Spheroid cells show a more aggressive growth pattern.

<p>(A) Spheroid cells had higher proliferation potential than their parent cells. Both spheroid cells and parental cells were cultured in CM for 24 h, then 5000/well cells were cultured in CM in 96 well plates. Half of media was changed every 3 days. Cell proliferations were detected from day 0 to day 17 by CellTiter-Glo Luminescent Cell Viability Assay. (B) Spheroid cells had higher migration ability than parent cells. Parental and spheroid EOC cells were treated with RPMI only medium for 24 hrs. In a transwell plate, 2×10⁵/well cells were put into insert with 0.2%BSA-RPMI medium, the lower chamber contains no cells except RPMI medium with 2.5% FBS. After culture for 1, 2, and 3 days, the numbers of cells that passed through membrane as well as cells fall into chamber were calculated under microscope. (C) Spheroid cells recovered ‘scratch’ made by 200-µL pipette tip after 24 hours more efficient than their parent cells. Experiments were repeated three times with similar results. (D) More spheroid cells invaded into matrigel than parent cells as shown by microscopy. Spheroid cells and parental cells were treated with serum-free medium RPMI for 24 h. In a transwell plate, 40000/well of cells were put into insert coated with matrigel with 0.2% BSA, while the chamber had only media with 5% FBS, after culture for 1, 2, and 3 days, The number of cells invading into matrigel were counted after 0.1% crystal blue staining. (E) Invasion tests were done in duplicate and average and SD were shown. *,p<0.01. (F) Spheroid cells formed colonies in top agar detected in anchorage-independent growth assay (AIG) as shown under microscopy. 500 cells were seeded in 6 well plates containing a top layer of 0.3% soft agar and 0.5% agar base in DMEM, 10% FBS. 24 hrs later, the number of cells seeded was determined by counting cells under the light microscope. After 3 weeks of growth in soft agar, the number of colonies (Colonies >5 cells) were counted; 10 different fields were quantified per well and the average number of colonies per field was calculated. The AIG index was expressed relative to the number of cells seeded. More than 70% of seeded spheroid cells formed colonies while none of seeded parent cells formed colonies. Experiments were repeated twice with similar results. *,p<0.01. Error bars: SD.