Flow cytometry-assisted quantification of cell cycle arrest in cancer cells treated with CDK4/6 inhibitors.

Cyclin-dependent kinase 4 (CDK4) and CDK6 inhibitors (i.e., palbociclib, abemaciclib, and ribociclib) are well known for their capacity to mediate cytostatic effects by promoting cell cycle arrest in the G₁ phase, thus inhibiting cancer cell proliferation. Cytostatic effects induced by CDK4/6 inhibitors can be transient or lead to a permanent state of cell cycle arrest, commonly defined as cellular senescence. Induction of senescence is often associated to metabolic modifications and to the acquisition of a senescence-associated secretory phenotype (SASP) by cancer cells, which in turn can promote or limit antitumor immunity (and thus the efficacy of CDK4/6 inhibitors) depending on SASP components. Thus, although accumulating evidence suggests that anti-cancer effects of CDK4/6 inhibitors also depend on the promotion of antitumor immune responses, assessing cell cycle arrest and progression in cells treated with palbociclib remains a key approach for investigating the efficacy of CDK4/6 inhibitors. Here, we describe a method to assess cell cycle distribution simultaneously with active DNA replication by flow cytometry in cultured hormone receptor-positive breast cancer MCF7 cells.