Targeting αvβ3 and αvβ5 integrins inhibits pulmonary metastasis in an intratibial xenograft osteosarcoma mouse model

Osteosarcoma is an aggressive bone cancer that has a high propensity for metastasis to the lungs. Patients with metastatic disease face a very poor prognosis. Therefore, novel therapeutics, efficiently suppressing the metastatic process, are urgently needed. Integrins play a pivotal role in tumor cell adhesion, motility and metastasis. Here, we evaluated αvβ3 and αvβ5 integrin inhibition with cilengitide as a novel metastasis-suppressive therapeutic approach in osteosarcoma. Immunohistochemical analysis of αvβ3 and αvβ5 integrins expression in a tissue microarray of tumor specimens collected from osteosarcoma patients revealed that αvβ5 integrin is mainly found on tumor cells, whereas αvβ3 is predominantly expressed by stromal cells. In vitro functional assays demonstrated that cilengitide dose-dependently inhibited de novo adhesion, provoked detachment and inhibited migration of osteosarcoma cell lines. Cilengitide induced a decline in cell viability, blocked the cell cycle in the G1 phase and caused anoikis by activation of the Hippo pathway. In a xenograft orthotopic mouse model cilengitide minimally affected intratibial primary tumor growth but, importantly, suppressed pulmonary metastasis. The data demonstrate that targeting αvβ3 and αvβ5 integrins in osteosarcoma should be considered as a novel therapeutic option for patients with metastatic disease

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 coexpressing 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 Relevance of CD44 and hyaluronan interaction in osteosarcoma progression and metastasis

Formation of metastases in the lungs is the major cause of death in patients suffering from osteosarcoma (OS), the most common primary bone cancer in children and adolescents. Significant clinical improvements over the past several decades through the use of combination chemotherapy and surgery have led to a dramatic increase in the survival of patients with localized disease. However, patients with metastatic or recurrent disease continue to have a very poor prognosis, with <20% long term survival. The failure of treatment in these patients is often associated with gained resistance of tumor cells to chemotherapy. Therefore, it is of substantial relevance to identify molecular markers associated with the increased metastatic potential or chemoresistance, which may serve as diagnostic or prognostic factors. Acquiring insight into the basic biology of OS progression will make the identification of such new therapeutic targets possible with the final goal to develop treatment strategies that eradicate metastases and thereby improve the survival of OS patients. Metastasis is a complex multistep process which involves detachment of tumor cells from primary tumor mass, invasion of local stroma, intravasation, survival during transport in the circulation, arrest at distant organ sites, extravasation into corresponding parenchyma, adaptation to this new foreign tissue microenvironment and finally colonization in the distant organ. CD44 has been linked with increased metastatic spread in various types of cancer, however, its function in sarcomas has been only superficially addressed. CD44 and its numerous splice variants constitute a family of widely distributed type I transmembrane glycoproteins that serve as cell-cell and cell-matrix adhesion molecules and as principal receptors for hyaluronan (HA), a major component of the extracellular matrix in many tissues including bone. In this thesis, the prognostic value of CD44 expression for OS patients’ outcome and the biological relevance of CD44/HA interactions for in vitro malignant properties of OS tumor cells and for in vivo OS progression and metastasis in orthotopic xenograft OS mouse models were investigated. Our tissue microarray analysis of OS tumor specimens identified CD44 expression as an additional indicator of poor prognosis together with metastasis and resistance to chemotherapy, the two best established predictors of poor OS patient’s outcome. In vitro, CD44 expression correlated with the adhesion to HA and with cell migration. Moreover, cells with forced CD44 expression were more resistant to cisplatin. In vivo, our studies in different human xenograft OS mouse models revealed that CD44 gene products may play a dual role in regulating OS progression and metastases, depending on the cellular background. However, in the context of osteoblastic OS, the most common type of OS, our study demonstrated for the first time that CD44 promotes OS growth and dissemination in a HA-dependent manner and that CD44 expression is associated with in vitro enhanced migration rates and chemoresistance to cisplatin. This is also consistent with patient’s outcome as observed with tissue microarray analysis. On the other hand, in a subgroup of OS, where Ras signaling is increased, CD44 may act as a tumor suppressor probably by upregulation of merlin. Taken together, the findings presented in this thesis underscore the important role of CD44s/HA interaction in determining tumor malignancy in experimental OS. In conclusion, our results highlight CD44/HA interaction as a promising target for therapeutic intervention in this highly aggressive cancer type

A bispecific antibody targeting IGF-IR and EGFR has tumor and metastasis suppressive activity in an orthotopic xenograft osteosarcoma mouse model

