Pancreatic cancer is one of the most life-threatening cancers and its prognosis has not been improved despite advances in diagnostic and therapeutic strategies. The reasons for resistance against conventional therapy and re-growth of untreatable tumor are now attributed to the existence of cancer stem cells (CSC), which occupy only a small part of the entire cancer tissue. CSC has characteristic features such as chemoresistance, establishment of metastasis and reconstruction of a hierarchical population of tumor cells. Specific surface markers have been identified, enabling the purification of CSC fractions from cell lines or clinical samples, serving to dissect the nature of CSCs. In addition, tumor stromal cells also contribute to the malignant behavior of cancer cells by promoting an invasive phenotype and the development of metastasis. Among those stromal cells, a mesenchymal stem cell (MSC) has unique contributions to the cancer tissue since this type of cell could accumulate in the tumor from distant organs such as bone marrow. MSC also acts as a defender of cancer cells by infiltrating into the tumor. This unique feature is now applied to generating cancer-specific delivery systems by utilizing MSC as a vehicle of therapeutic agents. The tumor microenvironment, which is created by the complex interaction between cancer cells and stromal cells, yields cancer promoting effects, especially induction of a hypoxic environment. Hypoxia- induced signals activate cellular adaptation machinery such as increased cell survival or enhanced stemness in pancreatic cancer cells. Tumor stromal cells also support engraftment of metastatic nodules which provide a partial CSC niche. Targeting the tumor stroma, CSCs, and MSCs will possibly lead to the development of novel therapeutic methods against pancreatic cancer.

Introduction

Pancreatic cancer is a characteristic cancer by its dismal prognosis and resistance against conventional therapies such as chemotherapy or radiotherapy. Only ~20% of pancreatic cancer patients are eligible for surgical resection, which is the only curative therapy, though the 5-year survival in these patients is less than 25% (1). Gemcitabine is a standard chemotherapeutic agent against pancreatic cancer, while the efficacy of treatment stays palliative in patients with unresectable disease (2). The reasons for such clinical course are due to the regrowth of therapy-resistant tumor after therapeutic interventions. The concept of CSC, which attracts great attention in the past decade, accounts for these phenomena as a small subset of therapy-resistant cells which give rise to the untreatable tumors. In pancreatic cancer, several CSC markers such as CD24/CD44/ESA triple positive (3) or CD133 (4) are identified and these CSC fractions revealed higher tumorigenicity, metastatic capability and chemoresistance.

Pancreatic cancer manifests distinct histological characteristics referred to as a desmoplastic reaction which consists of dense fibrotic stroma surrounding tumor cells (5). Establishment of this fibrotic tissue results from inflammation caused by cancer cells which activate intra- and/or extra-pancreatic pro- inflammatory cells (6). It is noteworthy that the inflammatory process can recruit bone marrow derived cells into the tumor tissue (7, 8). It has been suspected that the existence of a desmoplastic reaction might contribute to pancreatic cancer progression. Recent research uncovered a significant role of the tumor stromal cells (including MSC) in pancreatic cancer such as protection from chemotherapeutic agents (9) or establishment of distant metastasis (10). Interaction between cancer cells and stromal cells is a dynamic process which involves production of multiple cytokines and activation of various signaling pathways. Stromal cells also contribute to the maintenance of CSC function (11) which is the latest target for novel therapeutic intervention against CSC.

This chapter reviews the characteristic feature of CSC in pancreatic cancer, interaction of cancer cells and tumor stromal cells, and contribution of stromal cells during cancer progression.

Normal pancreatic stem cell and pancreatic CSC

Recent research has uncovered that tissue patterning of normal organ development requires normal tissue stem cells to give rise to a wide variety of cells (12). These normal tissue stem cells are capable of self-renewal while providing progenitor cells for asymmetrical division (13). The CSC concept was established as a counterpart to normal tissue stem cells. As shown in figure 1, normal tissue stem cells give rise to various types of cells. In contrast, CSC has self-renewal capacity and manifests several characteristics such as initiation of metastasis, chemoresistance, and hierarchical reconstitution of the entire tumor. The origin of cancer is possibly attributed to the normal tissue stem cell due to the similarity of normal stem cells and CSCs in regards to possessing varying degrees of differentiation (14). However, normal tissue stem cells and CSC are not always identical. Normal tissue stem cells reside in the assigned tissue structure while CSC are capable of migrating from the original tumor to the establishment of distant metastasis (“migrating CSC” concept) (15). This concept also indicates the partial independence of CSC from stem-cell niche which plays an indispensable role in the maintenance of the normal tissue stem cell (16).

