Selectin binding is essential for peritoneal carcinomatosis in a xenograft model of human pancreatic adenocarcinoma in pfp--/rag2-- mice.

BACKGROUND AND OBJECTIVE: E- and P-selectins expressed on the luminal surface of mesodermally derived endothelial cells play a crucial role in the formation of haematogenous metastases in a number of malignancies. As peritoneal mesothelial cells are also derived form the mesoderm, it was hypothesised that selectins are also of importance in peritoneal tumour spread. METHODS: Immunohistochemistry was used to identify selectin expression on normal human peritoneum and isolated mesothelial cells. E- and P-selectin interactions with human pancreatic adenocarcinoma cells were investigated in dynamic flow assays and flow cytometry; the latter was also used to determine the main selectin ligands on pancreatic adenocarcinoma cell lines PaCa 5061, BxPC-3 and PaCa 5072, and selectin expression on human mesothelial cells. All cell lines were xenografted into the peritoneum of E- and P-selectin-deficient pfp/rag2 mice and selectin wild-type controls. Peritoneal carcinomatosis was quantified using MRI or a scoring system. RESULTS: E- and P-selectin were constitutively expressed on human mesothelial and endothelial cells in the peritoneum. PaCa 5061 and BxPC-3 cells interacted with E- and P-selectins in dynamic flow assays and flow cytometry, with CA19-9 (Sialyl Lewis a) being the main E-selectin ligand. For xenografted PaCa 5061 and BxPC-3 cells, peritoneal metastasis was significantly reduced in E- and P-selectin double knockout mice compared with wild-type pfp/rag2 animals. In contrast, PaCa 5072 cells were almost devoid of selectin binding sites and no intraperitoneal tumour growth was observed. CONCLUSION: Interactions of tumour cells with peritoneal selectins play an important role in the peritoneal spread of pancreatic adenocarcinoma.

Modeling Spontaneous Bone Metastasis Formation of Solid Human Tumor Xenografts in Mice.

The majority of cancer-related deaths are due to hematogenous metastases, and the bone marrow (BM) represents one of the most frequent metastatic sites. To study BM metastasis formation in vivo, the most efficient approach is based on intracardiac injection of human tumor cells into immunodeficient mice. However, such a procedure circumvents the early steps of the metastatic cascade. Here we describe the development of xenograft mouse models (balb/c rag2-/- and severe combined immunodeficient (SCID)), in which BM metastases are spontaneously derived from subcutaneous (s.c.) primary tumors (PTs). As verified by histology, the described methodology including ex vivo bioluminescence imaging (BLI) even enabled the detection of micrometastases in the BM. Furthermore, we established sublines from xenograft primary tumors (PTs) and corresponding BM (BM) metastases using LAN-1 neuroblastoma xenografts as a first example. In vitro "metastasis" assays (viability, proliferation, transmigration, invasion, colony formation) partially indicated pro-metastatic features of the LAN-1-BM compared to the LAN-1-PT subline. Unexpectedly, after s.c. re-injection into mice, LAN-1-BM xenografts developed spontaneous BM metastases less frequently than LAN-1-PT xenografts. This study provides a novel methodologic approach for modelling the spontaneous metastatic cascade of human BM metastasis formation in mice. Moreover, our data indicate that putative bone-metastatic features get rapidly lost upon routine cell culture.

Selectin-deficiency reduces the number of spontaneous metastases in a xenograft model of human breast cancer.

Metastasis formation is a complex process still poorly understood. Previous work in a colon cancer xenograft model showed that E(ndothelial) and P(latelet) selectins mediate spontaneous metastasis to the lungs. To investigate the functional role of selectins in breast cancer, human DU4475 breast cancer cells were injected subcutaneously into pfp-/-rag2-/- mice and in all their selectin-deficient variants (EP-/-, E-/- and P-/-). Pfp-/-rag2-/- mice as well as all their selectin-deficient variants developed primary tumours and spontaneous metastases. Compared with the wild-type mice, disseminated tumours cells were significantly lower (74% reduction, P=0.046) in the bone marrow of selectin-deficient mice. Pfp-/-rag2-/- mice developed significantly higher numbers of lung metastases (6644.83±741.77) than the E-/- (4053.33±112.58; P=0.002) and the EP-/- pfp-/-rag2-/- mice (4665.65±754.50; P<0.001). The results indicate that E- and P-selectins play a role in spontaneous metastasis formation both into bone marrow and lungs. However, spontaneous metastasis was not completely abrogated, hence additional cell adhesion molecules must be involved in the metastatic spread.

Human prostate cancer in a clinically relevant xenograft mouse model: identification of ß(1,6)-branched oligosaccharides as a marker of tumor progression.

PURPOSE: To establish xenograft mouse models of metastatic and nonmetastatic human prostate cancer and to apply these models to the search for aberrant glycosylation patterns associated with tumor progression in vivo and in patients. EXPERIMENTAL DESIGN: Prostate cancer cells (LNCaP, PC-3, LuCaP 23.1, and DU-145) were xenografted subcutaneously into immunodeficient pfp(-/-)/rag2(-/-) mice. Tumor growth and metastasis formation were quantified and as altered glycosylation patterns have been associated with metastasis formation in several other malignancies, prostate cancer cells were profiled by a quantitative real-time PCR (qRT-PCR) glycosylation array and compared with normal human prostate cells. The activity of upregulated glycosyltransferases was analyzed by their sugar residues end products using lectin histochemistry on primary tumors and metastases in the animal experiments and on 2,085 clinical samples. RESULTS: PC-3 cells produced the largest number of spontaneous lung metastases, followed by LNCaP and LuCaP 23.1, whereas DU-145 was nonmetastatic. qRT-PCR revealed an upregulation of ß1,6-N-acetylglucosaminyltransferase-5b (Mgat5b) in all prostate cancer cell lines. Mgat5b products [ß(1,6)-branched oligosaccharides] were predominantly detectable in metastatic xenografts as shown by increased binding of Phaseolus vulgaris leukoagglutinin (PHA-L). The percentage of prostate cancer patients who were PHA-L positive was 86.5. PHA-L intensity correlated with serum prostate-specific antigen and a cytoplasmic staining negatively affected disease-free survival. CONCLUSION: We show a novel xenograft mouse model for human prostate cancer respecting the complete metastatic cascade. Specific glycosylation patterns reveal Mgat5b products as relevant markers of both metastatic competence in mice and disease-free survival in patients. This is the first description of Mgat5b in prostate cancer indicating a significant biologic importance of ß(1,6)-branched oligosaccharides for prostate cancer progression.

Premutations in the FMR1 gene as a modifying factor in Parkin-associated Parkinson's disease?

Premutations in the FMR1 gene may be associated with some cases of parkinsonism. To test this hypothesis, we determined the CGG repeat number in FMR1 in 673 individuals with and without parkinsonism and detected 3 premutation carriers (2 patients, 1 control). Of note, 1 of the affected premutation carriers had a heterozygous Parkin mutation.