Sander M. Botter

Recent advances in osteosarcoma.

Although osteosarcoma (OS) is a rare malignancy, it is ranked among the leading causes of cancer-related death in the pediatric age group. The cancers low prevalence and its large tumor heterogeneity make it difficult to obtain meaningful progress in patient survival. In this review we present an overview of current clinical trials which largely focus on stimulation of the immune system or rely on the inhibition of kinases such as Src and mTOR. The potential efficacy of tumor-targeted TNFalpha is discussed, as well as the importance of preclinical validation of new targets. To improve the success of future clinical trials, clinicians and basic researchers need to intensify their exchange. Finally, a case is made for individualized treatment of OS patients, based on interdisciplinary cooperation in dedicated Sarcoma Centers.

RGD-modified anti-CD3 antibodies redirect cytolytic capacity of cytotoxic T lymphocytes toward αvβ3-EXPRESSING endothelial cells

To redirect the lytic activity of cytotoxic T lymphocytes (CTL) toward tumor vascular endothelial cells, we prepared bifunctional proteins with specificity for both αvβ3 and CD3. Monocyclic RGD peptides (cRGDfK) were covalently coupled to an anti‐CD3 monoclonal antibody at different peptide:protein ratios. The resulting RGDpep‐anti‐CD3 conjugates bound specifically to αvβ3‐expressing endothelial cells. Displacement studies with radiolabeled αvβ3 ligand demonstrated that the αvβ3 binding affinity of RGDpep‐anti‐CD3 conjugates was elevated as compared to the non‐conjugated RGDpep. IC50 values ranged from 150–1.1 nM, depending on the amount of coupled RGDpep molecules per antibody molecule. RGD modification did not affect the ability of anti‐CD3 antibodies to bind to CTL. Furthermore, RGDpep‐anti‐CD3 was fully capable of activating T cells upon CD3 binding as was shown in a Jurkat/NFAT reporter‐gene activation assay. All RGDpep‐anti‐CD3 conjugates were able to induce RGDpep, CD3‐dependent lysis of human primary endothelial cells by anti‐CD3/IL‐2 activated human peripheral blood mononuclear cells (PBMC), with a significant induction of cytotoxicity observed at an E/T ratio as low as 10. Redirecting cytolytic activity reached up to 50% cytotoxicity using the conjugate with the highest RGD peptide load. Combining the good accessibility of tumor blood vessel endothelium for CTL with the efficiency of target cell killing warrants further investigations on anti‐tumor effects of this type of conjugates in vivo.

Genes Regulated in Metastatic Osteosarcoma: Evaluation by Microarray Analysis in Four Human and Two Mouse Cell Line Systems

Osteosarcoma (OS) is a rare bone neoplasm that affects mainly adolescents. It is associated with poor prognosis in case of metastases formation. The search for metastasis predicting markers is therefore imperative to optimize treatment strategies for patients at risk and important for the search of new drugs for the treatment of this devastating disease. Here, we have analyzed by microarray the differential gene expression in four human and two mouse OS cell line systems consisting of parental cell lines with low metastatic potential and derivatives thereof with increased metastatic potential. Using two osteoblastic cell line systems, the most common OS phenotype, we have identified forty-eight common genes that are differentially expressed in metastatic cell lines compared to parental cells. The identified subset of metastasis relevant genes in osteoblastic OS overlapped only minimally with differentially expressed genes in the other four preosteoblast or nonosteoblastic cell line systems. The results imply an OS phenotype specific expression pattern of metastasis regulating proteins and form a basis for further investigation of gene expression profiles in patients samples combined with survival analysis with the aim to optimize treatment strategies to develop new drugs and to consequently improve the survival of patients with the most common form of osteoblastic OS.

Prognostic value of tumor suppressors in osteosarcoma before and after neoadjuvant chemotherapy

BackgroundPrimary bone cancers are among the deadliest cancer types in adolescents, with osteosarcomas being the most prevalent form. Osteosarcomas are commonly treated with multi-drug neoadjuvant chemotherapy and therapy success as well as patient survival is affected by the presence of tumor suppressors. In order to assess the prognostic value of tumor-suppressive biomarkers, primary osteosarcoma tissues were analyzed prior to and after neoadjuvant chemotherapy.MethodsWe constructed a tissue microarray from high grade osteosarcoma samples, consisting of 48 chemotherapy naïve biopsies (BXs) and 47 tumor resections (RXs) after neoadjuvant chemotherapy. We performed immunohistochemical stainings of P53, P16, maspin, PTEN, BMI1 and Ki67, characterized the subcellular localization and related staining outcome with chemotherapy response and overall survival. Binary logistic regression analysis was used to analyze chemotherapy response and Kaplan-Meier-analysis as well as the Cox proportional hazards model was applied for analysis of patient survival.ResultsNo significant associations between biomarker expression in BXs and patient survival or chemotherapy response were detected. In univariate analysis, positive immunohistochemistry of P53 (Pu2009=u20090.008) and P16 (P16; Pu2009=u20090.033) in RXs was significantly associated with poor survival prognosis. In addition, presence of P16 in RXs was associated with poor survival in multivariate regression analysis (Pu2009=u20090.003; HRu2009=u20090.067) while absence of P16 was associated with good chemotherapy response (Pu2009=u20090.004; ORu2009=u200974.076). Presence of PTEN on tumor RXs was significantly associated with an improved survival prognosis (Pu2009=u20090.022).ConclusionsPositive immunohistochemistry (IHC) of P16 and P53 in RXs was indicative for poor overall patient survival whereas positive IHC of PTEN was prognostic for good overall patient survival. In addition, we found that P16 might be a marker of osteosarcoma chemotherapy resistance. Therefore, our study supports the use of tumor RXs to assess the prognostic value of biomarkers.

