Immunofluorescent microscopy to quantify and characterize chromosomal instability (CIN)

Chromosomal instability (CIN) is the continuous missegregation of chromosomes or chromosome fragments over multiple cell divisions and is present to some degree in nearly all cancer types. Chromosome missegregation causes aneuploidy, or daughter cells with an abnormal chromosome content, which is a hallmark of cancer. CIN can be caused by altered expression of mitotic checkpoint genes, defects in chromosome congression to the metaphase plate, hyperstable kinetochore-microtubule attachments, defective error correction, replication stress, defects in sister chromatid cohesion, and telomere crisis, among other intrinsic factors. CIN can also be induced by exogenous factors such as DNA tumor viruses, ionizing radiation, and microtubule stabilizing drugs. CIN has been shown to promote or suppress tumors or have no effect depending on the rate of chromosome missegregation. However, increasing CIN above a maximally tolerated threshold of chromosome loss leads to cell death, implying that altering the rates of CIN could be a potential therapeutic strategy. Furthermore, CIN leads to the formation of micronuclei, which can release cytosolic DNA and activate the cGAS-STING pathway, leading to increased innate and adaptive immune responses depending on the chronicity of CIN. Here, we detail a simple method to quantify and characterize CIN and micronuclei in cells by immunofluorescent microscopy. The technique described here can be implemented and optimized for human or murine cell lines or tissues.