Histopathological evaluation of tumor tissue traditionally focuses on the epithelial elements when determining histologic type, grade, and stage for prognostic purposes. The stroma is qualitatively appreciated but not conceptualized as an integral extension of the tumor, or considered as a quantitative disease marker. In this study, we systemically combined traditional pathology review and second harmonic generation imaging to interrogate stromal collagen structure and organization in normal and diseased pancreatic tissue. Our quantitative data demonstrate that a characteristic collagen topology exists in the pancreatic ductal adenocarcinoma stroma compared with that of normal ducts and ducts in the setting of chronic pancreatitis. At the histologic level, the profuse stroma production in chronic pancreatitis distorts the normal conformation of ducts, causing them at times to appear histologically similar to infiltrating pancreatic ductal adenocarcinoma. Our data showing a characteristic collagen topography in pancreatic ductal adenocarcinoma compared with chronic pancreatitis may be useful for the development of an ancillary histologic tool to help differentiate these two types of tissue changes. More importantly, the data suggest an important role for collagen organization during pathogenesis and progression of cancer.
To date, the specific pathophysiologic processes driving collagen reorganization in PDAC remain unclear. It has recently been shown that both the cellular and non-cellular composition of the periductal stroma changes dynamically during pancreatic ductal adenocarcinoma progression. Shi et al characterized the periductal stroma of ducts in chronic pancreatitis and pancreatic intraepithelial neoplasms, which are known to be precursor lesions to pancreatic ductal adenocarcinoma, through immunohistochemical staining of collagen, periostin, infiltrating macrophages, and activated alpha-smooth muscle actin-positive pancreatic stellate cells. They demonstrated that low-grade pancreatic intraepithelial neoplasms show a marked increase in periductal collagen deposition without a large increase in periostin deposition or pancreatic stellate cell activation. As the pancreatic intraepithelial neoplasms evolve to higher grade lesions, the collagen density remains steady, but there is an increase in the number of activated pancreatic stellate cells. This stromal composition contrasts with that of chronic pancreatitis, where the stroma is known to be uniformly rich in periostin, collagen, macrophages, and pancreatic stellate cells. It is possible that increased activation of pancreatic stellate cells may result in the type of collagen remodeling in pancreatic ductal adenocarcinoma that our study demonstrates. In fact, many studies have shown that pancreatic stellate cells are crucial contributors to pancreatic ductal adenocarcinoma progression and are the principle source of fibrillar collagens, other extracellular matrix proteins, and soluble factors that promote the growth, invasion, metastasis, and survival of cancer cells.[31–36] In addition, the detection of pancreatic stellate cells immediately adjacent to pancreatic ductal adenocarcinoma epithelium correlates with adverse clinicopathological parameters.[6,7,37] Our group and others have shown that highly contractile pancreatic stellate cells can produce aligned collagen matrices in vitro, and the resultant topography can facilitate pancreatic ductal adenocarcinoma cancer cell migration.[38–40] Our immunohistochemical analysis suggests a positive correlation between alpha-smooth muscle actin expression and aligned collagen in the pancreatic ductal adenocarcinoma periductal stroma, which should be further validated on a larger patient cohort for potential mechanistic insight (Figure 4, Supplementary Figure S3 https://www.nature.com/modpathol/journal/v28/n11/suppinfo/modpathol201597s1.html?url=/modpathol/journal/v28/n11/full/modpathol201597a.html). As second harmonic generation can be directly applied to histopathology specimens beyond those stained with hematoxylin and eosin, collagen topology analysis could be readily paired with other disease markers currently under investigation in pancreatic ductal adenocarcinoma for nuanced insight into pathogenesis.[41–43]
The pathological impact of changes in pancreatic ductal adenocarcinoma stroma collagen topology also remains unclear. Accumulating evidence suggests that focally aligned collagen at the stroma–cancer interface guides the persistent migration of cancer cells away from the tumor and toward vasculature during the metastatic cascade.[20,44–46] In addition, the detection of aligned collagen signatures in routine histopathology slides can predict disease recurrence and patient survival in breast cancer patients. In pancreatic ductal adenocarcinoma, cancer cells in contact with collagen type I show enhanced migratory capabilities through upregulation of Snail, a regulator of epithelial-to-mesenchymal transition.[47–51] Epithelial-to-mesenchymal transition is a process that dissemination-competent cells undergo. It has been shown to be prevalent in pancreatic ductal adenocarcinoma tissue and to negatively impact patient prognosis. The potential functional link between collagen reorganization and increased pancreatic ductal adenocarcinoma cancer cell migration has yet to be elucidated.
Another potential pathological consequence of collagen reorganization in pancreatic ductal adenocarcinoma is increased matrix tension. Collagen is the preeminent scaffolding element in the human body, and alignment and cross-linking have been shown to increase tumor tissue stiffness levels.[53,54] Cells, in turn, are able to sense changes in the mechanical properties of the local environment and respond to them in ways that deviate from behaviors under normal tissue homeostasis.[55,56] Therapeutic targeting of lysyl oxidase in mouse models has shown promise in reducing collagen cross-linking and tumor stiffness, and ultimately in reversing the malignant phenotypes of cells. Likewise, ablating mechanically interacting aligned collagen tracks between cancer cells results in a similar phenotype reversal. Also related to increased tissue stiffness is the inability to systemically deliver therapeutics throughout pancreatic ductal adenocarcinoma tumors. Enzymatic and small molecule targeting of the periductal stroma has shown promise in remodeling the architecture, softening the tumor, decreasing interstitial pressure, and improving the efficiency of systemic delivery in pancreatic ductal adenocarcinoma.[59,60]
As last, disease markers that forecast which patients are most likely to experience accelerated progression or recurrence based on intrinsic tumor biology are critically needed to inform patient expectations, guide patient surveillance measures, and tailor personalized treatment regimens. There is emerging interest to stratify patients on the basis on stromal characteristics.[61,62] Stromal collagen topology has already been shown to correlate with breast tumors that have a greater propensity to metastasize and negatively impact prognosis. It is likely that a diversity of collagen topology profiles exist in pancreatic ductal adenocarcinoma tumors, which may prove useful as an ancillary disease marker to subtype patients during histological evaluation. It is conceivable that collagen topology could be characterized from tissue obtained preoperatively (via endoscopic fine-needle aspiration or computer tomography-guided core needle biopsy) or post-resection using our validated methodology. This is currently an active area of study and is expected to add to our overall understanding of stroma–epithelial interactions during pancreatic ductal adenocarcinoma progression.
This study was supported by the University of Wisconsin School of Pharmacy with additional funding from the University of Wisconsin Laboratory for Optical and Computational Instrumentation. The authors thank the University of Wisconsin Translational Research Initiatives in Pathology Laboratory, in part supported by the University of Wisconsin Department of Pathology and Laboratory Medicine and the University of Wisconsin Carbone Cancer Center grant P30 CA014520, for use of its facilities and services. We also thank Dr Jens Eickhoff (Department of Biostatistics & Medical Informatics, University of Wisconsin–Madison) for providing guidance on statistical analysis.
Mod Pathol. 2015;28(11):1470-1480. © 2015 Nature Publishing Group