A more complete understanding of the effect of colonic NECs on the SC population in the normal colon is crucial to understanding SC function and cell proliferation and will likely provide insight into the mechanism underlying SC overpopulation that drives CRC growth. It is known that somatostatin and SSTR expression is found in the normal colon and SSTR1+ cells are located in close proximity to SCs within the crypt SC niche (Additional file 1: Figure S1) [5, 21]. Being in close proximity to each other, we surmised that the interactions and communication between these cell types may be crucial to regulation of normal SCs. Research has shown that the SCs of the colonic crypt are relatively quiescent as SC do not divide as rapidly as other cells in the epithelium and dysregulation of the crypt homeostasis is what leads to CRC development [2, 22]. Focus has been placed on different molecular factors involved in colonic SC regulation such as microRNAs [23], transcription factors [24], and growth factors [25] that can regulate SC function. However, we suggest that in addition to such molecular factors, attention should be placed on cellular mechanisms such as the cell-cell interactions between NECs and SCs.
In our study, we first looked at the expression of ALDH and SSTR1 in different CRC cell lines, as well as, in matched normal and tumor tissue samples (Fig. 1). In addition, we found that SSTR1+ cells from HT29 and SW480 cell lines also express other neuroendocrine markers (Additional file 5: Figure S5). We observed differential expression of ALDH and SSTR1 in the CRC cell lines and were able to identify that in fact these markers identify two separate subpopulations of cells found in human tissue samples and in CRC cell lines. Next, we showed expression of mRNA encoding SST and its receptors is present in matched normal and tumor tissue samples (Fig. 2). Interestingly, SST was expressed in all the normal tissue samples, but not expressed in the matched tumor tissues. When we analyzed additional normal tissue samples (ones without matched tumor), the results still showed presence of SST expression in all samples analyzed (data not shown). In addition, expression of SSTR1 was present in both primary normal and matched tumor colon samples, as well as, in the normal only tissue samples analyzed (data not shown). SST receptors have also been reported to be differentially expressed in various tumor types [26], and our data from different CRC tissue samples supports the expression of SSTR1 [27].
Next, the expression of SST mRNA and its receptors was analyzed in the SW480 and HT29 CRC cell lines. The results showed a trend similar to the pattern of expression in normal and tumor tissue samples. The HT29 cells, which were originally derived from a low grade, well-differentiated tumor, expressed SST and all five of its receptors, but the SW480 cells, which were originally derived from a high grade undifferentiated tumor, did not express SST or SSTR5. Of note, at the protein level, SSTR1+ cells were expressed in all CRC cell lines and normal and tumor colon tissue samples. That SST and SSTR1 were always expressed in the normal colonic epithelial cells, and SST was completely lost in the tumor cells while SSTR1 was also expressed in the tumor cells of some patients is an important finding. Interestingly, a recent study similarly reported that SST expression is lost in CRC yet expressed in the normal colonic epithelium, thus supporting our findings [28].
Along with identifying the presence of ALDH+ and SSTR1+ subpopulations in the CRC cell lines and human tissue samples, we tested the growth abilities of HT29 and SW480 cell lines in terms of cell proliferation and self-renewal. A major aim of this study was to study the “stemness” characteristics of the HT29 and SW480 cells relative to the proportion of ALDH+ and SSTR1+ cells. Accordingly, several CRC cell lines were screened that were originally derived from CRCs having differences in histological grade, stage and differentiation. Within the CRC cell lines, we predicted that the relative proportion of ALDH and SSTR1+ cells in any given cell line will correlate with a certain rate of cell proliferation and self-renewal. Therefore, the CRC cell line that has a higher proportion of ALDH is predicted to show more characteristics of “stemness” (slower proliferation and smaller spheres). First, we obtained the cell proliferation rate of various cell lines over a five day time course. Our data on the in vitro growth of the cell lines showed that the proliferation rate inversely correlates with ALDH/SSTR1 quotient (Fig. 3). For example, HT29 cells (having a low ALDH/SSTR1 quotient) had the fastest growth rate (doubling time of 2.5d), while the SW480 cells (having a high ALDH/SSTR1 quotient) the slowest growth rate (doubling time of 4d).
