Mechanical stimuli and matrix properties modulate cancer spheroid growth in three-dimensional gelatin culture

Abstract

Most patients who succumb to cancer have metastases to bone that contribute to their death. Cancer cells that metastasize to bone are regularly subjected to mechanical stimuli that may affect their proliferation, growth and protein expression. Understanding why some cancer cells thrive in this environment could provide insight into new approaches to prevent or treat metastasis to bone. We used 4T1 cells as a model of breast cancer cells, and implanted them in gelatin hydrogels with moduli of 1 or 2.7 kPa to mimic the properties of bone marrow. The constructs were subjected to either perfusion of media through the hydrogel or combined perfusion and cyclic mechanical compression for 1 h d −1 for 4 d. Controls were cultured in free-swelling conditions. The cells formed spheroids during the 4 d of culture, with larger spheroids in the statically cultured constructs than in perfusion or compressed constructs. In stiffer gelatin, smaller spheroids formed in compressed constructs than perfusion alone, while compression had no effect compared to perfusion in the softer gelatin. Immunostaining indicated that the spheroids expressed osteopontin, parathyroid hormone-related protein and fibronectin, which are all hallmarks of bone metastasis. The proliferative marker Ki67 was present in all spheroids on day 4. In the 1 kPa gelatin, Ki67 staining intensity was greater in the statically cultured, free-swelling constructs than in bioreactor culture, regardless of dynamic compression. By contrast, proliferation was higher in the compressed gelatins compared to perfusion alone in the 2.7 kPa constructs, although the spheroids were smaller, on average. This suggests the stiffer gelatin may restrict spheroid growth at the same time that it enhances mechanobiological signalling during compression. Taken together, 4T1 breast cancer cells are mechanically sensitive, and mechanical stimuli can alter their proliferation and protein expression within soft materials with mechanical properties similar to bone marrow. As such, both in vivo and in vitro models of cancer metastasis should consider the role of the mechanical environment in the bone.

What do cellular responses to acidity tell us about cancer?

“…ionic composition [ 16 – 18 ]) and physical properties (e.g. matrix stiffness [ 19 – 21 ]) of the tumour [ 22 , 23 ].…”

The effect of marrow secretome and culture environment on the rate of metastatic breast cancer cell migration in two and three dimensions

“…, 2015 ). In 3D culture models, the properties of the surrounding matrix also alter the size and the protein expression of spheroids formed by 4T1 cancer cells ( Curtis et al. , 2020b ).…”

“…Computational models have shown that these stiffness mismatches between cell types result in protection of smaller cells from mechanical stress during bone loading (Vaughan et al, 2015). In 3-D culture models, the properties of the surrounding matrix also alters the size and the protein expression of spheroids formed by 4T1 cancer cells (Curtis et al, 2020b).…”

Designing Hydrogel-Based Bone-On-Chips for Personalized Medicine

“…The microenvironment is soft with rigid struts of bone matrix. These changes of stiffness and mechanical stimulation were simulated in vitro by Curtis et al on a breast cancer cell line, 4T1, laden in a gelatin scaffold and showed changes in the proliferation and protein expression (PTHrP and OPN) within soft materials with mechanical properties similar to bone marrow [56]. This showed evidence of mechanosensitivity of cancerous cells to their microenvironment.…”