Stroma and pancreatic ductal adenocarcinoma: An interaction loop

Biochimica et Biophysica Acta 1826 (2012) 170–178

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Review

Stroma and pancreatic ductal adenocarcinoma: An interaction loop Guopei Luo 1, Jiang Long 1, Bo Zhang, Chen Liu, Jin Xu, Quanxing Ni, Xianjun Yu ⁎ Department of Pancreas & Hepatobiliary Surgery, Shanghai Cancer Center, Shanghai, 200032, China Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, 200032, China

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Article history: Received 31 January 2012 Received in revised form 6 April 2012 Accepted 8 April 2012 Available online 13 April 2012 Keywords: Pancreatic ductal adenocarcinoma Stroma Tumor microenvironment Targeted therapy

a b s t r a c t Pancreatic ductal adenocarcinom a (PDA) has two exceptional features. First, it is a highly lethal disease, with a median survival of less than 6 months and a 5-year survival rate less than 5%. Second, PDA tumor cells are surrounded by an extensive stroma, which accounts for up to 90% of the tumor volume. It is well recognized that stromal microenvironment can accelerate malignant transformation, tumor growth and progression. More importantly, the interaction loop between PDA and its stroma greatly contributes to tumor growth and progression. We propose that the extensive stroma of PDA is closely linked to its poor prognosis. An improved understanding of the mechanisms that contribute to pancreatic tumor growth and progression is therefore urgently needed. Targeting the stroma may thus provide novel prevention, earlier detection and therapeutic options to this deadly malignancy. Accordingly, in this review, we will summarize the mechanism of PDA stroma formation, the role of the stroma in tumor progression and therapy resistance and the potential of stroma-targeted therapeutics strategies. © 2012 Elsevier B.V. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Mechanism of stroma formation and its role in PDA development and progression 2.1. Components of the stroma . . . . . . . . . . . . . . . . . . . . . 2.1.1. PSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2. Fibroblasts . . . . . . . . . . . . . . . . . . . . . . . . 2.1.3. Immune cells . . . . . . . . . . . . . . . . . . . . . . . 2.1.4. Source of stromal cells . . . . . . . . . . . . . . . . . . . 2.2. Growth factors . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.1. TGF-β . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2.2. Shh . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Causes of extensive stroma formation . . . . . . . . . . . . . . . . 2.3.1. Chronic pancreatitis . . . . . . . . . . . . . . . . . . . . 2.3.2. Environmental/lifestyle factors . . . . . . . . . . . . . . . 2.3.3. Aging . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.4. EMT process . . . . . . . . . . . . . . . . . . . . . . . . Role of stroma in PDA therapy resistance . . . . . . . . . . . . . . . . . . 3.1. Failure of surgery . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. Chemoresistance . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. Radioresistance . . . . . . . . . . . . . . . . . . . . . . . . . .

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Abbreviations: PDA, pancreatic ductal adenocarcinoma; PSCs, pancreatic stellate cells; ECM, extracellular matrix; NGFs, nerve growth factors; α-SMA, α-smooth muscle actin; PDGF, platelet derived growth factor; TGF-β, transforming growth factor β; FGF, fibroblast growth factor; TNF-α, tumor necrosis factor α; IL, interleukin; MMPs, matrix metalloproteinases; TIMPs, tissue inhibitor of metalloproteinases; COX-2, cyclooxygenase-2; FAP, fibroblast activation protein; CAFs, cancer-associated fibroblasts; SDF-1, stromal cell-derived factor 1; IGF1, insulin-like growth factor 1; HGF, hepatocyte growth factor; M-CSF, macrophage colony stimulating factor; TAMs, Tumor-associated macrophages; EGF, epidermal growth factor; EMT, epithelial-mesenchymal transition; MSCs, mesenchymal stem cells; Shh, Sonic hedgehog; SPARC, secreted protein acidic and rich in cysteine; VEGFA, vascular endothelial growth factor; PanIN, pancreatic intraepithelial neoplasia; ZO-1, zonula occludens-1; ZEB1, zinc-finger E-box binding homeobox 1 ⁎ Corresponding author at: No. 270, Dong'An Road, Xuhui District, Shanghai, 200032, China. Tel.: + 86 21 64175590; fax: + 86 21 64174774. E-mail address: [email protected] (X. Yu). 1 These authors made equal contributions to this article. 0304-419X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. doi:10.1016/j.bbcan.2012.04.002

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Stromal therapy in PDA . . . . . . . . . . . . . . 4.1. Targeting the tumor signals to the surrounding 4.2. Targeting the stromal signals to the tumor . 4.3. Eliminating the stroma . . . . . . . . . . . 4.4. Special drug delivery system . . . . . . . . 5. Conclusions . . . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . .

. . . . stroma . . . . . . . . . . . . . . . . . . . . . . . .

