The stroma of the pancreas undergoes evident qualitative and quantitative modifications playing a particularly important role in pancreatic ductal adenocarcinoma (PDAC). In PDAC the stroma is profoundly modified being this carcinoma is characterized by an intense “desmoplastic reaction”, consisting of the abnormal accumulation of stromal components, mostly collagen fibers. The stroma in the tumor microenvironment contains extracellular matrix (ECM) components, growth factors and soluble mediators, and different stromal cells including fibroblasts, and inflammatory and pancreatic stellate cells, thus influencing cancer cell phenotype, behavior, and chemoresistance.
ECM components of the stroma act as a physical scaffold, facilitating interactions between different cell types, providing survival and differentiation signals, and affecting resistance to anticancer drugs. Tumor stroma is therefore a dynamic environment where both cellular and extracellular matrix components reciprocally interact with tumor cells, thus modulating their phenotype and behavior.
Anti-stroma therapies have appeared to be a promising therapeutic approach for the treatment of pancreatic cancer, to reduce the stroma and tumor volume, and to improve drug delivery. Recently, new data suggested that the microenvironment is responsible for a dual and opposite effect on tumor cells, and the actions of the stroma in PDAC may be therefore context-dependent.
The comprehension of the very complex tumor-stroma cross-talk and plasticity will contribute to the comprehension of PDAC biology and to the identification of new therapeutic targets.
The Tumor Microenvironment in Pancreatic Cancer
The pancreas is a solid organ, playing the role of an exocrine and endocrine gland. The parenchyma of the exocrine pancreas is classified as a compound acinar or tubuloacinar serous gland, containing secretory units that release secretory products in ducts branching in the organ. The parenchyma is supported and organized by the connective tissue of the stroma, mostly containing collagen fibers and stromal cells.
The stroma of the pancreas undergoes evident qualitative and quantitative modifications and plays a particularly important role in pancreatic ductal adenocarcinoma (PDAC), an increasingly common carcinoma accounting for the fourth leading cause of cancer mortality in the United States, with an overall 5-year survival of less than 7%, due to the high incidence of recurrence and metastases dissemination.
In PDAC the stroma is profoundly modified being this carcinoma is characterized by an intense “desmoplastic reaction”, defined as the host fibrotic response to the invasive carcinoma, consisting in the abnormal accumulation of stromal components, mostly collagen fibers. The desmoplastic reaction is the histological hallmark of PDAC, often constituting 50-80% of the tumor volume, and is the microenvironment where cancer cells are embedded.
The stroma in the tumor microenvironment contains extracellular matrix (ECM) components, growth factors and other soluble mediators, and different stromal cells including fibroblasts, inflammatory and pancreatic stellate cells, and plays a key role as a modulator of cancer cell phenotype, behavior, and chemoresistance. A variety of stromal cells in the surrounding environment are recruited to tumors, and these not only enhance the growth of primary cancer but also facilitate its metastatic dissemination to distant organs.
Among the stromal cell types that have been implicated in tumor promotion are endothelial cells, which comprise the blood and lymphatic circulatory systems, pericytes, fibroblasts, and various bone marrow-derived cells (BMDCs), including macrophages, neutrophils, mast cells, myeloid cell-derived suppressor cells (MDSCs) and mesenchymal stem cells (MSCs).
Fibroblasts are cells living in the connective tissue of the pancreatic cancer stroma, playing a key role in modulating the malignant progression of transformed epithelial cells. Co-culture experiments showed that normal fibroblasts prevented the growth of initiated prostatic epithelial cells, and could even reverse the malignant phenotype of neoplastic epithelial cells. The predominant mesenchymal cell is believed to be the pancreatic stellate cell (PSC), accounting for about 4% of all pancreatic cells. In normal pancreas, they are quiescent, while they become activated and play a critical role within the tumor stroma since they are largely responsible for the desmoplastic reaction: when activated, PSCs secrete large amounts of ECM components, mostly collagen type I. They also release mediators inducing collagen deposition, but also influencing tumor cell behavior. In vitro studies demonstrated that, in turn, tumor cells are able to activate PSCs. Interestingly, PSCs by themselves might metastasize to distant organs and from the primary tumor accompanying cancer cells to the metastatic sites, supporting their colonization.
The tumor microenvironment contains cells of the immune system, such as T and B lymphocytes, and tumor-associated macrophages as well. Even within individual cell types, there are opposing functions; for example, CD4+ T-cells, macrophages, and natural killer (NK) T-cells have either tumor-suppressive or tumor-promoting properties depending on the tissue context and cellular stimuli. Many researchers focused their attention on macrophages since they can either impede or promote tumor progression, depending on their functional state. In vivo studies in animal models revealed that they are able to promote tumor angiogenesis, invasion, intravasation and metastasis. Tumor-associated macrophages (TAMs) exhibit the M1 and M2 functional states: the former are tumoricidal, while the last facilitate tumor progression. TAMs are recruited to hypoxic areas were they likely to promote tumor angiogenesis.
