Human prostate cancer ARCaPE
is a unique cell line for studying EMT (16
). Because of the tight correlation between EMT and tumor malignant potential, detailed investigation of ARCaPE
cells may lead to a mechanistic elucidation of molecular events underlying EMT in prostate cancer progression and metastasis.
In the present study, we used the previously described mouse model of human prostate cancer progression and metastasis (15
) to assess the extent of EMT in an ARCaPE
clone following successive orthotopic inoculation of ARCaPE
cells. We tagged the cancer cells with red fluorescence protein to sensitively detect tumor cell metastasis in the mouse host, and to identify cancer cells that adopted significant morphologic changes. This study revealed that through successive in vivo
tumor formation and metastasis, substantial numbers of ARCaPE
cells underwent drastic changes, adopting a mesenchymal stromal morphology and expression profile (, and ). Assisted by red fluorescence tracking, we isolated representative clones and demonstrated that ARCaPE
cells with stromal morphology have altered gene expression and increased malignancy. The study has potentially significant implications for the role of EMT in prostate cancer progression and metastasis.
This study revealed that EMT is a general phenomenon of ARCaPE
tumor cells during in vivo
xenograft growth. In a previous study (15
), we noticed the presence of large cells bearing stromal morphologies in ARCaPE
tumors (), but were not sure whether those cells were derived from the ARCaPE
lineage, since cells of the mouse host can be transformed by tumor cells (21
). In the present study, we used red fluorescence protein tagging to confirm that these cells are descendents of the ARCaPE
cells. Moreover, red fluorescence protein tagging revealed that substantial numbers of tumor cells have undergone EMT. Especially after a second round of inoculation, from 17% to 24% of the tumor cells were seen with mesenchymal stromal features (). EMT is thus a trend for ARCaPE
tumor cells when subjected to in vivo
growth and metastasis.
In clinical specimens, prostate cancer cells in the transition state of EMT are difficult to identify and the relevance of EMT to prostate cancer is controversial (14
). On the other hand, histopathologic analyses demonstrate that prostate tumor cells often lose epithelial properties and adapt mesenchymal stromal gene expression. Loss of the epithelial marker E-cad, for example, is a common observation in prostate cancer specimens (5
), while enhanced expression of the stromal cell intermediate filament protein vimentin is frequently seen (12
). Loss of E-cad and activated vimentin expressions are the two most informative markers of EMT (22
). Moreover, the EMT-like phenotype is correlated to progression and metastasis (14
), suggesting that EMT is an integral aspect of prostate cancer progression. In this study, we documented the progressive conversion of a clone of ARCaPE
human prostate cancer cells from epithelial to mesenchymal stroma-like cells (, , and ). Results from this study confirmed that prostate cancer cells have the capability of undertaking an EMT-like process. Most importantly, by evaluating isolated clones in athymic mice upon intracardiac inoculation, we demonstrated that cancer cells with EMT-like properties had increased tumorigenic potency ().
The tumor microenvironment plays a dominant role in determining the fate of tumor cells (23
). During prostate cancer progression and metastasis, infiltrating tumor cells come into direct contact with mesenchymal stromal cells. Tumor cells may have to be assisted by stromal cells for growth and survival by cancer-stromal interaction, and adopting stromal properties through EMT may provide tumor cells with advantages in migration, invasion and metastasis. The red fluorescent ARCaPE
-R1 cells used in this study were from a recently established clone, and were mostly morphologically homogeneous (). When subjected to cloning again in vitro
, all the clones maintained the epithelial morphology. In contrast, during xenograft tumor formation and metastasis in vivo
, these cells would frequently adopt stromal morphologies, which became more pronounced after successive inoculation. This study indicated an obligatory role of the host tumor microenvironment in promoting EMT-like changes. Further investigation is needed to define how interactions with the tumor microenvironment could result in such drastic morphologic and behavioral changes in cancer cells.
Results from this study suggest that mesenchymal stromal features are fixed permanently to xenograft tumor cells. ARCaPE
cells have the tendency to undergo EMT following a variety of extracellular and intracellular cues. We have reported that soluble factors, including EGF, IGF-1, TGFβ1, and β2-microglubulin, could induce EMT in ARCaPE
cells by receptor-mediated signal transduction (16
). The factor-induced EMT, however, was mostly transient and reversible, because the treated cells would resume epithelial morphology once the inducing factor was removed. ARCaPE
cells can also commit to EMT by endogenous and genetic changes. We have reported that stable overexpression of a constitutively active SNAIL mutant leads to EMT in ARCaPE
), and the same is true with stable overexpression of LIV-1 (16
). Importantly in both cases, the genetic manipulations resulted in permanent and irreversible EMT in these cells. Since cancer cells can acquire genomic and genetic changes through interaction with mesenchymal stromal cells (34
), it is likely that the permanent EMT observed in this study is caused by genetic changes. The representative clones have been isolated from ARCaPE
tumors and identification of the causal factors is currently underway.
Finally, this study demonstrated that fluorescence protein tagging is a sensitive technique to track xenograft tumor cells metastasis. Following a second inoculation, orthotopic tumors showed increased growth rates and distant metastasis. Due to tumor burden, the animals were sacrificed before metastatic bone lesions could be detected by conventional methods (data not shown). Nonetheless, with red fluorescence protein tagging, metastatic tumor cells could be detected and cloned from blood, bone marrow, ascites, and lung because tumor cells could be detected and distinguished from mouse host cells by red fluorescence ( and ). Similarly, we expect that red fluorescence may facilitate quantification and comparison of metastatic cells in these tissues. We are currently testing to quantify these cells with flow cytometric methods.