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1. Introduction Pancreatic ductal adenocarcinoma (PDA) is a very aggressive and severe disease with the highest mortality rate worldwide, which is usually diagnosed at an advanced stage [1,2]. Although these tumors represent less than 2% of cancer cases, they are the fourth leading cause of cancerrelated death in the United States and other industrialized countries [3]; the median survival rate for those with clean microscopic surgical margins is approximately 2 years, with a 5-year survival of 15–20% [4]. One of the most prominent histological features of PDA is an extensive stroma that surrounds the tumor cells and accounts for up to 90% of the tumor volume (Fig. 1) [5]. There is a growing understanding of the contribution of the stromal microenvironment to the mechanisms responsible for malignant transformation and progression [6–17]. The poor prognosis of PDA is highly correlated with the presence of an extensive matrix of stromal cells [18]. Additionally, mutations in protooncogenes and tumor suppressor genes [14] as well as distinctive epigenetic changes [19] are observed in both the tumor epithelium and the surrounding stromal cells. Whereas normal stroma can delay or prevent tumorigenesis, abnormal stromal components can promote tumor growth [20]. Because PDA is exceptional in both its lethality and the extent and compact of the stroma surrounding the tumor cells, researchers have wondered whether the stroma is at least partly responsible for its poor prognosis [21]. Recently, several studies have shown promising results by reducing the amount of stroma surrounding PDA tumors [18,22,23]. However, the pathophysiological mechanisms underlying the regulation and perpetuation of the stroma in PDA remain poorly understood [24,25]. An improved understanding of the mechanisms that contribute to pancreatic tumor growth and progression is therefore urgently needed, and this knowledge will open new avenues for tumor prevention, earlier detection and improved therapy [26]. Accordingly, in this review, we will summarize the formation mechanism of PDA stroma, the role of the stroma in tumor progression, therapy resistance and the potential of stroma-targeted therapeutics in PDA.

Fig. 1. Immunofluorescence of human PDA showing PDA neoplastic ductal cells (blue and red merge) surrounded by typical dense, fibrotic stroma (green).

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2. Mechanism of stroma formation and its role in PDA development and progression Cancer cells can alter their adjacent stroma to form a permissive and supportive environment for tumor progression by producing stromamodulating growth factors [27]. These factors act in a paracrine manner to induce the inflammatory response and activate surrounding stromal cells, such as pancreatic stellate cells (PSCs) and fibroblasts, smoothmuscle cells and adipocytes. This activation leads to the secretion of additional growth factors, cytokines and proteases (Fig. 2) [27], which in turn activate both cancer cells and the surrounding stromal cells, indicating the existence of an interaction loop. This interaction results in a positive feedback effect on the growth and progression of PDA.

2.1. Components of the stroma The desmoplastic stroma is a complex structure composed of an extracellular matrix (ECM), PSCs, fibroblasts, macrophages, blood and lymphatic vessels, pericytes, marrow-derived stem cells and other inflammatory cells [28,29]. The transition from normal to invasive carcinoma is preceded or accompanied by the activation of local host stroma [30]. The ECM can serve many functions, such as providing support and anchorage for cells and facilitating cell contacts. The stroma also contains nerve fibers that release nerve growth factors (NGFs) promoting PDA cell growth and spreading [31], and bone-

Fig. 2. Interaction loop between PDA cells and stroma and potential therapeutic strategies. Multiple cancer cell-derived factors, including TGF-β, Shh and HGF/Met, mediate stroma production through autocrine and paracrine mechanisms. In response to the changes in the cancer cells, there is an altered gene expression profile in the cancer-associated stroma, including Integrin, COX-2, VEGFA and collagen I. Crosstalk between epithelial tumor cells and cells of the stromal compartment by means of these factors will result in the acquisition and enhancement of the pancreatic tumor abilities, such as cancer development, migration and invasiveness. Potential therapeutic strategies targeted to the stroma of PDA including: 1) targeting the tumor signals to the surrounding stroma, 2) targeting the stroma signals to the tumor, 3) eliminating the stroma, and 4) employing special drug delivery system.

