Single Cell tracking of breast cancer cells enables the prediction of sphere formation from early cell divisions

Published: 21 September 2018| Version 1 | DOI: 10.17632/bb96mh7pvd.1
Contributor:
patrick bailey

Description

We hypothesized that you could predict sphere formation using only information from early cell division. When analyzed, this data shows its possible to predict day 14 sphere formation at only 24 hours with 98% accuracy in these two cell lines. This data was gathered by visually tracking almost two thousand cells by eye over 14 days. This is the raw tracking data for the paper published under the same name as the description. It contains excel files of each individual cell that we followed by eye over the course of 14 days. The tables are set up to be easily understandable and labeled according to the wells of a 96 well plate. E.g. A1-12 etc .

Files

Steps to reproduce

Mammosphere culture and cell tracking: Cells were trypsinized, triturated repeatedly and passed through a 40um cell strainer to enrich for single cells. Cells were counted and visualized on a hemacytometer. If cell clumps were observed, cells were passed through a 25-gauge needle 10 times. For density dilution assays cells were seeded in 1 mL Mammocult media supplemented with heparin, penicillin/streptomycin and hydrocortisone per manufacturers instruction (complete media) in 24 well Ultra-low Attachment plates and incubated at 37oC 5%CO2 for 7 days. Each density was seeded in triplicate for one experimental replicate (N) for a total of three replicates. For tracking and predictive experiments cells were diluted to 2 cells/300 µL in complete Mammocult media. Cells were seeded in the center 12 wells of 96 well Ultra-Low Attachment plates at a volume of 300µL per well. The outside edges of the plates were filled with PBS to prevent evaporation of media. Each experiment used 8 plates with 12 wells seeded. Cells were allowed to settle in an incubator for 2 hours, after which light microscopy was employed to visualize the position of every cell in the plate (No specialized equipment required). These initial positions were marked for subsequent tracking. Manual manipulation with a 10µL pipette was employed to move cells that were either too numerous or too close together. Every day for 7 days and on days 10 and 14 PreSp position, count, size and morphology were recorded. Both tracking and predictive experiments were repeated three times. NOTE: MCF-7 spheres are counted as objects over 50um T47D spheres are counted as objects over 35um at day 7 and 40um at day 14 (This is based on tracking data comparing spheres at day 7 to spheres at day 14. Ad hoc definitions of sphere size limit are arbitrary and include both spheres that eventually die off and also exclude smaller clonal growths that eventually grow into spheres. The values above are based on a value that minimizes those errors)