Evidence suggests that the Extra Cellular Matrix (ECM) stiffness may be a driving factor in tumor progression and may sensitize epithelial cells towards the invasive phenotype. In fact, high mammographic density is linked to a higher risk of tumor. In vitro, stiffness in naturally derived ECMs is commonly increased via changing concentration or cross-linking. Either process will change ligand density, porosity, architecture, and diffusivity. Therefore, it is difficult to determine whether the resulting tumor phenotype is due to stiffness changes or other factors. ECM stiffness is conventionally measured via parallel plate rheology or AFM. But, while one only provides an average ensemble of the entire sample, the other lacks the axial resolution required to measure deep within the gels.
We use our stiffness gradient device (SGD) to tune stiffness independent of concentration or crosslinking. In the SGD, the ECM is polymerized around a post, which is rotated to induce strain, and a stiffness gradient is established. Stiffness can be tuned from 100 - 1000 Pa within one dish, the range of normal and malignant breast tissue. We have combined SGD with active microrheology (AMR) to investigate the role of ECM stiffness in sensitizing epithelial cells towards an invasive phenotype. AMR allows deep measurements within the sample to map ECM stiffness in 3D and around cells. We can correlate these maps with the function and signaling of mechanoreceptors such as integrins.
Materials and Methods
Cells were cultured inside hydrogels (Collagen or Matrigel) containing 2 um silica beads to be used as probes for AMR. AMR was conducted to calculate the complex shear modulus (G*). G* is a measure of material stiffness (G’) and loss (G’’).
MCF10A.ErbB2 cells (courtesy of the Muthuswamy lab) were cultured inside 3D hydrogels within the SGD. These cells can be driven towards malignancy with the addition of a synthetic dimerizer that activates the ErbB2/Her2 pathway. The cells were allowed to reach their growth arrested acini phase, at which time the appropriate matrices were stiffened. After an additional 2 weeks, dimerizer was added to the culture. Non-linear imaging in regions of high stiffness and low stiffness reveals acini morphology and molecular distributions.
Results and Discussion
In dimerizer-free culture, we observed an organized tubule network connecting acini, which lost their growth control only in stiffened regions. In the non-stiffened regions we saw growth arrested hollow acini. With the addition of dimerizer, in soft regions we observed, consistent with published results, thin disorganized tubules. Interestingly, in the stiffened region, we saw a partial rescue of the organized morphology suggesting that stiffness may play an orthogonal role to Her2 signaling. Further observations suggested that a stiff ECM sensitizes mammary epithelial cells to the actions of the Her2 pathway.
Citation Format: Abhishek Kurup, Elliot Botvinick. Mapping matrix mechanics in 3D to study the role of stiffness in breast tumor progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1099. doi:10.1158/1538-7445.AM2013-1099