Altered CXCL12 expression reveals a dual role of CXCR4 in osteosarcoma primary tumor growth and metastasis.

PURPOSE: Better understanding of the molecular mechanisms of metastasis-the major cause of death in osteosarcoma (OS)-is a key for the development of more effective metastasis-suppressive therapy. Here, we investigated the biological relevance of the CXCL12/CXCR4 axis in OS. METHODS: We interfered with CXCL12/CXCR4 signaling in CXCR4-expressing human 143-B OS cells through stable expression of CXCL12, of its competitive antagonist P2G, or of CXCL12-KDEL, designed to retain CXCR4 within the cell. Intratibial OS xenograft mouse model metastasizing to the lung was used to assess tumorigenic and metastatic potential of the manipulated cell lines. RESULTS: Constitutive expression of native CXCL12 promoted lung metastasis without affecting tumor growth. Stable expression of P2G or CXCL12-KDEL significantly accelerated tumor growth but diminished lung metastasis. Tumors grown from P2G- or CXCL12-KDEL-expressing cells contained higher levels of CXCR4-encoding mRNA going along with a higher percentage of CXCR4-expressing tumor cells. Lung metastases of all groups were predominantly enriched with CXCR4-expressing tumor cells. CONCLUSION: Higher abundance of CXCR4 possibly contributed to increased local retention of tumor cells by bone marrow-derived CXCL12, reflected in the increased primary tumor growth and decreased number of lung metastases in P2G and CXCL12-KDEL groups. Higher percentage of CXCR4-expressing lung metastatic cells compared to the corresponding primary tumors point to important functions of the CXCL12/CXCR4 axis in late steps of metastasis. In conclusion, based on the here reported results, local treatment of lung metastases with novel CXCR4-targeting therapeutics might be considered and favored over anti-CXCR4 systemic therapy.

CXCR4 antibody treatment suppresses metastatic spread to the lung of intratibial human osteosarcoma xenografts in mice.

Current combined surgical and neo-adjuvant chemotherapy of primary metastatic osteosarcoma (OS) is ineffective, reflected by a 5-year survival rate of affected patients of less than 20 %. Studies in experimental OS metastasis models pointed to the CXCR4/CXCL12 homing axis as a novel target for OS metastasis-suppressive treatment. The present study investigated for the first time the CXCR4-blocking principle in a spontaneously metastasizing human 143B OS cell line-derived orthotopic xenograft mouse model. The highly metastatic 143B cells, unlike the parental non-metastatic HOS cells, express functional CXCR4 receptors at the cell surface, as revealed in this study by RT/PCR of gene transcripts, by FACS analysis with the monoclonal anti CXCR4 antibody 12G5 (mAb 12G5) and by CXCL12 time- and dose-dependent stimulation of AKT and ERK phosphorylation. A significantly (p < 0.05) higher CXCL12 dose-dependent chemotactic response of 143B compared to HOS cells in a Boyden chamber trans-well migration assay suggested a crucial role of the CXCL12/CXCR4 homing axis in 143B cell lung metastasis. Repetitive treatment of mice with 143B cell-derived intratibial tumors given intravenous bolus injections of mAb12G5 indeed inhibited significantly (p < 0.01) the number of X-gal-stainable lung micrometastases of lacZ-transduced 143B cells. Antibody treatment had also a mild inhibitory effect on primary tumor growth associated with remarkably less osteolysis, but it did not affect the number of developing lung macrometastases. In conclusion, these results demonstrate considerable potential of high-affinity CXCR4-blocking agents for OS tumor cell homing suppressive treatment in metastasizing OS complementary to current (neo)-adjuvant chemotherapy.

Expression of the chemokine receptor CXCR7 in CXCR4-expressing human 143B osteosarcoma cells enhances lung metastasis of intratibial xenografts in SCID mice.

More effective treatment of metastasizing osteosarcoma with a current mean 5-year survival rate of less than 20% requires more detailed knowledge on mechanisms and key regulatory molecules of the complex metastatic process. CXCR4, the receptor of the chemokine CXCL12, has been reported to promote tumor progression and metastasis in osteosarcoma. CXCR7 is a recently deorphanized CXCL12-scavenging receptor with so far not well-defined functions in tumor biology. The present study focused on a potential malignancy enhancing function of CXCR7 in interaction with CXCR4 in osteosarcoma, which was investigated in an intratibial osteosarcoma model in SCID mice, making use of the human 143B osteosarcoma cell line that spontaneously metastasizes to the lung and expresses endogenous CXCR4. 143B osteosarcoma cells stably expressing LacZ (143B-LacZ cells) were retrovirally transduced with a gene encoding HA-tagged CXCR7 (143B-LacZ-X7-HA cells). 143B-LacZ-X7-HA cells co-expressing CXCR7 and CXCR4 exhibited CXCL12 scavenging and enhanced adhesion to IL-1ß-activated HUVEC cells compared to 143B-LacZ cells expressing CXCR4 alone. SCID mice intratibially injected with 143B-LacZ-X7-HA cells had significantly (p<0.05) smaller primary tumors, but significantly (p<0.05) higher numbers of lung metastases than mice injected with 143B-LacZ cells. Unexpectedly, 143B-LacZ-X7-HA cells, unlike 143B-LacZ cells, also metastasized with high incidence to the auriculum cordis. In conclusion, expression of the CXCL12 scavenging receptor CXCR7 in the CXCR4-expressing human 143B osteosarcoma cell line enhances its metastatic activity in intratibial primary tumors in SCID mice that predominantly metastasize to the lung and thereby closely mimic the human disease. These findings point to CXCR7 as a target, complementary to previously proposed CXCR4, for more effective metastasis-suppressive treatment in osteosarcoma.

