Purpose: Human fibroblast activation protein (FAP)/seprase is a 97-kDa surface glycoprotein expressed on tumor associated fibroblasts in the majority of epithelial cancers including colon adenocarcinomas. FAP overexpression in human tumor cells has been shown to promote tumor growth in animal models, and clinical trials targeting FAP enzymatic activity have been initiated. The primary objective of this study was to evaluate the clinical significance of stromal FAP in human colon cancers by immunohistochemisty. Experimental Design: Sections of paraffin-embedded resected primary human colon cancer specimens from 1996 through 2001 within the Fox Chase Cancer Center tumor bank were stained with D8 antibody directed against FAP/seprase. Xenotransplanted human colorectal tumors in mice were examined similarly for stromal FAP in tumors of different sizes. Overall percentage of stromal FAP staining of the primary tumor was assessed semiquantitatively (0, 1+, 2+, 3+) and staining intensity was also graded (none, weak, intermediate, strong). Survival time and time to recurrence data were analyzed using Kaplan-Meier plots, log-rank tests, and Cox proportional hazards models. Results: One hundred thirty-eight patients with resected specimens were available for study (mean follow-up, 1,050 days) with 6 (4%) stage I, 52 (38%) stage II, 43 (31%) stage III, and 37 (27%) stage IV patients. FAP was detected in >93% of specimens. Semiquantitative staining was scored as 1+ in 28 (20%), 2+ in 52 (38%), and 3+ in 49 (35%). FAP staining intensity was graded as weak in 45 (33%), intermediate in 48 (35%), and dark in 36 (26%). Stromal FAP was found to correlate inversely with tumor stage (semiquantitative, P = 0.01; intensity, P = 0.009) and with tumor size of the tumor xenograft model (correlation coefficient, À0.61; P = 0.047), suggesting that stromal FAP may have a greater role in the early development of tumors. Furthermore, greater stromal FAP for patients with known metastatic disease was associated with a decreased survival. Conclusion: Our data indicate that patients whose colon tumors have high levels of stromal FAP are more likely to have aggressive disease progression and potential development of metastases or recurrence. This study affirms the rationale for ongoing clinical investigations using FAP as a therapeutic target to disrupt FAP-driven tumor progression in patients with metastatic disease. It also suggests that the effects of FAP inhibition should be investigated in earlier-stage tumors, given its high levels and potential effect earlier in the course of the disease.

BackgroundAlterations 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.MethodsWe 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.ResultsWe 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.ConclusionCancer 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.

Mutations in the genes encoding endothelin receptor-B (Ednrb) and its ligand endothelin-3 (Edn3) affect the development of two neural crest-derived cell types, melanocytes and enteric neurons. EDNRB signaling is exclusively required between E10.5 and E12.5 during the migratory phase of melanoblast and enteric neuroblast development. To determine the fate of Ednrb-expressing cells during this critical period, we generated a strain of mice with the bacterial beta-galactosidase (lacZ) gene inserted downstream of the endogenous Ednrb promoter. The expression of the lacZ gene was detected in melanoblasts and precursors of the enteric neuron system (ENS), as well as other neural crest cells and nonneural crest-derived lineages. By comparing Ednrb(lacZ)/+ and Ednrb(lacZ)/Ednrb(lacZ) embryos, we determined that the Ednrb pathway is not required for the initial specification and dispersal of melanoblasts and ENS precursors from the neural crest progenitors. Rather, the EDNRB-mediated signaling is required for the terminal migration of melanoblasts and ENS precursors, and this pathway is not required for the survival of the migratory cells.

Hirschsprung disease (HSCR) is a multigenic, congenital disorder that affects 1 in 5,000 newborns and is characterized by the absence of neural crest-derived enteric ganglia in the colon 1 . One of the primary genes affected in HSCR encodes the G protein-coupled endothelin receptor-B (EDNRB) 2,3 . The expression of Ednrb is required at a defined time period during the migration of the precursors of the enteric nervous system (ENS) into the colon 4 . In this study, we describe a conserved spatiotemporal ENS enhancer of Ednrb. This 1-kb enhancer is activated as the ENS precursors approach the colon, and partial deletion of this enhancer at the endogenous Ednrb locus results in pigmented mice that die postnatally from megacolon. We identified binding sites for SOX10, an SRY-related transcription factor associated with HSCR 5 , in the Ednrb ENS enhancer, and mutational analyses of these sites suggested that SOX10 may have multiple roles in regulating Ednrb in the ENS.Mice and individuals with HSCR with mutations in the EDNRB-mediated pathway have megacolon because of the absence of enteric neurons in the distal gut 1 . This regional specificity of aganglionosis could be explained by a temporal requirement for Ednrb between embryonic day (E) 11 and E12.5 (ref. 4), when vagal neural crest-derived ENS progenitors are populating the hindgut during mouse embryogenesis, such that in the absence of EDNRB the migratory wavefront is delayed near the ileocecal junction (Fig. 1a) [6][7][8] . To elucidate the molecular mechanisms for Ednrb expression in the ENS, we dissected the Ednrb genomic region. We isolated a 78-kb P1 genomic clone encompassing Ednrb (Fig. 1b) and used it to create four independent transgenic lines. When we crossed the individual transgenic lines into the Ednrb-null mice, all the lines rescued postnatal death from megacolon (Fig. 1c). Although three of the lines did not rescue the melanocyte defect in the Ednrb-null mice (Fig. 1c), one line (when homozygous with respect to the transgene) partially rescued the pigmentation defect that resembles the hypomorphic Ednrb s allele 9 (data not shown). These results suggested that the P1 clone contained the necessary information for expression of Ednrb in ENS progenitors. In addition, we mapped a main transcription start site Ednrb -/-Tg P5

T lymphocytes develop into two major lineages characterized by expression of the ab and cd T cell receptor (TCR) heterodimers. Within each major lineage, further specialization occurs, resulting in distinct subsets that differ in TCR specificity, phenotype and functional attributes. Thus, in the murine thymus, two distinct subsets of mature (CD24À) cd cells have been identified, that is NK1.1 þ cells, which are enriched for Vc1.1 usage and selectively produce IFNc on stimulation, and CCR6 þ cells, which are enriched for Vc2 usage produce IL17. The upstream signals and transcriptional pathways that promote development of these distinct cd subsets remain relatively poorly understood. Here, we show that the Zn-finger transcription factor ThPOK has a critical function in the development of cd thymocytes. Thus, lack of functional ThPOK causes a marked reduction in the percentage and absolute number of mature cd thymocytes, and a particularly severe reduction of NK1.1 þ cells. Conversely, constitutive ThPOK expression leads to a striking increase in mature NK1.1 þ cd thymocytes. Further, we show that ThPOK induction in cd thymocytes is induced by strong TCR signals mediated by engagement with antibody or high-affinity endogenous ligands, and that an important ThPOK cis-acting element, the distal regulatory element (DRE), is sufficient for this TCR-dependent induction. These results show that ThPOK expression in cd thymocytes is regulated in part by the strength of TCR signalling, identify ThPOK as an important mediator of cd T cell development/maturation, and lend strong support to the view that development of a significant fraction of cd T cells depends on TCR engagement/ signalling.