Generation and Isolation of Cell Cycle-arrested Cells with Complex Karyotypes

Abstract

Chromosome mis-segregation leads to aneuploidy, a condition in which cells harbor an imbalanced chromosome number. Several lines of evidence strongly indicate that aneuploidy triggers genome instability, ultimately generating cells with complex karyotypes that arrest their proliferation. Isolation and characterization of cells harboring complex karyotypes are crucial to study the impact of an imbalanced chromosome number on cell physiology. To date, no methods have been established to reliably isolate such aneuploid cells. This paper provides a protocol for the enrichment and analysis of aneuploid cells with complex karyotypes utilizing standard, inexpensive tissue culture techniques. This protocol can be used to analyze several features of aneuploid cells with complex karyotypes including their induced senescence-associated secretory phenotype, pro-inflammatory properties, and ability to interact with immune cells. Because cancer cells often harbor imbalances in chromosome number, it is crucial to decipher how aneuploidy impacts cell physiology in normal cells, with the ultimate goal of uncovering both its pro- and anti-tumorigenic effects.

Aneuploid senescent cells activate NF‐κB to promote their immune clearance by NK cells

“…We removed the drug once the cells had undergone one round of aberrant mitosis due to SAC inhibition. 72 h after inducing chromosome mis‐segregation, we exposed cells to the spindle poison nocodazole for 12 h, which allowed us to remove dividing cells by mitotic shake‐off (Santaguida et al , 2017 ; Wang et al , 2018 ). We repeated the mitotic shake‐off 4 more times to ensure the removal of all cycling cells.…”

“…We conclude that in the assay we developed here, highly aneuploid RPE1‐hTERT cells are more effectively recognized and eliminated by NK92‐MI cells in vitro than their euploid counterparts. Since we had developed robust protocols to generate the aneuploid cell population using RPE1‐hTERT cells (Santaguida et al , 2017 ; Wang et al , 2018 ), we decided to focus on this cell line to investigate the effects of karyotype alterations on NK cell‐mediated immune clearance.…”

“…By inducing high levels of chromosome mis‐segregation followed by continuous culturing, we previously generated cells with abnormal complex karyotypes that eventually cease to divide and enter a senescent‐like state. We have named such cells arrested with complex karyotypes (ArCK) cells (Santaguida et al , 2017 ; Wang et al , 2018 ). Prior work indicated that ArCK cells upregulate gene expression signatures related to an immune response that render them susceptible to elimination by natural killer (NK) cells in vitro (Santaguida et al , 2017 ).…”

Cell cycle–dependent localization of the proteasome to chromatin

“…To examine whether nuclear proteasome abundance is increased in quiescent cells, we performed a colcemid-challenge test on TIG-3(WT) cells in order to remove cycling cells 26 . The cells were thus cultured in the presence of the mitotic inhibitor colcemid for 12 h on each of 5 days, after which those that had passed through mitosis were "shaken off. "…”

“…A colcemid-challenge test were performed as previously described 26 . In brief, TIG-3 cells were treated with 2 mM thymidine for 18 h, released for 8 h, and incubated with 40 nM colcemid (N-desacetyl-N-methylocolchicine, Thermo Fisher Scientific) for 12 h, after which mitotic cells were removed by shake-off treatment.…”