| Résumé | Background: Circulating tumor cells (CTCs) are released in blood from the primary tumor, but although very heterogeneous both in size and marker expression, are very rare and provide information not available from the primary tumor. The identification of CTC cells and clusters could advance our understanding of metastasis and help personalize therapy.1,2 Notably, CTC clusters were shown to have a higher metastatic potential than single cells³ but the process remains poorly understood. We present a multistage filtration system with pore sizes from 20 down to 8 μm for the size-selective enrichment of Ov90 ovarian cancer cells and clusters from blood. Each captured cell population was released, cultured and characterized independently. Methods: We developed a 3D printed multistage filtration cartridge and polymer filters with 20, 15, 12, 10 and 8 μm-diameter pores. Filters were stacked from 20 (top) to 8 μm (bottom) and used to enrich and sort Ov-90 cells spiked in 1:6 mL of blood:PBS. Captured cells were released by removing individual filters from the cartridge, reverse flowing OSE medium, and then cultured separately. Results: Ov-90 clusters were found mostly on the top filter (20 μm) and interestingly, few small clusters (3-4 cells) were found on the 8 and 10 μm filters, suggesting alignment of cluster cells as they pass through the pores.⁴ Cell and nucleus diameters were measured, and a general correlation was found between filter pore size and cell and nucleus size. Interestingly, nucleus size was found to be the single most significant parameter in determining passage of single cells and small clusters through pores. Following cell culture, two distinct phenotypes were observed: cell captured on small pore filters (8-12 μm) grew primarily in a monolayer. Cells captured on filters with larger pores (15-20 μm) first grew as monolayer, but rapidly formed cell aggregates that subsequently detached from the surface. Staining for E-Cadherin, a cell-cell adhesion protein, revealed a loss of expression of cells from filter with larger pores. Conclusion: We developed a new multistage filtration method and selectively enriched and sorted cells based primarily on their nucleus size. We identified two Ov-90 populations with different growth behaviors with low E-cadherin expression on the cells forming clusters, which is known to correlate with metastasis. The application of multistage filters may also reveal different CTC populations based on nucleus and cell size. |
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