Involvement of Host Stroma Cells and Tissue Fibrosis in Pancreatic Tumor Development in Transgenic Mice

Itai Spector,Olga Genin,Arnon Nagler,Adi Lavy,Mark Pines,Yael Zilberstein,Donald Gullberg

doi:10.1371/journal.pone.0041833

Abstract

IntroductionStroma cells and extracellular matrix (ECM) components provide the pivotal microenvironment for tumor development. The study aimed to evaluate the importance of the pancreatic stroma for tumor development.MethodsPancreatic tumor cells were implanted subcutaneously into green fluorescent protein transgenic mice, and stroma cells invading the tumors were identified through immunohistochemistry. Inhibition of tumor invasion by stroma cells was achieved with halofuginone, an inhibitor of TGFβ/Smad3 signaling, alone or in combination with chemotherapy. The origin of tumor ECM was evaluated with species-specific collagen I antibodies and in situ hybridization of collagen α1(I) gene. Pancreatic fibrosis was induced by cerulean injection and tumors by spleen injection of pancreatic tumor cells.ResultsInhibition of stroma cell infiltration and reduction of tumor ECM levels by halofuginone inhibited development of tumors derived from mouse and human pancreatic cancer cells. Halofuginone reduced the number only of stroma myofibroblasts expressing both contractile and collagen biosynthesis markers. Both stroma myofibroblasts and tumor cells generated ECM that contributes to tumor growth. Combination of treatments that inhibit stroma cell infiltration, cause apoptosis of myofibroblasts and inhibit Smad3 phosphorylation, with chemotherapy that increases tumor-cell apoptosis without affecting Smad3 phosphorylation was more efficacious than either treatment alone. More tumors developed in fibrotic than in normal pancreas, and prevention of tissue fibrosis greatly reduced tumor development.ConclusionsThe utmost importance of tissue fibrosis and of stroma cells for tumor development presents potential new therapy targets, suggesting combination therapy against stroma and neoplastic cells as a treatment of choice.

Highlights

Stroma cells and extracellular matrix (ECM) components provide the pivotal microenvironment for tumor development
[4] The predominant stroma cells infiltrating tumors and responsible for ECM synthesis are myofibroblasts that can switch from tumor-suppressing to tumor-promoting functions during carcinogenesis. [5,6,7] The importance of myofibroblasts in tumor progression was demonstrated by co-inoculation of tumor cells with myofibroblasts in breast [8,9] and pancreas xenografts, [10] which resulted in increases in tumorigenicity and tumor size
Tumor stromal myofibroblasts were more effective in promoting carcinogenesis than equivalent fibroblasts extracted from noncancerous tissue of the same individual or from healthy donors. [11,12] Collagen type I, the major ECM component produced by myofibroblasts, functions as a scaffold for the tissue and regulates the expression of genes associated with cellular signaling and metabolism, and gene transcription and translation

Summary

Introduction

Stroma cells and extracellular matrix (ECM) components provide the pivotal microenvironment for tumor development. The microenvironment of a developing tumor comprises of proliferating tumor cells, stroma cells, blood vessels and infiltrating inflammatory cells. It is a unique environment, which emerges during tumor progression as a result of tumor/host interactions; it is created by, and at all times is shaped and dominated by the tumor, which orchestrates molecular and cellular events taking place in surrounding tissues. [4] The predominant stroma cells infiltrating tumors and responsible for ECM synthesis are myofibroblasts (cancer- or tumor-associated fibroblasts) that can switch from tumor-suppressing to tumor-promoting functions during carcinogenesis. It affects fundamental cellular processes that are essential for tumor progression, such as cell survival, apoptosis and cell invasion. [13,14,15] ECM in general, and collagen type I in particular, can promote epithelial–mesenchymal transformation (EMT) [16,17], which is an additional source of myofibroblasts. [6,18] in various malignancies, tumor-dependent transformation of fibroblasts to myofibroblasts enhances neoplastic progression, and the presence of desmoplastic stroma enriched in myofibroblasts was associated with unfavorable prognoses. [14,19,20,21] The fibroblast-to-myofibroblast transition is driven especially by transforming growth factor-beta (TGFb), secreted either by the stroma cells [22], or by the tumor itself via the cancer exosomes. [23] In addition to the increase in ECM synthesis, the fibroblasts that acquire an activated phenotype are characterized by expression of contractile genes such as a smooth-muscle actin (aSMA) and transgelin (SMA22a), and exhibit a highly proliferative and migratory phenotype. [24].

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