Osteosarcoma is a highly aggressive bone cancer and the second most frequent cause of cancer-associated death in childhood and adolescence. Pulmonary metastases account for the high mortality rate in osteosarcoma patients. Therefore, novel therapeutic approaches, efficiently restraining the metastatic disease, are mandatory for a significant improvement of the currently poor patients' survival. Although initial studies with antibodies targeting insulin-like growth factor receptor (IGF-IR) showed promising potential for the treatment of patients with bone and soft tissue sarcomas, phase II clinical trials revealed variable results, which implied activation of alternative signaling pathways leading to therapy resistance. Since a cross-talk between IGF-IR and the epidermal growth factor receptor (EGFR) has been demonstrated in several cancer types, co-targeting of these two receptors was considered in the present study as a valuable therapeutic strategy to overcome single-agent treatment resistance in osteosarcoma. The effects of IGF-IR and/or EGFR targeting by intraperitoneal administration of the monospecific IGF-IR antibody R1507 or the EGFR antibody Cetuximab or the bispecific IGF-IR/EGFR antibody XGFR* on primary tumor growth and pulmonary metastasis were investigated in an intratibial human xenograft osteosarcoma mouse model. In vitro functional assays demonstrated that targeting IGF-IR and EGFR didn't affect osteosarcoma cell viability, but inhibited ligand-activated intracellular signaling and cell migratory capacity. The blocking potential of ligand-induced signaling in vitro was similar for all antibodies, but, in vivo, only XGFR* treatment significantly inhibited intratibial primary tumor growth and pulmonary metastasis. The therapeutic response to XGFR* was associated with an infiltration of innate immune system effector cells into the tumor microenvironment. Taken together, our study highlights the bispecific anti-IGF-IR/EGFR antibody XGFR* as an innovative promising effective candidate for the treatment of metastatic osteosarcoma and provides the rationale for future clinical studies

A new time dimension in the fight against metastasis

Despite advances in uncovering vulnerabilities, identifying biomarkers, and developing more efficient treatments, cancer remains a threat because of its ability to progress while acquiring resistance to therapy. The circadian rhythm governs most of the cellular functions implicated in cancer progression, and its exploitation therefore opens new promising directions in the fight against metastasis. In this review we summarize the role of the circadian rhythm in tumor development and progression, with emphasis on the circadian rhythm-regulated elements that control the generation of circulating tumor cells (CTCs) and metastasis. We then present data on chronotherapy and discuss how circadian rhythm investigations may open new paths to more effective anticancer treatments.ISSN:0962-8924ISSN:0968-0039ISSN:1879-308

Circulating tumour cells in gastrointestinal cancers: food for thought?

Gastrointestinal (GI) cancers account for 35% of cancer-related deaths, predominantly due to their ability to spread and generate drug-tolerant metastases. Arising from different locations in the GI system, the majority of metastatic GI malignancies colonise the liver and the lungs. In this context, circulating tumour cells (CTCs) are playing a critical role in the formation of new metastases, and their presence in the blood of patients has been correlated with a poor outcome. In addition to their prognostic utility, prospective targeting of CTCs may represent a novel, yet ambitious strategy in the fight against metastasis. A better understanding of CTC biology, mechanistic underpinnings and weaknesses may facilitate the development of previously underappreciated anti-metastasis approaches. Here, along with related clinical studies, we outline a selection of the literature describing biological features of CTCs with an impact on their metastasis forming ability in different GI cancers.ISSN:0007-0920ISSN:1532-182

Travelling under pressure-hypoxia and shear stress in the metastatic journey

Cancer cell invasion, intravasation and survival in the bloodstream are early steps of the metastatic process, pivotal to enabling the spread of cancer to distant tissues. Circulating tumor cells (CTCs) represent a highly selected subpopulation of cancer cells that tamed these critical steps, and a better understanding of their biology and driving molecular principles may facilitate the development of novel tools to prevent metastasis. Here, we describe key research advances in this field, aiming at describing early metastasis-related processes such as collective invasion, shedding, and survival of CTCs in the bloodstream, paying particular attention to microenvironmental factors like hypoxia and mechanical stress, considered as important influencers of the metastatic journey.ISSN:1573-7276ISSN:0262-089

Inhibition of the activin receptor signaling pathway: A novel intervention against osteosarcoma

Osteosarcoma is a cancer of pathological bone remodeling with high mortality and severe comorbidity. New therapies are urgently needed. Activin A, a member of the transforming growth factor β (TGFβ) superfamily, has been suggested to stimulate proliferation and invasion of osteosarcoma cells in vitro, thus representing a potential therapeutic target. In this study, inhibition of the activin receptor signaling pathway was explored as a therapy for osteosarcoma. In a murine intratibial osteosarcoma xenograft model, two types of inhibitors were tested: (a) a soluble activin type IIA decoy receptor (ActRIIA‐mFc), or (b) a modified variant of follistatin (FSTΔHBS‐hFc), either alone or in combination with a bisphosphonate. Both inhibitors reduced primary tumor development by nearly 50% compared to vehicle treatment. When ActRIIA‐mFc was combined with bisphosphonate, the effect on tumor size became even more pronounced (78% reduction vs. vehicle). Moreover, FSTΔHBS‐hFc increased body weight in the face of tumor progression (14% increase vs. vehicle), and ActRIIA‐mFc reduced the number of lung metastases when combined with bisphosphonate. The present study demonstrates a novel approach to treating osteosarcoma and encourages further investigation of inhibition of the activin receptor signaling pathway as an intervention against the disease