Tumor stroma is a dynamic environment where both cellular and extracellular matrix components reciprocally interact with tumor cells, thus influencing their phenotype.
The ECM is particularly relevant in PDAC, characterized by an intense desmoplastic reaction. The ECM plays many functions, including acting as a physical scaffold, facilitating interactions between different cell types, providing survival and differentiation signals, and affecting resistance to anticancer drugs. ECM proteins of the tumor microenvironment have been determined to be an important mediator of cancer cell behavior, influencing tumor cell proliferation and migration and tissue homeostasis. The ECM also influences cell polarity and angiogenesis. At the same time, the bidirectional cross-talk between cancer cells and the stroma occurs, driven by a complex network of cytokines, chemokines, and growth factors released in the tumor microenvironment, that, in turn, promote the desmoplastic reaction and activation of quiescent fibroblasts, therefore playing a key role in promoting all stages of carcinogenesis.
Key molecules involved in the desmoplastic reaction have been identified, such as collagen type I, IV, and V, fibronectin, laminin, matrix metalloproteinases (MMPs) and their inhibitors (TIMPs), and transforming growth factor-β1 (TGF-β1). Collagen Type I has been associated with increased integrin-mediated cell-cell adhesion, proliferation, and migration of PDAC cells. Basement membrane collagen Type IV and laminin provide a proper microenvironment for pancreatic cancer cells decreasing the cytotoxicity of anti-cancer drugs and inducing the up-regulation of cancer cell growth. The role of type V collagen, a minor component of ECM, remains poorly understood since it triggers opposite cellular responses depending on the cell type. The evidence of large quantities of fibronectin in both chronic pancreatitis and pancreatic cancer suggests that this protein may facilitate the development of pancreatic cancer.
Cross-talk between stromal and cancer cells occurs through soluble factors mutually influencing cell behavior, but also by direct cell-to-cell contact. Moreover, also cell-matrix interactions are involved in determining cell behavior such as proliferation and migration. In the ECM matricellular proteins, such as the Secreted Protein Acidic and Rich in Cysteine (SPARC), are involved in the modulation of cell-ECM interactions. SPARC has de-adhesive effects and is over-expressed during both ECM remodeling and tumor progression. Increased invasiveness of PDAC cells in the presence of exogenous SPARC was reported, which could explain the decreased survival rates of patients with stromal SPARC expression. A strong expression of SPARC was detected predominantly in the stromal fibroblasts adjacent to tumor cells, likely influencing tumor progression at the site of the tumor-host interface.
Stromal ECM is not static but is a dynamic component undergoing a finely regulated turnover by matrix metalloproteinases (MMPs). The loosening of cell contacts facilitates cancer cell migration, either as individual cells or in groups, and protease degradation or turnover of proteins in the ECM and basement membrane enables invasive cells to migrate into the surrounding tissue and vasculature. MMPs break down basement membrane proteins and allow cancer cells to enter lymphatic or blood vessels. MMP-2 is produced by both tumor and stromal cells of pancreatic cancer28, and a strong correlation between MMP-2 expression and the invasive potential of pancreatic cell lines has been shown. MMP-9 expression is correlated with lymph node involvement and occurrence of distant metastases, and a correlation between MMP-9 expression and the worse prognosis in PDAC patients was found. The stroma increases its production in adjacent pancreatic cancer cells, therefore stromal influences may be a key facilitator of tumor invasion.
In this context, the prevailing paradigm of the tumor microenvironment field is that the stroma supports tumor growth and progression. As a consequence, anti-stroma therapies have appeared to be a promising therapeutic approach for the treatment of pancreatic cancer, to reduce the stroma and tumor volume, and to improve drug delivery 32. But this concept has been remodeled by the paradoxical results of recent studies by Rhim, demonstrating that the complexity of the microenvironment is responsible for a dual and opposite effect on tumor cells: some components of the stroma have a tumor-promoting role, while other components could be tumor-suppressive, and the final effect is dependent on the differentiation grade of cancer cells. The actions of the stroma in PDAC may be therefore context-dependent.
In this very complex scenario, characterized by high complexity and plasticity, many actors mutually and bidirectionally interact, thus the development of experimental models to be easily manipulated in order to characterize the role of each actor is crucial. Understanding how cells and ECM of the tumor microenvironment influence cancer cell behavior, and how cancer cells interact with the tumor microenvironment will lead to the comprehension of PDAC biology and to the identification of unique targets that may finally improve the treatment of patients with pancreatic cancer.