Real‐time cell cycle imaging during melanoma growth, invasion, and drug response

Abstract

Solid cancers are composed of heterogeneous zones containing proliferating and quiescent cells. Despite considerable insight into the molecular mechanisms underlying aberrant cell cycle progression, there is limited understanding of the relationship between the cell cycle on the one side, and melanoma cell motility, invasion, and drug sensitivity on the other side. Utilizing the fluorescent ubiquitination-based cell cycle indicator (FUCCI) to longitudinally monitor proliferation and migration of melanoma cells in 3D culture and in vivo, we found that invading melanoma cells cycle actively, while G1-arrested cells showed decreased invasion. Melanoma cells in a hypoxic environment or treated with mitogen-activated protein kinase pathway inhibitors remained G1-arrested for extended periods of time, with proliferation and invasion resuming after re-exposure to a more favorable environment. We challenge the idea that the invasive and proliferative capacity of melanoma cells are mutually exclusive and further demonstrate that a reversibly G1-arrested subpopulation survives in the presence of targeted therapies.

Imaging- and Flow Cytometry-based Analysis of Cell Position and the Cell Cycle in 3D Melanoma Spheroids

“…There are several methods of producing tumor spheroids, this protocol uses the non-adherent growth method, where cells are cultured on agar or agarose 3,7,9 . Figure 1 shows an example of a C8161 melanoma spheroid after 3 days on agar.…”

“…In order to visualize the cell cycle of individual cells within the 3D spheroid model, C8161 melanoma cells were transduced with the FUCCI system 7,15 . Due to the large size of the spheroids (up to 1 mm in diameter after 3 days on agarose and 24 hr growth in collagen for C8161), sectioning is the best option for visualizing cells in the center of the spheroid.…”

“…Take a z-slice image of a middle FUCCI spheroid section using a 10X or 20X objective as previously described 7 . Make sure no overlap or crosstalk occurs between the Kusabira Orange2 and Azami Green.…”

“…Once the assay has been optimized and Hoechst penetration validated via confocal microscopy, run cells without fixing on a flow cell sorter as previously described 7,16 for further analysis of live cell sub-populations.…”

“…Spheroid models of many types of cancer are able to mimic the growth characteristics, drug sensitivity, drug penetration, cell-cell interactions, restricted availability of oxygen and nutrients and development of necrosis that is seen in vivo in solid tumors [6][7][8][9][10][11] . Spheroids develop a necrotic core, a quiescent or G1 arrested region surrounding the core, and proliferating cells at the periphery of the spheroid 7 . The development of these regions may vary depending on the cell density, proliferation rate and the size of the spheroid 12 .…”