each subfolder within this zip-file contains a series of brightfield images through the equatorial plane of a Luminal-B breast cancer tumoroid (tumor tissue sample). The tumoroids are embedded in a three-dimensional 1.2mg/ml collagen gel, together with micron-sized fiducial markers. Images were taken at an interval of 10min, spanning a period of 24h in total. The pixel-size of the image series is 1.02µm.

A172-15000-spheroids.zip

This zip-file contains three subfolders, each of which represents an independent experiment (biological replication). Each subfolder contains series of brightfield images through the equatorial plane of multiple spheroids (technical replication) created from 15,000 A172 glioblastoma cancer cells. The spheroids are embedded in a three-dimensional 1.2mg/ml collagen gel, together with micron-sized fiducial markers. Images were taken at an interval of 5min, spanning a period of 12h in total. The pixel-size of the image series is 1.29µm.

U87-15000-spheroids.zip

This zip-file contains three subfolders, each of which represents an independent experiment (biological replication). Each subfolder contains series of brightfield images through the equatorial plane of multiple spheroids (technical replication) created from 15,000 U87 (full name: U-87 MG) glioblastoma cancer cells. The spheroids are embedded in a three-dimensional 1.2mg/ml collagen gel, together with micron-sized fiducial markers. Images were taken at an interval of 5min, spanning a period of 12h in total. The pixel-size of the image series is 1.29µm.

U87-7500-spheroids.zip

This zip-file contains three subfolders, each of which represents an independent experiment (biological replication). Each subfolder contains series of brightfield images through the equatorial plane of multiple spheroids (technical replication) created from 15,000 U87 (full name: U-87 MG) glioblastoma cancer cells. The spheroids are embedded in a three-dimensional 1.2mg/ml collagen gel, together with micron-sized fiducial markers. Images were taken at an interval of 5min, spanning a period of 12h in total. The pixel-size of the image series is 1.29µm.