It is becoming increasingly clear that normal tissue stem cells are localized in a defined microenvironment that provides specific factors for the maintenance of the properties of the stem cells as well as for the regulation of a balance between proliferation, differentiation and quiescence of these cells (33
). In prostate cancer, there is strong evidence that signals originating from the cancer-associated fibroblasts (CAFs) could significantly enhance the tumorigenicity of cancer cells. As a central role for CSCs is being ascribed for tumor homeostasis and progression (37
), we wished to inquire if CAFs may regulate the biology of prostate CSCs. This is a critical question for the hypothesis that terminally differentiated cancer cells may have only limited proliferation ability and CSCs, with asymmetric division to both self-renew and differentiate may indeed be responsible for the growth and progression of the tumor. In this report, we describe a mouse model of prostate adenocarcinoma from which CSC-enriched epithelial cells were derived to examine the effects of CAFs that were also generated from the tumors of the same model.
Interest in this study is three-fold. First, a modified method is described for the isolation of epithelial cell fractions retaining a small number of cells with properties of putative CSCs. A cell fraction from this tumor model is shown to possess self-renewal and spheroid-forming abilities along with multipotentiality for differentiation in vitro, and the ability to form tumor-like glandular structures in vivo under appropriate conditions. The selection for tumor cells (T-LSChi) with high levels of expression of both Sca-1 and CD49f surface markers appears to discriminate between these cells from those with high Sca-1 and medium CD49f levels (T-LSCme). While a cell fraction contained in the T-LSChi subpopulation displays spheroid-forming ability and the capability to generate prostate glandular structures, the T-LSCme subpopulation is practically devoid of these capabilities. The CSC-enriched T-LSChi is still mostly composed of non-CSC epithelial cells as evident from the efficiency of in vitro spheroid-forming ability, although it is likely that viability of all cells in the subfractions may not withstand the steps used for the isolation. The bulk of the cells in the T-LSChi compartment may represent transit-amplifying cells and terminally differentiated cells, and, thus, indicating that the markers used like Sca-1 and CD49f, are shared with non-CSC cells, but still, use of these markers in a quantitative manner, as shown here, does contribute to enrichment of prostate CSCs from the tumors of this mouse model.
Second, we have observed a significant difference in the pattern of relative expression of certain relevant genes in the T-LSChi
subpopulations. While expressions of basal cell markers CK5 and p63 are stronger in T-LSChi
relative to T-LSCme
, the T-LSCme
fraction is found to express higher levels of CK18 and AR as compared to T-LSChi
, although the level of AR is significantly reduced in both subgroups in comparison to the T-LSC−
cells. The same general pattern is found in the respective subpopulations from the normal mouse prostate. Thus, it appears that CSC-enriched fraction from the prostate tumor of the Pten
deletion model contain cells with the characteristics of the similarly enriched fraction from the normal mouse prostate. Two other putative epithelial stem cell markers were examined. The levels of each of CD44 and CD133 transcripts appear to be significantly higher in T-LSCme
subpopulation of the tumors relative to either T-LSChi
groups of cells, implying that these two markers may not characterize the CSCs of the tumors in the Pten
deletion mouse model. We also examined expression levels of three cancer-related genes: Survivin, Runx2
. Survivin, a member of the inhibitor of apoptosis (IAP) protein family, is highly expressed in human cancer (40
). In the conditional Pten
deletion mice, we demonstrated a strong correlation between increased levels of Runx2 transcription factor with the growth of the tumor (31
), an observation that is very similar to what we described for Survivin protein levels in the same model (30
). Moreover, Runx2 appears to be a major regulator of Survivin
gene transcription in prostate cancer cells (31
). GRP78, a major ER chaperone is reported to be highly induced in a wide range of tumors including prostate cancer (41
), and we described that loss of GRP78 in the prostatic epithelium can prevent prostate tumor formation in the Pten
deletion model (32
). Here, we find that while Grp78
expression is higher in the T-LSC−
population compared to either T-LSChi
subpopulations, the pattern is opposite in the case of Survivin or Runx2. Survivin
are expressed in both T-LSChi
groups of cells at levels even higher than the bulk of the cancer cells represented in the T-LSC−
fraction. Based on these results, we project that high levels of expression of Survivin and Runx2 might be associated with both CSCs and transit-amplifying cells as it is with many cancer cells. However, this contention remains to be tested at the level of individual cells, a task that is difficult at this time in the absence of definitive markers for the cell types under study.
Third, for the first time, we demonstrate that the spheroid-forming efficiency of the CSC-enriched cells is differentially influenced by the fibroblasts in co-cultures. The modified spheroid-forming co-culture system we used has the promise to be a powerful method to facilitate the studies of paracrine signalings in interactions between stromal fibroblasts and CSCs. Because fibroblasts are located on the insert above the matrigel layer, there is no direct cell-cell contact between the two cell groups in this system. An important finding from such analysis is that as compared to UGSM or NPFs, CAFs enhance spheroid formation in the first generation by approximately two-fold. In vivo, the grafts grown from the T-LSChi cells are found to contain multiple glandular structures in each case, although grafts formed with CAFs appear to exhibit higher proliferative index as compared to those formed with NPFs. The observations with CAFs underscore a role of CAFs in CSC biology, and open up possibilities for better identifying the responsible molecular interactions.
In summary, our study describes a process refined to enrich the putative CSC population using the surface marker phenotype of Lin−Sca-1hiCD49fhi from the prostate adenocarcinomas of the Pten deletion model and demonstrates that such cells have the capacity to form tumor-like structures in spheroids in vitro and grafts in vivo. The CSCs appear to retain properties of normal tissue stem cells, such as, the potential to self-renew and to generate differentiated progenies. Most notably, we present evidence that CAFs could enhance both the stemness and growth potentials of the CSCs. It is likely that these new clues could be further developed to better understand the biology of CSCs in prostate cancer and potentially, in cancers, in general.