Single Layer Paper Based Assay for the Cancer Cell Migration Assessment

Backgrounds Metastasis is the main reason for cancer-related deaths. Cell-based technologies to understand these mechanisms are critical for finding novel therapies. Cell migration is one of the initial steps of metastasis and can be studied by several techniques, including transwell migration and wound healing assays. However, these assays can oversimplify the 3D tumor environment so new approaches may be needed for assessment of cell migration. Here, we describe a single-layer paper-based migration assay, using commercial filter papers. Methods We developed a single-layer paper-based migration assay in which commercial filter papers with specific porosity (25 µm pore size) were seeded with breast cells: MCF10A normal breast cells, MCF7 non-invasive breast cancer cells, and MDA-MB-231 metastatic breast cancer cells. Their migration potential was validated using a wound healing assay, and then migration from the paper to the plate was monitored using MTT assay and cell counting after confirming cellular 3D arrangement within the paper by immunostaining and fluorescence microscopy. Results After establishing their 3D arrangement in the filter paper, MDA-MB-231 showed significantly higher migration from the paper to the plate compared to MCF7. MDA-MB-231 demonstrated a 15.4-fold increase in migration to the plate by day 7, which was significantly greater than the 3.8-fold increase observed for MCF7. Cell counting analysis confirmed these findings, with significantly more MDA-MB-231 present on the plate surface compared to MCF7 on both day 4 and day 7. In contrast, there was no detectable transfer of MCF10A from the paper to the plate, reflecting the differential invasiveness potential of the cells in the paper-based platform and consistent with the wound healing assay results. Conclusion Our results suggest that a single-layer paper-based culture system combined with MTT assay and/or cell counting can be used as an alternative, cost-effective, and easily prepared technique for in vitro migration analysis.