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marrow-derived stem cells that may have the ability to differentiate into various stromal cells [32]. Stroma proliferation is further enhanced by the recruitment of a cohort of inflammatory immune cells and the activation of endothelial cells and pericytes expressing high levels of profibrotic and proangiogenic factors to the periphery of pancreatic tumors [5,28]. 2.1.1. PSCs PSCs are fibroblast-like cells with close morphological and biochemical resemblance to hepatic stellate cells [33,34]. They stain positive for vimentin, desmin, and α-smooth muscle actin (α-SMA) and contain vitamin-A-storing lipid droplets in the cytoplasm, indicating that they are neither fibroblasts nor smooth muscle cells [33]. In a healthy pancreas, PSCs comprise approximately 4% of all pancreatic cells and show a periacinar distribution [35]. It has become increasingly clear that activated PSCs are the predominant source of extracellular matrix proteins in pancreatic fibrosis [35]. Interestingly, PSCs have also emerged as a class of pancreas-specific mesenchymal cells and key regulators of desmoplasia in PDA [36,37]. These cells significantly increased the tumor growth and invasiveness accompanied by a pronounced desmoplastic reaction [38,39]. Strikingly, PSCs were also detected in metastatic foci in the liver of nude mice, suggesting co-migration of PSCs with cancer cells to establish a potentially tumor-favorable microenvironment at distant sites [39]. Several profibrogenic mediators that regulate PSCs have been identified, such as platelet-derived growth factor (PDGF) [40], transforming growth factor β (TGF-β) [40], fibroblast growth factor (FGF) [40], activin A [41], reactive oxygen, tumor necrosis factor α (TNF-α) [40,42], interleukin (IL)-1 and IL-6 [42]. Among them, PDGF is considered to be the most effective mediator [40]. In response to these profibrogenic mediators, PSCs undergo an activation process that involves proliferation, exhibition of a myofibroblastic-like phenotype and enhanced secretion of excessive components of the ECM by activating the enzymes of mitogen-activated protein kinase family [43]. Activated PSCs, in turn, are capable of synthesizing cytokines such as TGF-β [44], activin A [41], matrix metalloproteinases (MMPs), tissue inhibitor of metalloproteinases (TIMPs) [45] and cyclooxygenase-2 (COX-2) [46], which may further potentiate an activated phenotype. These autocrine loops modulate the stroma and stimulate fibrosis. Taken together, PSCs are key cells in pancreatic fibrogenesis and represent a potential target for stroma therapy [47]. 2.1.2. Fibroblasts Fibroblasts are a type of cell that play a key role in the deposition of ECMs, the regulation of epithelial differentiation, the modulation of inflammation and the wound healing process [48]. They can be identified based on a combination of different markers, such as α-SMA, vimentin, desmin and fibroblast activation protein (FAP) [49]. Fibroblasts have a well-recognized role in the carcinogenic process [50,51]. Increasing evidence suggests that a subpopulation of fibroblasts – the so-called cancer-associated fibroblasts (CAFs) or myofibroblasts – constitutes a major portion of the reactive tumor stroma and is a key determinants in the malignant progression of cancer [27,52]. TGF-β and PDGF are known to mediate the interaction of cancer cells with CAFs [52]. The CAFs play a central role in promoting the growth of tumor cells through their ability to secrete stromal-cell-derived factor 1 (SDF-1) [53] and upregulate the expression of serine proteases and MMPs [54]. In addition, they produce of a range of growth factors and cytokines such as insulin-like growth factor 1 (IGF1) and hepatocyte growth factor (HGF), which promote tumor-cell survival as well as migration and invasion [55,56]. The structural and functional contributions of CAFs to the processes of malignant PDA transformation and progression are beginning to emerge. The invasive potential of PDA cells can be greatly enhanced by coculturing with stromal fibroblasts [57]. In 2004, Sato et al. [58] performed global gene expression profiling of PDA cells and stromal fibroblasts to determine gene expression changes induced by coculturing. They found

that COX-2 expression was markedly augmented in both cancer cells and fibroblasts [58]. In another study, Walter and colleagues [59] performed gene expression profiling of human pancreatic CAFs and non-neoplastic pancreatic fibroblasts, and they found that the Hedgehog receptor Smoothened (Smo) was upregulated in CAFs relative to control fibroblasts. Co-culture of Hedgehog-producing PDA cell lines with 10 T1/2 fibroblasts resulted in GLI reporter activity in the fibroblasts, demonstrating the capacity of tumor cells to induce paracrine signaling [60]. Moreover, CAFs have also been reported to have a prometastatic effect on PDA cells [36]. Taken together, this evidence suggests fibroblasts play an important role in the malignant progression of PDA and represent an important therapeutic target [9,61]. 2.1.3. Immune cells Immune cells, including macrophages, mast cells, granulocytes, dendritic cells, natural killer cells and lymphocytes, have complex and multifaceted functions in PDA growth and progression. Tumor cells secrete a series of factors that actively enhance the recruitment of immune cells [62]. Immune cells, in turn, produce cytokines and growth factors that can exert a direct effect on both the tumor and its stromal cells. For instance, tumor cells secrete macrophage colony stimulating factor (M-CSF), a potent chemoattractant for macrophages [63]. Tumorassociated macrophages (TAMs) are rich in growth factors, such as epidermal growth factor (EGF) and proteases, which can accelerate tumor cell proliferation, survival, angiogenesis and matrix remodeling, all of which can promote tumor progression [64]. Individuals suffering from chronic pancreatitis harbor an increased risk for pancreatic cancer development, owing partly to the progrowth environment generated by activated immune cells [65]. 2.1.4. Source of stromal cells The major source of stromal cells is poorly understood. Unlocking this major problem will help to explain the complex dynamics of tumor invasion and metastasis in PDA biology. Cells already present in the normal tissue stroma are obvious candidates for such conversion [66]. Alternatively, recent reports suggest that cancer cells themselves can partly form the surrounding stroma after epithelial–mesenchymal transition (EMT) [67]. It is worth mentioning that bone-marrowderived stem cells may be another potential source of non-resident cells [32,68–71]. Bone-marrow-derived stem cells can contribute to the PSC population in chronic pancreatitis [72]. In fact, data from animal models as well as from human breast cancers suggest that at least a subset of tumor-associated myofibroblasts is derived from hematopoietic precursor/stem cells [73]. Among them, mesenchymal stem cells (MSCs) have the great potential to be the source of tumor stroma. They can localize to solid tumors after intravenous administration and differentiate into a variety of cell types, including osteoblasts, chondrocytes and adipocytes, integrating into the tumor stroma [69,74]. 2.2. Growth factors At the molecular level, stroma production is mediated by the activation of multiple cancer-cell-derived genes/signaling pathways through autocrine and paracrine mechanisms. These genes/pathways include TGF-β [75], Sonic hedgehog (Shh) [76], HGF/Met [77], FGFs [78], IGF-1 [79], COX-2 [58], PDGF [40], NGFs [80], MMPs [81], Wnt signaling [82], neurotrophins [83], secreted protein acidic and rich in cysteine (SPARC) [84] and EGF [5]. In response to the changes observed in the cancer cells, there is an altered gene expression profile in the cancer-associated stroma, including the expression of MMPs [81], COX-2 [58], vascular endothelial growth factor (VEGFA) [85] and collagen I [86]. Crosstalk between epithelial tumor cells and cells of the stromal compartment by means of these factors will result in the acquisition and enhancement of pancreatic tumor abilities, such as angiogenesis, migration and invasiveness. The identification and characterization of factors involved in tumor-stroma interactions can thus identify potential