Figure 1.

Comparison of the normal pancreatic stem cell function and CSC function.

Marker of normal pancreatic stem cell and CSC

The precise location of normal tissue stem cells within pancreas is still controversial. The centroacinar cell is considered to be a possible pancreatic tissue stem cell according to its anatomical location and the expression of the transcriptional factor Hes1 which marks multipotent progenitor in the mouse pancreas (17). Recent research identified that the intestinal stem cell marker DCAMKL-1 was successfully isolated from a normal pancreatic stem cell fraction (18). Isolated cells formed spheroids in the non-adherent culture condition and expressed markers of exocrine pancreas in this study.

However, above mentioned molecules are not utilized to purify the CSC fraction from pancreatic cancer cell lines or surgical specimen. Instead distinct surface markers have been used to isolate CSCs. For example, side population cell is defined by the ability to excrete Hoechst dye from cell and identified as a hematopoietic stem cell marker (19). Side population cells from pancreatic cancer cell lines also contain CSC fraction evidenced by increased tumorigenicity and chemoresistance (20). The CD24/CD44/ESA triple positive cell population isolated from the surgical specimen also depicted the increased tumorigenicity in another study (3). The CD133/Prominin positive cell population revealed the capability to reconstruct the hierarchical population of tumor cells in an orthotopic implantation model (4). Furthermore, CD133/CXCR4 double positive subpopulation was identified to be essential for the establishment of metastatic lesions. Currently the CSC phenotype can be summarized by its tumorigenicity, chemoresistance, metastatic ability and reconstruction of hierarchy. These characteristics were based on the resultant cancer cell behavior after the isolation of specific population from entire cancer cells. Definitive CSC functions need to be elucidated for clarifying the nature of CSC.

Pancreatic cancer stem cell and therapy-resistance

Therapeutic strategies against pancreatic cancer suffer from frequent recurrence after radical surgery and resistance against conventional therapies such as chemotherapy and radiotherapy (21). A small number of CSCs can reconstruct the entire cancer tissue, leading to the recurrence after surgery. CSCs are reported to be highly resistant to the current chemotherapeutic agents (22). Currently available chemotherapeutic agents mainly target proliferating cancer cells and are unable to eliminate CSCs. The entire mechanism of chemoresistance in CSC is cryptic but recent researches clarified part of this picture.

Side population cells are reported to express high level of ATP-binding cassette (ABC) transporters. Expression levels of ABC transporters in cancer cells determine chemoresistant phenotype since chemotherapeutic agents such as etoposide, doxorubicin, vincristine and paclitaxel are direct substrates of ABC transporters (23, 24). Side population cells in pancreatic cancer also express ABCB1 and ABCG2 which play important role in chemoresistance of tumors in other organs (25). Expression of ABCG2 induces resistance against gemicitabine (26) though there is no clear evidence that ABC transporters directly efflux gemcitabine or its metabolites in pancreatic cancer cells. The cellular uptake of gemcitabine is mediated by nucleoside transporters including ENT1 (27) and expression levels of these molecules in side population cells or other CSC fractions are not fully explored. Detailed analysis will identify the mechanism of resistance in CSC of pancreatic cancer.

These reports mainly focused on the existing therapy-resistant population in cancer cells. Another report indicated that conventional chemotherapy itself could propagate the CSC population in pancreatic cancer. Long-term culture of pancreatic cancer cell line L3.6pl and AsPC-1 with increasing doses of gemcitabine enhances the CD24/CD44/ESA triple positive CSC fraction (28). These cells also revealed spindle-shaped appearance and decreased expression of E-cadherin which are the specific features of epithelial-mesenchymal transition (EMT) suggesting the acquisition of a metastatic phenotype. These observations indicate that conventional therapies could create selective pressure for more malignant phenotype of cancer cells.

