Fibroblast-Derived CCL2 Induces Cancer Stem Cells—Response
Authors: Akihiro Tsuyada, Shizhen Emily Wang
Journal: Cancer Research (2013)

We appreciate the interest of Gunaydin and colleagues in our study showing a role of fibroblast-derived CCL2 in inducing cancer stem cells. In a Letter to the Editor, they expressed the concerns about to what extent the sphere-forming property can reflect the cancer stem cell nature and the interpretation of the use of PKH dye in the sphere model.Sphere formation assays, which have been commonly used to retrospectively identify normal and cancer stem cells, indeed measure stem cell/early progenitor activity, not only in breast cancer, but in other solid cancers as well, such as epithelial ovarian cancer and head and neck squamous carcinomas (1–3). However, to the best of our knowledge and for solid tumors such as breast cancer, the terms "cancer stem cells," "cancer stem/progenitor cells," "cancer stem cell-like cells," and "cancer-initiating cells" are not yet clearly standardized for their definitions and are often used interchangeably by different groups to refer to the same fraction of cancer cells with the "cancer stem cell" potential. That is, the potential to initiate a new heterogeneous tumor that contains both a small fraction of cancer cells with the same properties as the initiators (self-renewal) as well as cells exhibiting diverging properties ("differentiation" or the ability to repopulate a heterogeneous entity). This tumor-initiating potential, although conceptually related to some properties of normal stem cells) has already diverged from a stem cell prototype and is difficult to be further categorized into a "stem" or "progenitor" population. Our study, along with many other studies characterizing cancer stem cells in solid tumor models, focuses on these tumor-initiating cells and their ability to repopulate a new tumor while maintaining themselves through self-renewal, a property commonly associated with "stem" or stem-like cells. So far, little is known about the presence of a quiescent state and induction to a proliferative state for cancer stem cells. The impact of different cancer stem cell states on tumor initiation and progression will be an interesting direction for future studies.Although the sphere formation assay provides an efficient and cost-effective way to assess cancer stem cell activity in vitro, it is apparently not a gold standard test for stem cells and needs to be combined with other functional tests. Therefore, we complemented our results with (i) PKH fluorescence intensity as a measure of the proliferative potential of the cells in the mammosphere as a means to identify the stem cell-like population, (ii) pathway analysis of CCL2-induced activation of the tumor-initiating population, showing that NOTCH1 was the major pathway of induction, a pathway which is known to regulate stem cells (4), (iii) in vivo tumor formation assay, and (iv) aldehyde dehydrogenase activity assay (5). Data from all 4 approaches suggest that fibroblast-derived CCL2 influences the tumor-initiating potential of cancer cells and the population of cancer cells exhibiting cancer stem cell-like properties through the activation of p38 and NOTCH1. In our original article (5), we also interpreted our sphere formation result using discreet terms such as "the stem cell-like mammosphere-forming phenotype."As Gunaydin and colleagues correctly concluded, our PKH dye-labeled sphere model suggested that CCL2 induces more cells to enter the stem cell-like, slow-proliferating, or quiescent state (PKHhigh) in a single-cell–originated mammosphere. We did not show the sphere formation data of the PKHmid population in the original article due to the difficulty of evaluating the proliferation history of these cells, as they can represent both cells that proliferate at a slower rate and cells that have remained non-proliferating for a segment of time during sphere formation. The sphere formation efficiency of these intermediate (PKHmid) cells ranged from 2% to 2.5%, whereas that of the PKHhigh cells ranged from 9% to 10% (Fig. 1, left). We did not detect a significant difference between CCL2-treated and nontreated spheres, suggesting that CCL2 does not significantly alter the corresponding sphere-initiating potential of PKHmid and PKHhigh cells. Expression of the stem cell markers Oct4 and Nanog were significantly higher in PKHhigh cells compared with the PKHmid cells (Fig. 1, right), further supporting the stem cell–like properties of PKHhigh cells. When sorted PKHmid and PKHhigh sphere cells were allowed to grow in 2D, we did not observe any significant difference in the morphology and proliferation rate between the 2 populations (data not shown). Therefore, although it remains possible that epithelial–mesenchymal transition (EMT) may occur in some of the sphere cells, the increase of secondary sphere formation as an effect of CCL2 likely reflects increase of the PKHhigh population in the primary spheres, suggesting an increase in the percentage of the tumor-initiating population.See the original Letter to the Editor, p. 1031No potential conflicts of interest were disclosed.