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targets for novel therapeutic strategies. Two factors, including TGF-β and sonic hedgehog, have a well-recognized role in the formation and development of PDA stroma [23,76,87–90]. 2.2.1. TGF-β TGF-β is a multifunctional factor that regulates many aspects of cellular functions such as epithelial cell growth and synthesis of extracellular matrixes [75,91]. It has been reported to be overexpressed in PDA tumors and is most predominant in the stroma [92]. In addition to its effect on tumor cells, there is increasing evidence for a paracrine, tumor-promoting role for TGF-β in modulating the stroma by altering the expression of ECM components and stimulating angiogenesis and immunosuppression [5,88,93]. TGF-β-transfected pancreatic tumor cells induced a rich stroma after orthotopic transplantation in the pancreas of nude mice [93]. In addition, TGF-β can lead to a reversible and time-dependent EMT in TGF-β-responsive PDA cell lines, characterized by a fibroblastoid morphology, an upregulation of mesenchymal markers and a downregulation of epithelial markers [89]. Furthermore, TGF-β can stimulate PSC activation (indicated by increased α-SMA expression) in a Smad2-dependent manner, whereas Smad3 was required for TGF-β-induced growth inhibition [94]. 2.2.2. Shh Hedgehog signaling plays a key role during the development and maintenance of many epithelial appendages, including derivatives of the gut [95]. Recently, the expression of Shh ligands and essential components of the pathway, including PTCH and Smo, has been reported in up to 70% of human PDA specimens, but is undetectable in normal human pancreatic ducts [76,96]. Hedgehog acts as an early and late mediator of PDA tumorigenesis [76]. Blockade of Hedgehog signaling can inhibit PDA invasion and metastasis [97]. Moreover, Shh has been identified as another mediator that promotes stromal desmoplasia [7,60,98]. It contributes to the formation of desmoplasia in PDA by affecting the differentiation and motility of human PSCs and fibroblasts [7]. Treating human pancreatic fibroblasts with Shh increases α-SMA expression, and overexpression of Shh ligand in xenotransplanted cells enhances collagen I and fibronectin expression in recruited host fibroblasts [7]. Shh mediates tumorigenesis through a paracrine paradigm, in which tumor cells secrete hedgehog ligand to induce tumor-promoting hedgehog target genes in the adjacent stroma [60,99]. Expression of SmoM2 in epithelial cells could not activate the pathway and had no impact on pancreatic development or neoplasia [99]. In contrast, activation of Smo in the mesenchyme led to hedgehog pathway activation, indicating that only the tumor stroma is competent to transduce the hedgehog signal [99]. 2.3. Causes of extensive stroma formation Compared with other types of cancer, PDA has an exceptionally extensive stroma. Early pancreatic intraepithelial neoplasia (PanIN) lesions, identified as precursor lesions to PDA, are associated with lower amounts of normal stroma surrounding the pancreatic ducts. When progressing towards invasive carcinoma, PDA cells are often accompanied by a readily evident increase in stroma formation that ultimately results in extensive stroma. Potential causes specific to PDA are explained here. 2.3.1. Chronic pancreatitis Chronic pancreatitis has long been recognized as a risk factor for human PDA [65,100]. In a pooled multivariable model, a history of pancreatitis was associated with a 7.2-fold increased risk estimate for PDA [101]. Chronic pancreatitis can dramatically sensitize adult mice to K-ras G12V-driven PanIN/PDA formation [102]. Its inflammatory environment, including cytokines, reactive oxygen species and mediators of the inflammatory pathway, may foster the transformation of epithelial pancreatic cells towards a neoplastic phenotype, eventually