Interaction of cancer cells and stromal cells

Pancreatic cancer cells affect stromal cell function and vice-versa. The interactions between pancreatic cancer cells and stromal cells were extensively studied but little was known about the detailed molecular mechanism so far. Detailed characterization of cell surface markers and/or cytokines has exposed intriguing mechanisms involved in these interactions. Numerous factors produced in pancreatic cancer stromal cells have tumor- promoting effect as inducers of cell migration and invasion of cancer cells. For instance, CD133 positive pancreatic cancer cells showed enhanced cellular migration and invasion after co-culture with stromal cells (11). The siRNA-based knockdown of CXCR4 in CD133 positive cells attenuated cellular migration and invasion which suggest that CSC-supporting function of stromal cells could be mediated by humoral factor such as SDF-1, the CXCR4 ligand. In addition, SPARC is also a secreted protein from tumor stromal cells and higher SPARC expression in pancreatic cancer stroma inversely correlated with overall survival. SPARC enhanced the cellular invasion of pancreatic cancer cells under the co-culture with human pancreatic stellate cells (hPSC) in vitro (29). These findings clearly demonstrate that cancer stromal cells enhance the metastatic potential of cancer cells which may also be a CSC function.

Pancreatic cancer cells are also capable of modulating stromal cell functions. Pancreatic stellate cells (PSCs) play a central role during the establishment of fibrosis after inflammation in the pancreas, though they normally reside within the pancreas in a quiescent state (30). PSC is activated by cytokines, reactive oxygen species and other stimuli caused by inflammation. Pancreatic cancer cells also activate PSC via humoral factors in a co-culture system (31) which indicates that pancreatic cancer cells can create a suitable microenviroment for their survival by activating stromal cells. A recent study indicated that the novel ligand named PAUF (pancreatic adenocarcinoma upregulated factor) from cancer cells could activate the Toll- like receptor 2 (TLR-2) and induce extracellular signal-regulated kinase phosphorylation in THP-1 cells (a human acute monocytic leukemia cell line) (32). This signaling pathway facilitates the production of tumor-promoting cytokines while inhibiting innate immune surveillance against cancer cells. Hedgehog signal is also reported to have a protective role in pancreatic cancer. This signaling pathway affects the neovascularization of pancreatic tumor via the alteration of Ang-1 and IGF-1 expression in bone marrow- derived pro-angiogenic cells (33). Further investigation should be carried out since CSC has a distinct expression profile of these stroma-modifying factors from other types of cells.

Role of MSC in pancreatic cancer

MSC was first identified within the bone marrow cells and their localization was confirmed around the perivascular area in various organs (34). MSCs can differentiate into mesenchymal tissues such as bone, adipose tissue, and cartilage (35). Recent research suggested its possible role during inflammation, immune response, wound healing, and cancer progression (36-39). MSCs, as well as other types of stromal cells, have been reported to contribute to pancreatic cancer progression. Tumor-derived growth factors such as platelet-derived growth factor, epidermal growth factor and VEGF induced migration of MSCs in in vitro experiments (40). Migrated MSCs could form spheroids with pancreatic cancer cells in vitro and these spheroids can also reside within orthotopically implanted tumors in an in vivo study. Increase in the vascularity was observed in these orthotopic pancreatic tumors with MSCs. This effect was attributed to the VEGF production by MSCs. Current knowledge suggests MSCs recruited into tumor facilitate cancer therapy resistance by promoting tumor growth.

However, active incorporation of MSCs into pancreatic tumors has now drawn attention to the possibility that this phenomenon could be a novel therapeutic target for specific delivery of anticancer agents. Genetically engineered labeled MSCs injected into tumor-bearing mice efficiently accumulate within the pancreas tumor (41). Another group indicated the possibility of adipose-derived MSC utilization for the tumor-specific delivery of tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) (42). A similar approach was also made by combining the TRAIL expressing MSC and knockdown of X-linked inhibitor of apoptosis protein in cancer cells by RNA interference (43). These approaches seem promising as tumor-specific delivery system which consist from sustainable resource by utilizing patients cells. More efficacious methods need to be developed to accumulate “engineered” MSCs into the tumor for clinical application.

