Development of a model for fibroblast-led collective migration from breast cancer cell spheroids to study radiation effects on invasiveness

Abstract

Background Invasiveness is a major factor contributing to metastasis of tumour cells. Given the broad variety and plasticity of invasion mechanisms, assessing potential metastasis-promoting effects of irradiation for specific mechanisms is important for further understanding of potential adverse effects of radiotherapy. In fibroblast-led invasion mechanisms, fibroblasts produce tracks in the extracellular matrix in which cancer cells with epithelial traits can follow. So far, the influence of irradiation on this type of invasion mechanisms has not been assessed. Methods By matrix-embedding coculture spheroids consisting of breast cancer cells (MCF-7, BT474) and normal fibroblasts, we established a model for fibroblast-led invasion. To demonstrate applicability of this model, spheroid growth and invasion behaviour after irradiation with 5 Gy were investigated by microscopy and image analysis. Results When not embedded, irradiation caused a significant growth delay in the spheroids. When irradiating the spheroids with 5 Gy before embedding, we find comparable maximum migration distance in fibroblast monoculture and in coculture samples as seen in unirradiated samples. Depending on the fibroblast strain, the number of invading cells remained constant or was reduced. Conclusion In this spheroid model and with the cell lines and fibroblast strains used, irradiation does not have a major invasion-promoting effect. 3D analysis of invasiveness allows to uncouple effects on invading cell number and maximum invasion distance when assessing radiation effects.

A deep learning-based pipeline for analyzing the influences of interfacial mechanochemical microenvironments on spheroid invasion using differential interference contrast microscopic images

“…Exploring the mechanisms behind migratory and invasive behaviors of cancer cells is critical for identifying strategies to counteract metastasis. While the image-based spheroid invasion assay provides many advantages for investigating cancer cell metastasis under in vitro conditions [ 57 , 58 ], the concurrent image processing and analysis during the assay period still present notable challenges. Due to the extensive number of time-lapse brightfield DIC images, the manual processing and analysis of these images is a time-intensive task.…”

Modeling the Role of Cancer-Associated Fibroblasts in Tumor Cell Invasion

“…CAFs can be incorporated into spheroids to generate co-culture spheroids ready for matrix embedding ( Figure 1 B, bottom). This is particularly useful with cancer cell lines that are unable to self-aggregate, as CAFs on their own will form tight spheroid structures and can help facilitate the generation of a spheroid in co-culture [ 56 ]. CAFs can also be seeded into the surrounding matrix of monoculture spheres (similar to the vertical gel set-up) to model the scenario of a tumor mass surrounded by CAF-populated stroma.…”

“…For example, Mei et al used 96-well, low-attachment, U-bottom plates, which allow for the generation of spheroids and the addition of a matrix to occur within the same plate. With this set-up, they investigated the protective effects of CAFs in co-culture spheroids on radiation and how it affected the CAF-mediated invasion of breast cancer cells [ 56 ]. To supplement the use of round-bottom plates, live-imaging systems (e.g., the Sartorius Incucyte ® ) now have image-acquisition programs optimized for the imaging of round bottom plates together with in-house analyses of the invasive area from spheroids, allowing for a more automated and seamless workflow [ 29 ].…”

Three-Dimensional in Vitro Models: A Promising Tool To Scale-Up Breast Cancer Research

“…This model showed that cancer cells follow invading fibroblasts closely. However, no promoting effect was observed in spheroid invasion following irradiation …”

“…However, no promoting effect was observed in spheroid invasion following irradiation. 27 Angiogenesis. Breast cancer spheroids have also been used to evaluate angiogenesis in breast cancer.…”