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resulting in PDA [103]. Inflammatory mediators, most notably NF-κB, are highly expressed in both chronic pancreatitis and PDA [28,65,100]. It is noteworthy that both chronic pancreatitis and PDA are accompanied by an organ fibrosis [104]. Additionally, at the histological level, the compositions of the stroma in chronic pancreatitis and in PDA are highly identical. Moreover, molecular profiling indicated many fewer differences between PDA and chronic pancreatitis, likely because of the shared stromal influences in the two diseases [105]. The roles of sustained activation of PSCs, fibroblasts and the EMT process in the fibrosis that is associated with both chronic pancreatitis and PDA are becoming increasingly appreciated, suggesting that a common pathway for PDA development may be through a chronic inflammatory process including stroma formation. 2.3.2. Environmental/lifestyle factors In addition to chronic pancreatitis, the pancreas experiences additional stresses, such as smoking, insulin, high-protein and high-fat diets, digestive enzymes, inflammation, diabetes, alcohol, coffee and acute pancreatitis. Over time, these stresses in the long run induce acute and chronic inflammation in the pancreas, leading to the formation of a stromal microenvironment favorable for the growth of PDA. For example, tobacco smoke is associated with chronic pancreatic inflammation with fibrosis and scarring of pancreatic acinar structures in rats [106]. Moreover, compared with non-smokers, cigarette smokers face an increase risk of pancreatic fibrosis [107]. Because of its location in the pancreas, stromal cells and PDA cancer cells are also exposed to high levels of insulin. These high insulin levels may exert mitogenic effects on the stromal cells and therefore contribute to excessive stroma formation [108]. 2.3.3. Aging There is a striking link between advanced age and increased incidence of cancer [109]. This association is the most prominent for PDA because it mostly occurs in old individuals. Only approximately 10% of patients develop PDA before the age of 50 [110]. Disease rates increase rapidly with age, as seen in data from the United States, where the rates for patients age 70–74 are approximately 57 per 100,000 per year compared with rates of 9.8 per 100,000 per year in patients aged 50–54 [110]. The age-related increase in cancer is accompanied by the accumulation of mutations and pro-oncogenic changes in the tissue stroma [111]. Cellular senescence is considered to contribute to aging [112]. For example, senescent human fibroblasts can stimulate premalignant and malignant epithelial cells, but not normal epithelial cells, to proliferate in culture and form tumors in mice [113]. With age, senescent cells accumulate and secrete factors (such as degradative enzymes, inflammatory cytokines and growth factors) that alter the tissue microenvironment and disrupt the tissue structure, which in turn allows the mutant epithelial cells to express their neoplastic phenotypes [114]. However, the exact molecular mechanisms involved in aging, tumor stroma and carcinogenesis remain to be defined. 2.3.4. EMT process EMT was originally identified as an essential normal developmental process including mesoderm formation and neural tube formation [115]. Later, it was found to be a source for converting epithelial cells into fibroblast-like cells in various tissues [116]. In cancer, EMT is a transient event that affects only a few cells in a tumor mass [117]. However, it generally induces an aggressive behavior of the tumor cells and is vital for tumor growth, motility and invasiveness [118]. During the processes of EMT, cells lose epithelial cell-cell junctions, actin cytoskeleton reorganization, and the expression of proteins that promote cell-cell contact such as E-cadherin, γ-catenin, and zonula occludens-1 (ZO-1), and they gain mesenchymal molecular markers such as vimentin, fibronectin, α-SMA, fibroblast-specific protein-1, and N-cadherin [119]. Thus, EMT can allow a nonmalignant stroma to facilitate epithelial tumor growth [67]. The EMT phenomenon was