Microenvironment of pancreatic cancer; Role of hypoxia

Pancreatic cancer is generally recognized as a less enhanced lesion by contrast-enhanced computed tomography which reflects hypovascularity in cancer tissue including dense stromal cells and a few cancer cells as mentioned earlier. Poor blood perfusion results in significant hypoxia of cancer cells compared to the normal tissue, and this status triggers cellular adaptation. The transcriptional factor HIF1α contributes to the gene expression required for the adaptation for hypoxia and angiogenesis upon hypoxic conditions, which rapidly degrade normoxia (44). HIF1α is frequently expressed in pancreatic cancer and higher expression correlates with poor prognosis (45). The HIF1α mediated signal influences both cancer cells and stromal cells. The HIF1α contributes to the production of VEGF which acts as a potent inducer of angiogenesis (46). In addition, the microRNA miR-210 is induced by hypoxic conditions in various cancer cells including pancreatic cancer in HIF1α dependent manner (47). Cellular adaptation to the hypoxic condition known as the “Pasteur effect”, switches energy production to glycolysis from oxidative phosphorylation. This is regulated by miR-210 which directly targets iron- sulfur cluster assembly proteins (ISCU1/2) in primary endothelial cells and transformed epithelial cells (48).

The detailed relationship between hypoxia and normal tissue stem cell or CSCs remained unclear until this decade. Recent progress in this field of research indicated the essential role of hypoxia during the maintenance of normal stem cell or CSC function. Long-term culture of human embryonic stem cell (hESC) under normoxic conditions frequently results in spontaneous differentiation while hypoxic culture condition significantly lowered this ratio, with sustained expression levels of Nanog and Oct4 (49). Hypoxia-mediated signals contribute to the maintenance of CSCs or MSCs in several reports. For example, CD133 expression levels in cancer cells turned out to be regulated by HIF1α as a downstream effector of the mTOR pathway (50). Furthermore, short term exposure of MSCs to hypoxic conditions resulted in increased expression of CX3CR1and CXCR, which increased the ability of cells to migrate after stimulation and xenotypic grafting into chicken embryo (51). This line of evidence indicates that hypoxia could contribute to the propagation of cancer cells with the CSC phenotype and expansion of cancer-promoting MSCs within a tumor.

Pancreatic cancer stromal cells as a possible CSC niche

Establishment of distant metastasis is a critical step of pancreatic cancer since it is deemed as an unresectable disease with extremely poor prognosis. Cancer metastasis requires multiple biological processes such as migration from the site of origin, invasion beyond the basement membrane, extravasation into the blood vessel or lymphatic vessel, and engraftment in distant organ. These processes also require dynamic cancer cell-stromal cell interactions. Contribution of stromal cells to the cancer cell migration and invasion was already discussed in the previous section. Several reports indicated that stromal cells also have a beneficial role in engraftment of cancer cells. Injection of pancreatic cancer cells combined with gender- mismatch PSC into mouse pancreas resulted in increased number of distant metastasis to liver, mesentery, or mediastinum (10). Metastatic nodules contained exogenous PSCs which were identified by the presence of Y chromosome. These results clearly demonstrated that stromal cells yield survival advantages to CSCs, particularly during metastasis as a niche since metastatic cancer cells are considered to harbor the CSC phenotype (15).

Inhibition of the cancer-stromal cell interactions revealed favorable capabilities to reduce tumor invasion and metastasis in experimental models. Contribution of hedgehog pathway to the cancer-stromal cell interactions were extensively studied for many years since small molecule inhibitors such as cyclopamine and its derivatives were available (52). A novel inhibitor of the hedgehog pathway, IPI-269609, was reported to reduce metastasis and invasion in an orthotopic implantation model (53). Treatment of xenograft tumor by this agent resulted in the decrease of aldehyde dehydrogenase- positive cells which contain CSCs. Another study indicated that alteration of tumor stromal structures by hedgehog inhibition (54) showed that CSCs are sensitive to modulating the pancreatic cancer stroma.

Closing remarks

Characteristic features of CSCs, cancer stromal cells, and MSCs were discussed in this chapter. Tumor stroma in pancreatic cancer is not a wasteland but a formidable defender of cancer cells and CSCs. Conventional therapies which ta rget cancer cells reach their limit, stressing the importance of targeting cancer associated tissues such as tumor stroma or vasculature. Possible therapeutic targets within the cancer cell-stromal cell interactions are summarized in figure 2. Molecular targeting of cancer storma or vasculature is now carried out and several compounds are undergoing clinical trials. Further investigation will provide novel therapeutic strategies into the pancreatic cancer treatment.

Figure 2.

Therapeutic targets within the cancer-stroma interaction.

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