Matthias J.E. Arlt

Cyr61 Expression in Osteosarcoma Indicates Poor Prognosis and Promotes Intratibial Growth and Lung Metastasis in Mice

Osteosarcoma is the most frequent primary malignant bone tumor in children and adolescents with a high propensity for lung metastasis, the major cause of disease‐related death. Reliable outcome‐predictive markers and targets for osteosarcoma metastasis‐suppressing drugs are urgently needed for more effective treatment of metastasizing osteosarcoma, which has a current mean 5‐year survival rate of approximately 20%. This study investigated the prognostic value and the biological relevance of the extracellular matrix‐associated growth factor Cyr61 of the CCN family of secreted proteins in osteosarcoma and metastasis. The prognostic value of Cyr61 was assessed with Kaplan‐Meier analyses based on Cyr61 immunostaining of a tissue microarray of osteosarcoma biopsies collected from 60 patients with local or metastatic disease. Effects of Cyr61 overexpression on intratibial tumor growth and lung metastasis of the low metastatic human SaOS‐2 osteosarcoma cell line were examined in severe combined immunodeficiency (SCID) mice. Cyr61‐provoked signaling was studied in vitro in nonmanipulated SaOS‐2 cells. Cyr61 immunostaining of osteosarcoma tissue cores correlated significantly (p = 0.02) with poor patient survival. Mice intratibially injected with Cyr61‐overexpressing SaOS‐2 cells showed faster tumor growth and an increase in number and outgrowth of lung metastases and consequently significantly (p = 0.0018) shorter survival than mice injected with control SaOS‐2 cells. Cyr61‐evoked PI‐3K/Akt/GSK3β signaling in SaOS‐2 cells resulted in a subcellular redistribution of the cell cycle inhibitor p21Cip1/WAF1. Cyr61 has considerable potential as a novel marker for poor prognosis in osteosarcoma and is an attractive target for primary tumor‐ and metastases‐suppressing drugs.

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.

LacZ transgene expression in the subcutaneous Dunn/LM8 osteosarcoma mouse model allows for the identification of micrometastasis†

More effective treatment of patients with metastasizing osteosarcoma (OS) with a mean 5‐year survival rate of <20% requires more detailed knowledge on the complex mechanisms of metastasis for the design of new drugs, which selectively target metastasizing cells. Moreover, novel diagnostic imaging technology for early detection of metastases is needed. Mouse models, which reproduce human metastasizing OS and allow visualization of single metastatic cells are instrumental for preclinical testing of new pharmaceuticals and diagnostic instruments. Here, the low metastatic Dunn cell line and its highly metastatic LM8 subline, both equipped with a constitutively expressed lacZ gene, were used to improve the well‐established OS models in syngeneic C3H mice to achieve ex vivo visualization of single metastatic cells in affected organs by X‐gal staining. These models, combined with a technique for in situ high quality lung tissue‐maintaining perfusion revealed, as a novel finding, single metastasizing Dunn cells in lung and liver. Importantly, constitutive lacZ gene expression did not affect in vitro and in vivo tumorigenic and metastatic properties of Dunn and LM8 cells. Thus, these improved Dunn and LM8 OS mouse models will in the future serve as a benchmark for the development of new metastasis‐targeting drugs and metastasis‐imaging technology.

Antimetastatic activity of honokiol in osteosarcoma

Metastasizing osteosarcoma has a mean 5‐year survival rate of only 20% to 30%. Therefore, novel chemotherapeutics for more effective treatment of this disease are required.

Matrix Metalloproteinase 1 promotes tumor formation and lung metastasis in an intratibial injection osteosarcoma mouse model

Proteolytic degradation of the extracellular matrix (ECM) is an important process during tumor invasion. Matrix Metalloproteinase 1 (MMP-1) is one of the proteases that degrade collagen type I, a major component of bone ECM. In the present study, the biological relevance of MMP-1 in osteosarcoma (OS) tumor growth and metastasis was investigated in vitro and in vivo. Human OS cells in primary culture expressed MMP-1 encoding mRNA at considerably higher levels than normal human bone cells. In addition, MMP-1 mRNA and protein expression in the highly metastatic human osteosarcoma 143-B cell line was remarkably higher than in the non-metastatic parental HOS cell line. Stable shRNA-mediated downregulation of MMP-1 in 143-B cells impaired adhesion to collagen I and anchorage-independent growth, reflected by a reduced ability to grow in soft agar. Upon intratibial injection into SCID mice, 143-B cells with shRNA-downregulated MMP-1 expression formed smaller primary tumors and significantly lower numbers of lung micro- and macrometastases than control cells. Conversely, HOS cells stably overexpressing MMP-1 showed an enhanced adhesion capability to collagen I and accelerated anchorage-independent growth compared to empty vector-transduced control cells. Furthermore, and most importantly, individual MMP-1 overexpression in HOS cells enabled the formation of osteolytic primary tumors and lung metastasis while the HOS control cells did not develop any tumors or metastases after intratibial injection. The findings of the present study reveal an important role of MMP-1 in OS primary tumor and metastasis formation to the lung, the major organ of OS metastasis.