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also identified in several PDA cell lines and surgically resected PDA [120,121]. Importantly, increased fibronectin or vimentin and decreased E-cadherin were correlated with poor survival in PDA, suggesting that EMT is associated with poor survival [122]. The TGF-β signaling pathway is a key regulator of EMT, as TGF-β initiates and maintains both developmental and carcinogenic EMT in different systems in vitro and in vivo [89]. 3. Role of stroma in PDA therapy resistance 3.1. Failure of surgery Currently, surgical resection, chemotherapy and radiotherapy remain the major therapeutic tools for PDA. Clearly, surgical resection is the only potentially curative technique for managing PDA [123]. However, local invasion and distant metastases prohibit approximately 80% of patients with PDA from receiving a curative resection. The contribution of the stromal microenvironment to malignant transformation, local invasion and distant metastasis has been well recognized [9,36]. Local recurrence and distant metastases are the major reasons for the failure of surgical resection of PDA tumors. 3.2. Chemoresistance Chemotherapy is another major treatment option for PDA [124]. PDA remains one of the most lethal human malignancies to treat, due in part to intrinsic and acquired poor responsiveness to chemotherapies [124]. In 2009, Farmer and colleagues [24] found that increased stromal gene expression predicts resistance to preoperative chemotherapy in individuals with estrogen-receptor-negative breast cancer. This finding identifies a previously undescribed resistance mechanism to chemotherapy and suggests that anti-stromal agents may offer new ways to overcome resistance to chemotherapy [24]. In PDA, the deficient vasculature was correlated with the presence of the dense stromal matrix that makes up the bulk mass of tumors [5]. The resistance of PDA tumors to systemic chemotherapies is at least partly due to the lack of effective drug delivery mechanisms to tumor [125]. There is increasing evidence that paracrine hedgehog signaling plays a crucial role in supporting pro-tumorigenic communication between tumor epithelium and stroma [7,60,99]. In 2009, Olive et al. [23] used a Hedgehog cellular signaling pathway inhibitor and gemcitabine, a first-line PDA chemotherapeutic agent, in an accurate mouse model of PDA. They found that the combination therapy produced a transient increase in intratumoral vascular density and intratumoral concentration of gemcitabine, leading to transient stabilization of the disease [23]. Therefore, inefficient drug delivery may be an important contributor to chemoresistance in PDA [23]. Emerging lines of evidence suggest that there is a molecular and phenotypic association between EMT phenotype and chemoresistance [125,126]. Gemcitabine-resistant PDA cells that have an acquired EMT phenotype, including the spindle-shaped morphology, appearance of pseudopodia, and reduced adhesion characteristic of transformed fibroblasts, also exhibited an increase in vimentin and a decrease in E-cadherin expression [126]. In addition, Notch-2 and its ligand, Jagged-1, are highly upregulated in gemcitabine-resistant cells, which are consistent with the role of the Notch signaling pathway in the acquisition of the EMT and cancer stem-like-cell phenotype [125]. The downregulation of Notch signaling led to partial reversal of the EMT phenotype, resulting in the mesenchymal epithelial transition, which was associated with decreased expression of vimentin, zinc-finger E-box binding homeobox 1 (ZEB1), Slug, Snail and NF-κB [125]. 3.3. Radioresistance Radiotherapy represents a major treatment option for many malignant tumors and has been frequently used in patients with PDA. Recently,

however, several lines of evidence have shown that irradiation promotes invasion and metastasis of cancer cells [127,128]. Ohuchida et al. [128] found that PDA cells co-cultured with irradiated pancreatic fibroblasts resulted in a more aggressive and invasive cancer than when nonirradiated fibroblasts were used. In addition, exposure of PDA cells to the culture media from irradiated fibroblasts resulted in increase in the phosphorylation of c-Met and the activity of mitogen-activated protein kinase [128]. Mantoni et al. [127] showed that PSCs could promote radioprotection and stimulate proliferation in PDA cells in a β1-integrindependent manner. Thus, radiation-induced cancer may not only result from deleterious mutations in the epithelia but also from alterations in the stroma [9]. 4. Stromal therapy in PDA The improved understanding of the genetic and molecular alterations that occur not only in tumor cells but also in the surrounding stromal cells has recently led to the development of novel therapeutic approaches specifically targeted to the stroma surrounding the tumor [129]. This is especially true for PDA because it has the most prominent tumor stroma. Four potential therapeutic targets will be discussed in the following sections (Fig. 2). 4.1. Targeting the tumor signals to the surrounding stroma Pro-fibrotic growth factors derived from cancer cells, such as TGF-β, EGFR, IGF-1 and PDGF, can be employed as potential therapeutic targets. TGF-β signaling plays a key role in PDA carcinogenesis and its stromal formation [27,89]. The loss of TGF-β responsiveness in fibroblasts resulted in intraepithelial neoplasia in the prostate and invasive squamous cell carcinoma of the forestomach, both of which are associated with an increased abundance of stromal cells [8]. In PDA, soluble type II TGF-β receptor inhibits TGF-β signaling in PDA cells in vitro and suppresses tumor formation and metastasis, suggesting that a soluble receptor approach can be used to block these tumorigenic effects of TGF-β [130]. The unclear role of the pathway in fully established tumors complicates the use of TGF-β pathway modulators [5]. EGFR is frequently overexpressed in PDA and is correlated with poor prognosis and disease progression [131]. EGFR signaling has also been shown to affect pancreatic fibrogenesis by activating PSCs [132]. The combination of gemcitabine and an EGFR-targeted agent, erlotinib, is the first combination therapy demonstrating survival benefits in PDA in a phase III trial (median survival 6.24 months vs 5.91 months) [133]. Overall, current targets of pro-fibrotic growth factors failed to significantly improve the prognosis for patients with PDA. Further investigations identifying crucial pro-fibrotic growth factors are urgently needed. 4.2. Targeting the stromal signals to the tumor In view of the supportive role of the stroma to PDA tumors, targeting factors from surrounding stromal cells may be another useful strategy. For example, MMPs are a large family of zinc-containing proteolytic enzymes involved in maintaining the extracellular environment in both physiological and pathological conditions [134]. They are expressed by PDA cancer cells as well as activated PSCs, fibroblasts, and immunocytes [81]. In 2003, Moore et al. conducted a phase III trial with BAY-12-9566, a specific inhibitor of MMP-2, MMP-3, MMP-9 and MMP-13 in patients with locally advanced or metastatic pancreatic cancer. These patients were treated with BAY-12-9566 or gemcitabine; however, the study was discontinued after the second interim analysis found that the new inhibitor was significantly inferior to gemcitabine (median overall survival 3.74 months vs 6.59 months) [135]. The ineffective of MMPs inhibitors may be due to the complexity in controlling of the extracellular matrix, and the inhibition of only a subset of MMPs is unlikely to have a significant impact on tumor growth and spread.

