In this manuscript we have taken advantage of an IEC immunolocalization marker that we call C2 to demonstrate two forms of cell fate divergence within IEC-18 culture. Using a combination of gene array screening and immunolocalization, we found that C2 is lost in over half the cells by the time of confluence and its loss is also associated with a down regulation of the APC gene, decreased APC protein abundance, decreased actin filament turn over, and reduced microvillar density. In short, there is an adenoma-like phenotype that fits with this genotype and this fits well with what is known about the function of the APC protein [12–15]. In addition, another cell genotype-phenotype correlate was detected by screening out the 5-HT2A gene and visualizing the cells that express its protein, i.e. neuroendocrine-like cells. These findings also fit well with cell phenotypes known to express 5-HT2A in the gut endoderm [11, 16, 17] and strongly argue against the dogma that IECs persist as a single lineage prior to reaching confluence.
While we think our findings have important implications for the existing IEC literature, the more important aspect of this manuscript may be in the methodology we have piloted. In many ways, cell culture, whether primary or immortalized, transformed or not, is by definition a model in flux. The progenitor lineage that a researcher starts with is rarely the hodge podge they end up with after a limited number of passages and it is common, if not expected that epigenetic drift will occur with each cell culture passage. However, what we describe in this manuscript is different in that IEC-18 cells are displaying a uniform trend in cell fate divergence – a trend that can be modulated with IGF. Many if not most gut epithelial cell lines are IGF (or high dose insulin) dependent for proliferation and thus are potentially vulnerable to this biological confounder. It will remain to be seen if other epithelial cell types have similar behaviors when examined in this fashion. Conversely, there is also a positive light to our findings; IEC-18 cells could be a compelling model for spontaneous adenomatous transformation because these adenoma-like cells are arising from a genetically competent progenitor prior to reaching confluence. To our knowledge, no such model with this property has been previously defined.
Our study has notable weaknesses and strengths. First, we have identified two divergent cell fates but have only partially characterized them because we were focused on developing a viable screening methodology (hence the ubiquitous use of the word "like" in this manuscript). Second, we are using a rat gene array chip that has approximately 9000 non-EST genes per chip. This is not an exhaustive survey of the rat genome and it is possible that there are other cell fates present in IEC-18 culture that we did not detect. Third, our phenotype assays are based on immunolocalization, which is a semi-quantitative technique with regards to assessing protein abundance. However, in this case we are actually combining cell-to-cell differences in protein abundance with distinguishing morphologic characteristics (e.g. loss of microvilli, flattening, bipolar shape, dendritic arbors, etc.) to delineate the phenotypes between adjacent cells. In short, what we are quantifying, in the case of adenoma-like cells, is the percentage of cells with a given phenotype. For this purpose, blinded immunolocalization is simple, quantitative and exceedingly efficient. The combined methodologies we chose result in a highly accurate technique for assessing divergence and their specificity can be bolstered by comparative studies of co-divergent markers (as we demonstrated with C2 and APC). As for other positives, the methodology is relatively rapid and can detect low prevalence phenotypes (as demonstrated by the anti-5-HT2A antibody). Additionally, we have demonstrated that a transcriptional marker is not required to create an effective screen. What is required, and what should probably prompt a researcher to employ this methodology, is a probe, a phenotype, or a pleiotropism that results in a consistently heterogeneous and quantifiable pattern (as C2 proved to be for us).
In closing, we point out one last caveat. Gene array investigation is an evolving science but there remain three potential pitfalls for every new application: experimental design flaws, data integrity issues and biological misassumptions [18–20]. In this study, we used a well-accepted screening principle (i.e. significant fold changes within individual genes in response to a treatment); we included a paired reference standard for each treatment condition (the SFM control); we increased the screening stringency by adding a requirement for parity in fold trend in accordance with changing cell compositions; and then confirmed our findings by phenotype assays. However, we demonstrated that a small minority of neuroendocrine-like cells were still able to significantly alter the outcome of our gene array screen (a possibility that we had thought to be remote, given our assay's stringency). We conclude that even low frequency epigenetic events can be a serious biologic confounder of gene array studies in cell culture.