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-1β-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.

The antineoplastic antibiotic taurolidine promotes lung and liver metastasis in two syngeneic osteosarcoma mouse models and exhibits severe liver toxicity

Osteosarcoma (OS) is the most frequent primary bone tumor. Despite multiagent neoadjuvant chemotherapy, patients with metastatic disease have a poor prognosis. Moreover, currently used chemotherapeutics have severe toxic side effects. Thus, novel agents with improved antimetastatic activity and reduced toxicity are needed. Taurolidine, a broad‐spectrum antimicrobial, has recently been shown to have antineoplastic properties against a variety of tumors and low systemic toxicity. Consequently, we investigated in our study the antineoplastic potential of taurolidine against OS in two different mouse models. Although both OS cell lines, K7M2 and LM8, were sensitive for the compound in vitro, intraperitoneal application of taurolidine failed to inhibit primary tumor growth. Moreover, it enhanced the metastatic load in both models 1.7‐ to 20‐fold and caused severe liver deformations and up to 40% mortality. Thus, systemic toxicity was further investigated in tumor‐free mice histologically, by electron microscopy and by measurements of representative liver enzymes. Taurolidine dose‐dependent fibrous thickening of the liver capsule and adhesions and atrophies of the liver lobes were comparable in healthy and tumor‐bearing mice. Liver toxicity was further indicated by up to eightfold elevated levels of the liver enzymes alanine transaminase, aspartate transaminase and GLDH in the circulation. Ultrastructural analysis of affected liver tissue showed swollen mitochondria with cristolysis and numerous lipid vacuoles in the cytoplasm of hepatocytes. The findings of our study question the applicability of taurolidine for OS treatment and may suggest the need for caution regarding the widespread clinical use of taurolidine as an antineoplastic agent.

Primary tumour growth in an orthotopic osteosarcoma mouse model is not influenced by analgesic treatment with buprenorphine and meloxicam

Little is known about the treatment of bone pain in animal models of bone cancer. In the present study, the orthotopic 143-B human osteosarcoma xenotransplantation model was used to address the following questions: (1) Can repetitive analgesic treatment extend the experimental period by prolonging the time to reach humane endpoints and (2) Does repetitive analgesic treatment affect bone tumour development and metastasis? The analgesics, buprenorphine and meloxicam, were either applied individually or in combination at 12 h intervals as soon as the animals began to avoid using the tumour cell injected leg. While control mice treated with NaCl showed continuous body weight loss, the major criterion previously for terminating the experiments, animals treated with analgesic substances did not. The control mice had to be sacrificed 26 days after tumour cell injection, whereas the groups of animals with the different pain treatments were euthanized after an additional eight days. Importantly, primary intratibial tumour growth was not affected in any of the experimental groups by any of the pain treatment procedures. Between days 26 and 34 after tumour cell injection an increase of about 100% of the number of lung metastases was found for the groups treated with buprenorphine alone or together with meloxicam, but not for the group treated with meloxicam alone. In summary, the results indicated that both buprenorphine and meloxicam are suitable analgesics for prolonging the experimental periods in an experimental intratibial osteosarcoma mouse model.

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

PurposeBetter 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.MethodsWe 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.ResultsConstitutive 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.ConclusionHigher 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.

Mammalian models of bone sarcomas

Animal models are indispensable for pre-clinical cancer research in general, but they are particularly important for research aiming at a better understanding of pathophysiological mechanisms and, consequently, more effective treatment of rare malignancies such as bone sarcomas. The shortage of sarcoma patients available for clinical drug trials and, consequently, slow progress in drug development contrasts with the urgent need to develop new drugs for patients with metastatic disease to further improve patient survival. Clinically relevant animal models, which, within a couple of weeks or months, reproduce human bone sarcomas as closely as possible, are therefore of critical importance for pre-clinical research aiming at careful selection of new drugs that promise fast clinical development and more effective treatment of bone sarcoma patients. In this chapter, we provide an overview of mammalian models that mimic the three most frequently diagnosed primary sarcomas of bone: osteosarcoma, chondrosarcoma and Ewing sarcoma. We discuss advantages and limitations of cell line-based models and highlight models in which tumors spontaneously form as a result of genetic engineering. Finally, we provide suggestions for further improvements of these models.