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4.3. Eliminating the stroma Because of the existence of an interaction loop between PDA and its stroma, eliminating the stroma may greatly improve the therapeutic effect. For example, Shh signaling plays a causative role in pancreatic tumorigenesis through a paracrine signaling mechanism received by tumor stromal cells [99]. In 2009, Olive et al. found that inhibition of the Shh signaling pathway resulted in a dramatic depletion of stromal components paralleled by an increase in intratumoral vascular density in an accurate mouse model of PDA [23]. Co-administration of gemcitabine and IPI-926, a Shh signal pathway inhibitor, resulted in a significantly enhanced intratumoral concentration of gemcitabine triphosphate, transient disease stabilization and a statistically significant prolongation of survival [23]. However, the pronounced stromal reaction and hypovascularity ultimately returned in the model, suggesting that the tumors can adapt to chronic Shh inhibition [23]. Activation of the TNF receptor superfamily member CD40 has been shown to be a key regulatory step in the development of T-celldependent antitumor immunity [136]. In 2011, Beatty et al. [18] tested the combination of an agonist CD40 antibody with gemcitabine chemotherapy in a small cohort of patients with surgically incurable PDA and observed tumor regression in some patients. They then reproduced this treatment effect in a genetically engineered mouse model of PDA and unexpectedly found that tumor regression required macrophages but not T cells or gemcitabine [18]. CD40-activated macrophages rapidly infiltrated tumors, became tumoricidal and facilitated the depletion of the tumor stroma [18]. 4.4. Special drug delivery system Compared with the normal pancreas, PDA has numerous circuitous small leaky blood vessels and capillaries. Special drug delivery systems can penetrate the tumor through the leaky blood vessels and release drugs in a controlled manner [2,137]. For example, in 2011, Von Hoff and colleagues [22] performed a phase I/II trial to identify the maximumtolerated dose of first-line gemcitabine plus nab-paclitaxel in metastatic PDA and to provide efficacy and safety data. They found that, at the maximum-tolerated dose, the response rate was 48%, with a median overall survival of 12.2 months and a 1-year survival rate of 48%. SPARC expression in the surrounding stroma, but not in the tumor, was correlated with improved survival. In mice with human PDA xenografts, nab-paclitaxel alone and with gemcitabine can deplete the desmoplastic stroma [22]. The intratumoral concentration of gemcitabine was increased 2.8-fold in mice receiving nab-paclitaxel in combination with gemcitabine compared with those receiving gemcitabine alone [22]. Therefore, special drug delivery systems may have the potential to serve as a highly effective therapeutic strategy to overcome the PDA challenge. 5. Conclusions The cardinal histological hallmark of PDA is an extensive stroma surrounded the tumor cells with hypovascular barrier, which may be closely related to the dismal prognosis of PDA. The interaction loop between PDA and its related stroma promotes tumor growth, invasion and metastasis, protects tumor from apoptosis and potentially impairs the delivery of therapeutic compounds [8,15,28,36,108,138]. Developing new strategies for “normalizing” the stromal microenvironment may ultimately provide novel and promising therapeutic options in PDA. In the future, we believe the treatment of pancreatic cancer will be divided into two steps. The first step will be the reduction of tumor stroma, which will make the prognosis of pancreatic cancer like other types of cancer with less amount of stroma, such as colon cancer, breast cancer and lung cancer. Second will be the cure of pancreatic cancer, which will be accompanied by the cure of all types of cancers. Currently, the translation of anti-stromal therapy into effective therapeutic approaches has been inefficient. It seems that only agents