The next step was to assess the ability to self-renew and form spheres from single cells embedded in soft agar. Our study showed the HT29 cells with a lower proportion of ALDH+ cells formed fewer but larger sized spheres than the SW480 cells (Fig. 3). This could be due to the fact that sphere formation assays measure the ability of a single SC to form many cells in culture [20, 29] so the more SCs in a culture the more spheres are formed. However, the difference between the sizes of the spheres could be due to the proliferation rate of the cell lines and the non-SC daughters with a faster doubling time could give rise to larger sized spheres. Our data indicates that the cell line with relatively more ALDH cells (SW480) showed more features of true quiescent- like SCs in terms of slower cell proliferation and smaller-sized spheres. On the other hand, the cell line with fewer ALDH+ cells (HT29) showed more features of transit amplifying or progenitor cells with a significantly higher proliferation rate and large-sized spheres.
That each CRC cell line has a unique ALDH+/SSTR1+ ratio that is maintained constant over multiple passages and that correlates with its growth dynamics in terms of proliferation and sphere-forming ability, suggests feedback mechanisms exist between ALDH+ and SSTR1+ cells that contribute to regulation of the ALDH+ population size.
To further explore the role of SST and its signaling via SSTR1 receptor, we chose to treat HT29 and SW480 cell lines with exogenous SST with the goal of understanding the effect on the ALDH population size. Already knowing the baseline proportions of ALDH+ to SSTR1+ cells, we could feasibly measure the changes to the ALDH+ population due to enhanced SSTR1 signaling. SST is a known to have anti-proliferative effects in normal dividing cells like intestinal mucosal [5] yet the mechanism involved is not well understood. SST can bind to all five SST receptors subtypes and exert its effects of anti-proliferation or secretion inhibition [5, 30, 31]. The dose of SST was determined using a concentration range of SST that is close to physiological levels. In treatment of both HT29 and SW480 cell lines with exogenous SST, it did not affect the ALDH population size in either line (Fig. 4). The fact that SST treatments did not change the ALDH population size or viability while decreasing proliferation (Fig. 4a-c) could be a result of prolonged cell cycle progression or delayed maturation.
On the other hand, the addition of cyclosomatostatin, a somatostatin receptor antagonist, to the SW480 and HT29 cells showed that when SST signaling was blocked, there was a significant decrease in the ALDH+ population in both cell lines. There was also a decrease in cell number after treatment, but no loss in cell viability. Given that upon inhibition of SSTR1 signaling, there is a significant decrease in the ALDH+ cell numbers, it seems that an appropriate level of SST and SSTR1 signaling is needed to maintain the ALDH+ cells in a slower cycling state and any more or less SSTR1 signaling can cause a significant change in the ALDH+ population size. Thus, results from both SST and cycloSST treatment suggests that just the right amount of SST signaling is necessary for maintenance of ALDH+ cells and generation of its proliferative progeny cell population.
Another important function of NECs in general is their ability to signal through paracrine or autocrine mechanisms [26]. To determine if SSTR1+ cell regulation of the ALDH+ cells (Additional file 4: Figure S4) might occur via paracrine signaling, we designed experiments in which ALDH+ and SSTR1+ cells were co-cultured in transwell dishes to avoid their direct cell-to-cell contact. In the HT29 cells, there were a reduced number of spheres formed and a significant difference in the average sizes, as compared to the co-cultures of ALDH+ cells with SSTR1 negative cells (Fig. 5a and c). In the SW480 cells, there was a significant decrease in the size and number of spheres formed between the ALDH+ and SSTR1 negative cells, and ALDH+ and SSTR1+ cells (Fig. 5b and d).
HT29 cells are fast growing colon cancer cells that contain few ALDH+ cells and more differentiated cell types. In comparison, SW480 cells are more undifferentiated and have a slower growth rate. Based on our data, co-culturing of ALDH+ cells with SSTR1+ cells had different effects depending on the cell line studied. Since HT29 cells have a faster growth rate (with a doubling time of less than 24 h), the effect of the SSTR1+ cells on ALDH+ cells was minimal. On the other hand, the effect of SSTR1+ cells on ALDH+ cells from the SW480 line exhibit a significant reduction in both sphere size and number. SSTR1+ cells affected the growth of the ALDH+ cells by limiting the number of spheres and size of spheres formed.
Collectively, our results indicate that SSTR1+ cells have a paracrine signaling type of interaction with colonic ALDH+ cells. Through our co-culture studies, there appears to be paracrine-mediated regulation via SSTR1+ cells to maintain the ALDH population size in a state of quiescence, leading to slower cell cycling. Since ALDH+ cells lack SST and SSTR1 expression and the ALDH negative cell population cells expressed both SST and SSTR1, we conjecture that SST signaling auto-regulates the rate of NEC maturation in a feedback manner as SCs mature along the NEC lineage, which contributes to quiescence of SCs and inhibition of proliferation.