The importance of the local tumor microenvironment to tumor progression has been recognized for many years and highlighted in several reviews (2
). Insight into the role of tumor-associated stroma in breast and prostate cancer has recently been described (4
); however, in contrast, there is little known about the possible contribution of the stroma to pancreatic cancer growth and metastasis. This is particularly unfortunate as pancreatic cancer has an especially abundant stroma. Using a new model of human cancer-associated stellate cells, we found that conditioned medium from HPSCs stimulated pancreatic tumor cell proliferation, migration, invasion, and anchorage-independent growth in a dose-dependent manner. Moreover, conditioned medium from HPSCs inhibited the response of tumor cells to chemotherapy and radiation. These observations indicate that soluble factors are produced by stellate cells that stimulate proliferation and survival of pancreatic cancer cells. We also observed that co-injection of stellate cells increased tumor incidence, growth, and metastasis in an orthotopic model of pancreatic cancer. Taken together, these studies indicate that the abundant stroma of pancreatic cancer plays an important role in the aggressiveness of this disease.
Previously, a relevant cell model to study pancreatic cancer–associated stromal fibroblasts did not exist. Although the pancreatic stellate cell has been identified as the cell responsible for stroma production in both chronic pancreatitis and pancreatic cancer (5
), few studies have used pancreatic stellate cells from human pancreatic adenocarcinoma samples due to the relative paucity of available fresh tumor specimens and the limited life span of primary cells. Immortalization of rat (10
) and human (29
) stellate cells has previously been found to be useful because primary cells grow slowly and eventually senesce after 10 to 15 passages. Our study is the first report of the use of immortalized primary HPSCs derived from pancreatic adenocarcinoma. Previous studies in other solid tumors have suggested that phenotypic differences exist between stromal cells derived from tumors and normal tissues (23
). Therefore, these tumor-derived cells are the most relevant cell model to study the role of pancreatic cancer–associated stromal fibroblasts.
We found that the effects of HPSCs on pancreatic cancer were dose dependent. In vitro
, this was manifest as a direct concentration dependence of the effects of conditioned media. In vivo
, a tumor-to-stroma ratio of at least 1:1 or higher resulted in the most dramatic increase in tumor growth and metastasis. The higher proportion of stromal cells compared with tumor cells is consistent with the histologic appearance of human pancreatic adenocarcinoma, in which the desmoplastic stroma is overwhelmingly abundant relative to neoplastic epithelial cells (16
). This is the first study to co-inject HPSCs with cancer cells in an orthotopic model of pancreatic cancer; however, other investigators have also found that the presence of pancreatic stellate cells increases growth of pancreatic cancer cells in s.c. xenograft models (12
The extensive stromal component of solid tumors has been postulated by other groups to influence tumor response to chemotherapy (32
). In the current study, we have confirmed this hypothesis. We observed HPSC-mediated inhibition of pancreatic cancer cell responses to gemcitabine and radiation. Interestingly, Ohuchida et al. (34
) also found that radiation treatment of stromal fibroblasts increases the invasiveness of pancreatic cancer cells even more than nonirradiated fibroblasts. Thus, by including the stromal contribution to pancreatic cancer, our xenograft model with co-injection of HPSCs and pancreatic tumor cells may be a more clinically relevant model of pancreatic adenocarcinoma than previous xenograft models of tumor cells alone.
The mechanism responsible for the increased survival of pancreatic cancer cells treated with conditioned medium from stellate cells is not certain. We observed that such treatments resulted in the activation of both proliferation related (Erks) and survival related (Akt) pathways; these processes may be mediated by interleukin-1β and transforming growth factor-β, both of which are secreted by pancreatic stellate cells (35
). Additional candidate molecules that may mediate tumor-stromal interactions have been identified by gene expression profiling of stromal fibroblast cocultured with CFPAC1 pancreatic cancer cells (36
). Of the genes identified, COX-2
was markedly augmented in both cancer cells and fibroblasts when cultured together and was shown to mediate invasion of CFPAC1 cells. In contrast, other stromal-derived factors may suppress tumor growth. Sato et al. (37
) found that SPARC was overexpressed in stromal fibroblasts in pancreatic cancer and exogenous SPARC decreased proliferation of pancreatic cancer cells in vitro
. Thus, the interactions between tumor and stromal fibroblasts are quite complex and further studies will be necessary to positively identify the factors and signaling mechanisms involved in these interactions.
Another important observation in the current study was that the presence of HPSCs increased the incidence of tumor formation when limiting numbers of cancer cells were injected. Inclusion of stellate cells allowed the injection of low numbers of cancer cells that otherwise were unable to form tumors. This supports the hypothesis that stellate cells provide a microenvironment that is particularly advantageous to pancreatic cancer cells. It is possible that stromal fibroblasts may interact with pancreatic cancer stem cells, which have recently been described by Li et al. (38
). The tumor vasculature, another important component of the tumor microenvironment, was recently shown to influence the number of brain cancer stem cells (nestin+
; ref. 39
). Thus, targeting the “niche” or unique microenvironment of the cancer stem cells may be an effective therapeutic strategy. The mechanisms responsible for this effect are not completely understood, but the observation suggests an important role for stellate cells in tumor initiation.
The current study is limited to a single HPSC line isolated from a single patient with pancreatic adenocarcinoma. The effects of stromal fibroblasts from different individuals may vary, as has been shown in prostate cancer (40
). Our lab has isolated HPSCs from several other patients with pancreatic adenocarcinoma but these have not yet been immortalized. Another potential limitation of this study is the two-dimensional nature of the in vitro
experiments. It is increasingly recognized that two-dimensional models of cancer can differ significantly from three-dimensional models in cell morphology and behavior of tumor and stromal cells (20
). It is likely that in three-dimensional cultures, HPSCs may express genes that were not identified in our study, either alone or in coculture with tumor cells. Finally, our study focuses on cancer-associated fibroblasts as the sole stromal element interacting with tumor cells. However, many other cell types in the stroma are known to influence tumor cells, of which the best studied are the endothelial cells and pericytes that compose the vascular compartment. Other cell types in the stroma, such as immune cells (44
) and adipocytes (45
), also interact with tumor cells to mediate malignant behavior. Thus, a more physiologically relevant model for human pancreatic adenocarcinoma would be one that includes all stromal cell types along with tumor cells in a three-dimensional environment, a model which is the subject of active investigation in our laboratory.
In conclusion, our data provide evidence that stromal fibroblasts and tumor cells interact to promote tumor progression in pancreatic cancer. The stroma also impairs tumor cell response to chemotherapy and radiation. Moreover, in an orthotopic model of pancreatic cancer, stromal fibroblasts enhanced tumor growth, metastasis, and initiation. Our lab is currently investigating several stroma-specific factors associated with pancreatic cancer and we hope to elucidate the specific mechanisms of action of these molecules. With this information, targeting the stroma in pancreatic cancer may not only be effective in treating the primary tumor and metastases but may also play a role in prevention of tumor development as well. The identity of these stellate cell derived factors and their mechanisms of action are the subject of ongoing investigations and may lead to novel therapies directed toward the microenvironment of pancreatic cancer.