Characterization of different osteosarcoma phenotypes by PET imaging in preclinical animal models.

UNLABELLED: The aim of this study was to characterize the different phenotypes of osteosarcoma by PET, comparing the uptake of 3 tracers ((18)F-FDG, (18)F-fluoromisonidazole [(18)F-FMISO], and (18)F-fluoride) in preclinical mouse models that reflect the heterogeneity of the human disease. METHODS: Mouse LM8 osteosarcoma, human 143B, and Caprin-1 stably overexpressing SaOS-2 cells were injected intratibially in C3H and severe-combined immunodeficient mice. PET imaging with (18)F-FDG, (18)F-FMISO, and (18)F-fluoride was performed in these mouse models, and a ratio between the standardized uptake value of the primary tumor and a control area of bone was calculated and compared among the models. Histology and immunohistochemistry were performed to confirm the PET findings. RESULTS: The pattern of tracer uptake differed among the primary tumors of the 3 models in accordance with the histology and immunohistochemistry on primary tumor sections. The osteolytic tumors in the 143B model showed the highest uptake of (18)F-FDG, an indicator of glucose metabolism, which was significantly higher (P < 0.05) than in the SaOS-2/Caprin-1 model and correlated with the percentage of Ki67-positive cells in the primary tumors. Hypoxia, indicated by (18)F-FMISO accumulation, was higher in the SaOS-2/Caprin-1 and 143B cell line-derived tumors (P < 0.01). Finally (18)F-fluoride, a marker of bone remodeling, correlated with the osteoblastic phenotype. The SaOS-2/Caprin-1 cell-derived tumors showed a significantly higher uptake than the moderately osteoblastic LM8 (P < 0.05) and the osteolytic 143B (P < 0.01) cell line-derived tumors. CONCLUSION: Differential PET imaging with tracers indicating metabolic activity, hypoxia, or bone remodeling will be helpful for the characterization of different osteosarcoma phenotypes and subsequent evaluation of more specific treatment modalities targeting the processes that are predominant in each specific tumor type or subtype.

Silencing of CD44 gene expression in human 143-B osteosarcoma cells promotes metastasis of intratibial tumors in SCID mice.

Osteosarcoma (OS) is the most frequent primary malignant bone cancer in children and adolescents with a high propensity for lung metastasis. Therefore, it is of great importance to identify molecular markers leading to increased metastatic potential in order to devise more effective therapeutic strategies that suppress metastasis, the major cause of death in OS. CD44, the principal receptor for the extracellular matrix component hyaluronan (HA), is frequently found overexpressed in tumor cells and has been implicated in metastatic spread in various cancer types. Here, we investigated the effects of stable shRNA-mediated silencing of CD44 gene products on in vitro and in vivo metastatic properties of the highly metastatic human 143-B OS cell line. In vitro, CD44 knockdown resulted in a 73% decrease in the adhesion to HA, a 57% decrease in the migration rate in a trans-filter migration assay, and a 28% decrease in the cells' capacity for anchorage-independent growth in soft agar compared to the control cells, implicating that CD44 expression contributes to the metastatic activity of 143-B cells. However, making use of an orthotopic xenograft OS mouse model, we demonstrated that reduced CD44 expression facilitated primary tumor growth and formation of pulmonary metastases. The enhanced malignant phenotype was associated with decreased adhesion to HA and reduced expression of the tumor suppressor merlin in vivo. In conclusion, our study identified CD44 as a metastasis suppressor in this particular experimental OS model.

CD44 enhances tumor formation and lung metastasis in experimental osteosarcoma and is an additional predictor for poor patient outcome.

Formation of metastases in the lungs is the major cause of death in patients suffering from osteosarcoma (OS). Metastases at presentation and poor response to preoperative chemotherapy are strong predictors for poor patient outcome. The elucidation of molecular markers that promote metastasis formation and/or chemoresistance is therefore of importance. CD44 is a plasma membrane glycoprotein that binds to the extracellular matrix component hyaluronan (HA) and has been shown to be involved in metastasis formation in a variety of other tumors. Here we investigated the role of CD44 expression on OS tumor formation and metastasis. High CD44 expression, evaluated with a tissue microarray including samples from 53 OS patients and stained with a pan-CD44 antibody (Hermes3), showed a tendency (p < 0.08) to shortened overall survival. However, nonresponders and patients with lung metastases and high CD44 expression had significantly poorer prognosis than patients with low CD44 expression. Overexpression of the standard CD44 isoform (CD44s) and its HA-binding defective mutant R41A in osteoblastic SaOS-2 cells resulted in HA-independent higher migration rates and increased chemoresistance, partially dependent on HA. In an orthotopic mouse model of OS, overexpression of CD44s in SaOS-2 cells resulted in an HA-dependent increased primary tumor formation and increased numbers of micrometastases and macrometastases in the lungs. In conclusion, although CD44 failed to be an independent predictor for patient outcome in this limited cohort of OS patients, increased CD44 expression was associated with even worse survival in patients with chemoresistance and with lung metastases. CD44-associated chemoresistance was also observed in vitro, and increased formation of lung metastases was found in vivo in SCID mice.