Cell Death Identification in Anticancer Therapy—Response
Authors: Santiago Rello-Varona, David Herrero-Martín, Roser López-Alemany, Oscar M. Tirado
Journal: Cancer Research (2015)

We would like to thank Brown and colleagues (1) for their effort and their valuable comments on our review about cell death mechanisms in anticancer therapy. We understand their view on assessing the persistence of clonogenic survival from quantification of death records in anticancer research.As it has been pointed out, the major form of cell death triggered in a cell culture relies on the genetics of tumor cells and is not equivalent to clonogenic survival. Nevertheless, the mechanism of cell death could provide significant information about tumor cell response. Indeed, when death records are accurately collected, by including not only apoptosis but also necrotic events and other phenotypes linked to less clonogenic potential (like micro or multinucleated cells), they become more reliable and close to clonogenic data (2). Furthermore, classic colony formation assays (when cells are extremely diluted prior to or after therapy) cannot measure the influence of cell-to-cell or paracrine communications, and its relevance decreases when used in settings apart from DNA-damaging agents (3).In order to perform valuable clonogenic endpoints, researchers should understand that the genetic background not only modulates the type and rate of cell death, as Brown and colleagues point out, but also determines the cell growth rate. Therefore, endpoints and valid colony criteria should be set appropriately to avoid confounding slow proliferation with lack of clonogenicity (4).Despite not being a cell death modality, senescence's relevance in cancer biology is being increasingly recognized. Unfortunately as a “permanent” condition, senescence is hard to study. Current knowledge in cancer progression admits the existence of long-dormant cells that can restart proliferation after years of latency (5). Such a behavior is almost impossible to address in current in vitro experimental settings.To successfully eradicate a tumor from a patient requires, besides cell killing, enforcing the abrogation of tumor regrowth. It is assumed that a very little fraction (less than 10%) of tumor cells are active clonogenic cells, but less consensus is met when considering if those “cancer stem cells” constitute a phylogenic separated population or are the result of a series of events of stem genesis within the tumor cell population (4, 6, 7).In our review, we advocate for an easier clonogenic endpoint setting consisting of keeping the cell culture for a time after drug washout to measure if the culture keeps dying, remains stable, or regrows. This setting keeps cell-to-cell and paracrine communications in closer degree than what would happen in actual tumors without inducing further stress (like trypsinization). In regrowth situations, we advise to repeat treatments, assessing then the possible acquisition of resistance. For authors who prefer colony formation assays, our counsel is to clearly specify the conditions of the experiments in their Materials and Methods sections and, when possible, to try different settings: cell dilution prior to treatment versus cell dilution after treatment (in this case being sure that already dead cells are not being seeded) and fresh medium versus conditioned medium (from a growing culture, thus enriched with tumor cell secretion products). In any case, endpoint times and cell number criteria for a valid colony to be counted have to be stated.We hope that both Brown and colleagues' remarks and our comments help cancer researchers to better refine their experimental in vitro settings to acquire the maximum valuable knowledge prior to embarking in animal testing.See the original Letter to the Editor, p. 3681No potential conflicts of interest were disclosed.