A Screening System for Evaluating Cell Extension Formation, Collagen Compaction, and Degradation in Drug Discovery

Abstract

Cell extension generation is vital for matrix remodeling during tissue invasion by cancer cells, yet current methods for identifying molecules that regulate this process are not well-suited for screening. To bridge this gap, we developed a grid-supported, floating collagen gel system (~100 Pa stiffness) that allows us to study cell extension formation, collagen compaction, and degradation within a single, integrated assay. This innovative approach offers a more nuanced view of the dynamic interactions between cancer cells and the extracellular matrix.

In our experiments using cultured diploid fibroblasts, a fibroblast cell line, and two cancer cell lines, we observed notable differences in cellular behavior when comparing the floating gels to more rigid attached collagen gels (~2800 Pa). Specifically, the mean number and length of cell extensions were significantly higher in the floating gels, underscoring the impact of the mechanical environment on cellular dynamics. This suggests that the physical properties of the matrix are crucial in regulating cell morphology and behavior.

To investigate the mechanisms behind cell extension formation, we examined specific processes and signaling pathways. Our findings revealed that, compared to control conditions, the number of cell extensions significantly decreased after treatment with latrunculin B, which disrupts actin polymerization, as well as with β1 integrin blockade and a formin FH2 domain inhibitor. These results highlight the critical roles of cytoskeletal dynamics and integrin signaling in cell extension formation.

To further identify regulatory molecules, we screened a kinase inhibitor library of 480 compounds using our floating gel assay. The screening results showed that several inhibitors, including SB431542, SIS3, Fasudil, GSK650394, and PKC-412, caused significant reductions in collagen compaction, pericellular collagen degradation, and the number of cell extensions compared to vehicle-treated cells. These outcomes not only demonstrate the efficacy of our assay in pinpointing potential therapeutic targets but also reveal the complex interplay between cell signaling pathways and the mechanical properties of the extracellular matrix.

In summary, our data indicate that the grid-supported floating collagen gel model is a powerful tool for screening inhibitors of cell extension formation and critical matrix remodeling processes associated with cancer cell invasion. This model offers promise for enhancing our understanding of cancer metastasis and identifying new therapeutic strategies to inhibit cancer cell invasiveness. Future research will build on these findings by incorporating additional molecular targets and exploring the interactions among various signaling pathways in the context of tissue invasion. By refining our approach, we aim to provide valuable insights GSK650394 that could ultimately contribute to the development of more effective cancer treatments.