Initial characterization of IEC-18 culture heterotypy using immunolocalization
We have recently discovered a novel antigen localization pattern by using anti-carboxyl IGF binding protein-2 (IGFBP-2) antibody (to an antigen which we call C2) to demonstrate a multivesicular pattern in some IEC-18 cells but not in others that are immediately adjacent (illustrated in Figure 1A). We note that anti-amino IGFBP-2 showed no staining in IEC-18 cells and that pretreatment with synthetic antigen abolished C2 staining (data not shown), confirming that C2 is a carboxyl fragment of IGFBP-2. For the purpose of this study, we wanted to determine whether or not this heterotypy represented cell fate divergence or systemic pleiomorphism.
To further characterize the observed heterotypy, we examined IEC-18 cells with anti-actin antibody in IEC-18 cells without performing antigen retrieval and allowing the detection reaction to proceed until only a limited amount of staining is seen. When used with the right antibody, this technique is a means to visualize dynamic portions of actin filaments, such as stress fibers and the proximal ends of microvilli, because they lack the actin binding proteins that obscure the antigen. If the reaction is allowed to continue, eventually all actin filaments will stain. In the version that we use, the assay is a qualitative assessment of cytoskeletal turnover when we are comparing two adjacent cells (because both cells had exactly the same conditions for detection of actin). Both stress fibers and microvilli are heavily stained and appear to have high turnover rates in cells that have C2 staining, but not in those without it (Figure 1B). We note that the punctate dots of filamentous-actin corresponded with small microvilli in C2 positive cells and that the C2 negative cells had a paucity of microvilli on their surface – which was confirmed by focusing up and down the shafts of the microvilli (data not shown).
IEC-18 cells spontaneously lose C2 staining prior to confluence, despite 10% FBS
When IEC-18 cells were plated at variable densities in 10% fetal bovine serum (FBS), we saw that all crypt cells were initially positive for C2 staining, but with increasing density there were increasing numbers of C2 negative cells (Figure 1C). We also saw that C2 positive cells had increased staining intensity as they approached confluence. This experiment demonstrates two important points: first, that loss of C2 staining begins before confluence and second, that it can occur with comparable prevalence in the presence of FBS as it does in serum free media (SFM).
C2 positive cells and C2 negative cells are both capable of phenotype preservation during proliferation
To determine if the C2 positive and negative IEC-18 cells were each capable of phenotype preservation during proliferation, IGFBP-2 stained cells on coverslips were searched for mitotic cells in the stage of cytokinesis and their images were captured by digital photomicroscopy (n = 6 cell pairs for each phenotype). In each case, all daughter cells had the same phenotype as their sister, confirming that both the C2 positive and C2 negative phenotypes are conserved in subsequent cycles of mitosis (representative examples in Figure 1D).
C2 positive cell abundance is increased in proportion to the efficacy of IGF agonist treatment
Because IEC-18 cells grow best in the presence of high dose insulin, we suspected that crypt cell proliferation was dependent upon IGF receptor stimulation. In general, differentiated and benignly transformed epithelial cells are less likely to proliferate upon reaching confluence, so we sought to preferentially drive crypt cell proliferation in a graded fashion using different IGF receptor agonists (Figure 2). IGF-II analog, a weak agonist, reduced the crypt cell abundance while NBI 31772 (an agent that displaces IGFs from IGFBPs) increased it significantly and R3-IGF-I doubled the number of crypt cells per 10X field (also highly significant) while none of the treatments significantly altered the abundance of C2 negative cells. This strategy allowed us to systematically skew the cell composition and use gene array analysis to determine whether C2 positive cells were epigenetically divergent.
Our gene array methodology identified four candidate genes as potential markers of cell fate divergence in IEC-18 culture
Eleven genes met our criteria for a significant fold change, four were positively correlated with crypt cell abundance and seven were inversely correlated (Figure 3). Of these first pass candidates, only six showed the appropriate fold trends across all treatment conditions, consistent with our hypothesis of constitutive expression that could reflect divergent cell fates. Of these six, one was found to have a significant difference between IGF-II analog and SFM, suggesting a direct treatment effect by IGF agonists but not by NBI 31772 (dithiolethione-inducible gene 1). Enzymatic glycosylation-regulating gene is known to be an insulin responsive gene and was also excluded because of the high probability of a direct effect by our IGF agonists [6]. Of the four remaining candidates, one (brain acyl CoA hydrolase) had absolute values that bordered on background levels in the SFM, IGF-II analog and NBI 31772 treatment conditions (defined as 10 arbitrary fluorescent units) and has had a relatively limited characterization in the literature [7–9]. We saw no obvious means for obtaining or generating an antibody to it and thought it unlikely to be a robust marker at the protein level, in large part because its message has only been found in brain thus far. Another had high homology with the EGF family and is currently a predicted protein based on genomic sequence [10]. However, the two remaining candidate genes we pulled out were well-characterized gut-related proteins (APC and 5-HT2A).
Immunolocalization for 5-HT2A and APC revealed 2 divergent phenotypes within IEC-18 cell culture
Western blots using antibodies against APC and 5-HT2A in IEC-18 cell lysates revealed staining of the appropriate sized band for each (Figure 4). In the case of APC, there were several discrete smaller bands which were inversely proportional in abundance to the 300 kD full-sized protein (consistent with proteolytic fragments) when compared across multiple samples (data not shown). In the case of 5-HT2A, there was a single 28 kD band that was faint, suggesting relatively low abundance. Both antibodies were deemed suitable for immunolocalization.
Immunolocalization for APC revealed that C2 positive cells also had intense APC staining whereas C2 negative cells either had limited or no APC staining (Figure 5A). This finding is in keeping with our gene array analysis and suggests that there is substantial and wide spread divergence of IEC-18 crypt cells away from the crypt cell phenotype and towards an adenoma-like transformation.
Immunolocalization for 5-HT2A demonstrated a second cell phenotype (Figure 5B,C and 5D). We had previously noted rare neuroendocrine-like morphology characterized by spindle-shaped cells with bipolar, dendritic arbors (unpublished findings), however with 5-HT2A immunostaining we found a mean of 5 positively stained cells per coverslip of confluent cells (n = 6 coverslips of confluent cells in SFM). 5-HT2A is present in high abundance within paneth and neuroendocrine cell types but is absent within small intestine crypt cells in vivo [11]. The cells that we observed appeared to be in transition, going from IECs to neuroendocrine-like cell morphology – with corresponding increases in staining intensity. While still a very low prevalence, the staining was quite intense in this small subset and was not detected in any of the adjacent cells.
Double labeling confirms that C2 negative cells have diminished APC abundance
Double immunolabeling with overlay technique (using C2 and actin antibodies) demonstrated that C2 negative cells have a paucity of microvilli in comparison to C2 positive cells (Figure 6A). Additionally, double labeling with C2 and APC demonstrated that C2 positive cells have uniform APC staining whereas C2 negative cell have variable and overall diminished staining in comparison (Figure 6B). These experiments provide an objective demonstration that C2 positive cells retain the IEC phenotype whereas C2 negative cell are undergoing transformation (which is an obligatory step associated with the loss of APC).