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can eliminate the amount of tumor stroma showing great potential [23]. This can partly be explained by the fact that PDA is highly complex. As PDA has different genetic alterations and individual alterations vary widely among the cancers, we lack consistent targets. Additionally, several potential targets, including TGF-β and MMPs, have both protumorigenic as well as tumor suppressive functions [81,139]. Furthermore, similar to traditional therapeutic agents, stroma targeted agents may be poorly delivered for the predominant desmoplastic stroma reaction and the pronounced hypovascularity of PDA [23]. It is worth to mention that patients with PDA may be benefit from treatments targeted to both the epithelial component and its stromal cells. In fact, in prostate cancer, simultaneous inhibition of epithelial tumor cells and stromal cells was extremely effective in reducing cancer cell growth [140]. Although the presence of prominent stroma has been noted for decades, the interest in research has been weak and consequently the knowledge of stroma is still fragmented. Several questions remain to be answered. What is the crucial governing pathway of PDA stroma? What are the differences among normal, inflammation and reactive tumor stroma? Why PDA has more prominent stroma and less vasculature compared with other type of cancers? An improved understanding of these key aspects for PDA will open new avenues for meaningful clinical progress. Acknowledgement This research was supported in part by the National Science Foundation of China (Grant No. 81101807). References [1] A. Jemal, F. Bray, M.M. Center, J. Ferlay, E. Ward, D. Forman, Global cancer statistics, CA Cancer J. Clin. 61 (2011) 69–90. [2] X. Yu, Y. Zhang, C. Chen, Q. Yao, M. Li, Targeted drug delivery in pancreatic cancer, Biochim. Biophys. Acta 1805 (2010) 97–104. [3] A. Jemal, R. Siegel, E. Ward, Y. Hao, J. Xu, T. Murray, M.J. Thun, Cancer statistics, 2008, CA Cancer J. Clin. 58 (2008) 71–96. [4] R.J. Geer, M.F. Brennan, Prognostic indicators for survival after resection of pancreatic adenocarcinoma, Am. J. Surg. 165 (1993) 68–72 discussion 72–63. [5] G.C. Chu, A.C. Kimmelman, A.F. Hezel, R.A. DePinho, Stromal biology of pancreatic cancer, J. Cell. Biochem. 101 (2007) 887–907. [6] D. Hanahan, R.A. Weinberg, Hallmarks of cancer: the next generation, Cell 144 (2011) 646–674. [7] J.M. Bailey, B.J. Swanson, T. Hamada, J.P. Eggers, P.K. Singh, T. Caffery, M.M. Ouellette, M.A. Hollingsworth, Sonic hedgehog promotes desmoplasia in pancreatic cancer, Clin. Cancer Res. 14 (2008) 5995–6004. [8] N.A. Bhowmick, A. Chytil, D. Plieth, A.E. Gorska, N. Dumont, S. Shappell, M.K. Washington, E.G. Neilson, H.L. Moses, TGF-beta signaling in fibroblasts modulates the oncogenic potential of adjacent epithelia, Science 303 (2004) 848–851. [9] N.A. Bhowmick, E.G. Neilson, H.L. Moses, Stromal fibroblasts in cancer initiation and progression, Nature 432 (2004) 332–337. [10] J.J. DeCosse, C.L. Gossens, J.F. Kuzma, B.R. Unsworth, Breast cancer: induction of differentiation by embryonic tissue, Science 181 (1973) 1057–1058. [11] I.J. Fidler, The pathogenesis of cancer metastasis: the 'seed and soil' hypothesis revisited, Nat. Rev. Cancer 3 (2003) 453–458. [12] R. Hill, Y. Song, R.D. Cardiff, T. Van Dyke, Selective evolution of stromal mesenchyme with p53 loss in response to epithelial tumorigenesis, Cell 123 (2005) 1001–1011. [13] A.E. Karnoub, A.B. Dash, A.P. Vo, A. Sullivan, M.W. Brooks, G.W. Bell, A.L. Richardson, K. Polyak, R. Tubo, R.A. Weinberg, Mesenchymal stem cells within tumour stroma promote breast cancer metastasis, Nature 449 (2007) 557–563. [14] F. Moinfar, Y.G. Man, L. Arnould, G.L. Bratthauer, M. Ratschek, F.A. Tavassoli, Concurrent and independent genetic alterations in the stromal and epithelial cells of mammary carcinoma: implications for tumorigenesis, Cancer Res. 60 (2000) 2562–2566. [15] A. Patocs, L. Zhang, Y. Xu, F. Weber, T. Caldes, G.L. Mutter, P. Platzer, C. Eng, Breast-cancer stromal cells with TP53 mutations and nodal metastases, N. Engl. J. Med. 357 (2007) 2543–2551. [16] I. Garrido-Laguna, M. Uson, N.V. Rajeshkumar, A.C. Tan, E. de Oliveira, C. Karikari, M.C. Villaroel, A. Salomon, G. Taylor, R. Sharma, R.H. Hruban, A. Maitra, D. Laheru, B. RubioViqueira, A. Jimeno, M. Hidalgo, Tumor engraftment in nude mice and enrichment in stroma- related gene pathways predict poor survival and resistance to gemcitabine in patients with pancreatic cancer, Clin. Cancer Res. 17 (2011) 5793–5800. [17] T.J. Wilson, R.K. Singh, Proteases as modulators of tumor–stromal interaction: primary tumors to bone metastases, Biochim. Biophys. Acta 1785 (2008) 85–95. [18] G.L. Beatty, E.G. Chiorean, M.P. Fishman, B. Saboury, U.R. Teitelbaum, W. Sun, R.D. Huhn, W. Song, D. Li, L.L. Sharp, D.A. Torigian, P.J. O'Dwyer, R.H. Vonderheide,

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