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Background: Alterations towards a permissive stromal microenvironment provide important cues for tumor growth, invasion, and metastasis. In this study, Fibroblast activation protein (FAP), a serine protease selectively produced by tumor-associated fibroblasts in over 90% of epithelial tumors, was used as a platform for studying tumor-stromal interactions. We tested the hypothesis that FAP enzymatic activity locally modifies stromal ECM (extracellular matrix) components thus facilitating the formation of a permissive microenvironment promoting tumor invasion in human pancreatic cancer., Methods: We generated a tetracycline-inducible FAP overexpressing fibroblastic cell line to synthesize an in vivo-like 3-dimensional (3D) matrix system which was utilized as a stromal landscape for studying matrix-induced cancer cell behaviors. A FAP-dependent topographical and compositional alteration of the ECM was characterized by measuring the relative orientation angles of fibronectin fibers and by Western blot analyses. The role of FAP in the matrix-induced permissive tumor behavior was assessed in Panc-1 cells in assorted matrices by time-lapse acquisition assays. Also, FAP+ matrix-induced regulatory molecules in cancer cells were determined by Western blot analyses., Results: We observed that FAP remodels the ECM through modulating protein levels, as well as through increasing levels of fibronectin and collagen fiber organization. FAP-dependent architectural/compositional alterations of the ECM promote tumor invasion along characteristic parallel fiber orientations, as demonstrated by enhanced directionality and velocity of pancreatic cancer cells on FAP+ matrices. This phenotype can be reversed by inhibition of FAP enzymatic activity during matrix production resulting in the disorganization of the ECM and impeded tumor invasion. We also report that the FAP+ matrix-induced tumor invasion phenotype is β1-integrin/FAK mediated., Conclusion: Cancer cell invasiveness can be affected by alterations in the tumor microenvironment. Disruption of FAP activity and β1-integrins may abrogate the invasive capabilities of pancreatic and other tumors by disrupting the FAP-directed organization of stromal ECM and blocking β1-integrin dependent cell-matrix interactions. This provides a novel preclinical rationale for therapeutics aimed at interfering with the architectural organization of tumor-associated ECM. Better understanding of the stromal influences that fuel progressive tumorigenic behaviors may allow the effective future use of targeted therapeutics aimed at disrupting specific tumor-stromal interactions., (© 2011 Lee et al; licensee BioMed Central Ltd.)

Objective: Due to a lack of experimental systems, little is known about ovarian stroma. Here, we introduce an in vivo-like 3-D system of mesenchymal stromal progression during ovarian tumorigenesis to support the study of stroma permissiveness in human ovarian neoplasias., Methods: To sort 3-D cultures into 'normal,' 'primed' and 'activated' stromagenic stages, 29 fibroblastic cell lines from 5 ovarian tumor samples (tumor ovarian fibroblasts, TOFs) and 14 cell lines from normal prophylactic oophorectomy samples (normal ovarian fibroblasts, NOFs) were harvested and characterized for their morphological, biochemical and 3-D culture features., Results: Under 2-D conditions, cells displayed three distinct morphologies: spread, spindle, and intermediate. We found that spread and spindle cells have similar levels of alpha-SMA, a desmoplastic marker, and consistent ratios of pFAKY(397)/totalFAK. In 3-D intermediate cultures, alpha-SMA levels were virtually undetectable while pFAKY(397)/totalFAK ratios were low. In addition, we used confocal microscopy to assess in vivo-like extracellular matrix topography, nuclei morphology and alpha-SMA features in the 3-D cultures. We found that all NOFs presented 'normal' characteristics, while TOFs presented both 'primed' and 'activated' features. Moreover, immunohistochemistry analyses confirmed that the 3-D matrix-dependent characteristics are reminiscent of those observed in in vivo stromal counterparts., Conclusions: We conclude that primary human ovarian fibroblasts maintain in vivo-like (staged) stromal characteristics in a 3-D matrix-dependent manner. Therefore, our stromal 3-D system offers a tool that can enhance the understanding of both stromal progression and stroma-induced ovarian tumorigenesis. In the future, this system could also be used to develop ovarian stroma-targeted therapies.

Tumor-associated fibroblasts are functionally and phenotypically distinct from normal fibroblasts that are not in the tumor microenvironment. Fibroblast activation protein is a 95 kDa cell surface glycoprotein expressed by tumor stromal fibroblasts, and has been shown to have dipeptidyl peptidase and collagenase activity. Site-directed mutagenesis at the catalytic site of fibroblast activation protein, Ser624 --> Ala624, resulted in an approximately 100,000-fold loss of fibroblast activation protein dipeptidyl peptidase (DPP) activity. HEK293 cells transfected with wild-type fibroblast activation protein, enzymatic mutant (S624A) fibroblast activation protein, or vector alone, were inoculated subcutaneously into immunodeficient mouse to assess the contribution of fibroblast activation protein enzymatic activity to tumor growth. Overexpression of wild-type fibroblast activation protein showed growth potentiation and enhanced tumorigenicity compared with both fibroblast activation protein S624A and vector-transfected HEK293 xenografts. HEK293 cells transfected with fibroblast activation protein S624A showed tumor growth rates and tumorigenicity potential similar only to vector-transfected HEK293. In vivo assessment of fibroblast activation protein DPP activity of these tumors showed enhanced enzymatic activity of wild-type fibroblast activation protein, with only baseline levels of fibroblast activation protein DPP activity in either fibroblast activation protein S624A or vector-only xenografts. These results indicate that the enzymatic activity of fibroblast activation protein is necessary for fibroblast activation protein-driven tumor growth in the HEK293 xenograft model system. This establishes the proof-of-principle that the enzymatic activity of fibroblast activation protein plays an important role in the promotion of tumor growth, and provides an attractive target for therapeutics designed to alter fibroblast activation protein-induced tumor growth by targeting its enzymatic activity.