Evaluation of intraarterial and intravenous cisplatin chemotherapy in the treatment of metastatic osteosarcoma using an orthotopic xenograft mouse model

BackgroundOsteosarcoma is the most common primary malignancy of bone. Its treatment relies on the administration of neoadjuvant and adjuvant chemotherapy combined with surgery. Alternative to common intravenous (i.v.) administration of chemotherapeutic drugs, clinical studies also evaluated the benefit of intraarterial (i.a.) administrations. However, conflicting results were obtained when both routes of administration of cisplatin (CDDP), a gold standard drug in osteosarcoma treatment, were compared. In order to overcome clinical confounding factors, we evaluated both routes of drug administration in a mouse model of experimental osteosarcoma.MethodsWe directly compared i.v. versus i.a. drug infusions of cisplatin (CDDP), in an orthotopic xenograft mouse model of metastatic osteosarcoma. We performed tumor monitoring using caliper and micro computed tomography and measured tumor perfusion using laser speckle contrast imaging. Histopathological changes were evaluated using hematoxylin and eosin staining as well as immunohistochemistry (cleaved PARP-1, CD31, HIF-1α).ResultsFirst, an effective concentration of 4xa0mg/kg i.a. CDDP was determined that significantly reduced primary tumor volume. We used this concentration of i.a. CDDP and compared it to infusions of i.v. CDDP. Systemic (i.v.) CDDP only showed minor suppression of tumor growth whereas local (i.a.) CDDP strongly inhibited tumor growth and destruction of cortical bone in the tumor-bearing hind limb. Inhibition of tumor growth was linked to a reduced blood perfusion and resulted in increased amounts of tumor necrosis after i.a. CDDP. After treatment with i.a. CDDP, remaining viable tumor tissue responded by increasing expression of HIF-1α. Side effects due to administration of CDDP were minor, showing no differences in kidney damage between i.v. and i.a. CDDP. However, increased epidermal apoptosis in the foot was an indirect marker for locally increased concentrations of CDDP.ConclusionsOur findings demonstrate the great potential of local administration of cytotoxic chemotherapeutics, such as CDDP. Consequently, we provide a preclinical basis for a renewed interest in the clinical use of i.a. chemotherapy in osteosarcoma therapy.

Foscan and Foslip based photodynamic therapy in osteosarcoma in vitro and in intratibial mouse models

Current osteosarcoma therapies cause severe treatment‐related side effects and chemoresistance, and have low success rates. Consequently, alternative treatment options are urgently needed. Photodynamic therapy (PDT) is a minimally invasive, local therapy with proven clinical efficacy for a variety of tumor types. PDT is cytotoxic, provokes anti‐vascular effects and stimulates tumor cell targeting mechanisms of the immune system and, consequently, has potential as a novel therapy for osteosarcoma patients. This study investigated the uptake and the dark‐ and phototoxicity and cytotoxic mechanisms of the photosensitizer (PS) 5,10,15,20‐tetrakis(meta‐hydroxyphenyl) chlorine (mTHPC, Foscan) and a liposomal mTHPC formulation (Foslip) in the human 143B and a mouse K7M2‐derived osteosaroma cell line (K7M2L2) in vitro. Second, the tumor‐ and metastasis‐suppressive efficacies of mTHPC formulations based PDT and associated mechanisms in intratibial, metastasizing osteosarcoma mouse models (143B/SCID and syngeneic K7M2L2/BALB/c) were studied. The uptake of Foscan and Foslip in vitro was time‐ and dose‐dependent and resulted in mTHPC and light dose‐dependent phototoxicity associated with apoptosis. In vivo, the uptake of both i.v. administered mTHPC formulations was higher in tumor than in healthy control tissue. PDT caused significant (Foscan pu2009<u20090.05, Foslip pu2009<u20090.001) tumor growth inhibition in both models. A significant (Foscan pu2009<u20090.001, Foslip pu2009<u20090.001) immune system‐dependent suppression of lung metastasis was only observed in the K7M2L2/BALB/c model and was associated with a marked infiltration of T‐lymphocytes at the primary tumor site. In conclusion, mTHPC‐based PDT is effective in clinically relevant experimental osteosarcoma and suppresses lung metastasis in immunocompetent mice with beneficial effects of the liposomal mTHPC formulation Foslip.

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.

Cytotoxic Efficacy of Photodynamic Therapy in Osteosarcoma Cells In Vitro

In recent years, there has been the difficulty in finding more effective therapies against cancer with less systemic side effects. Therefore Photodynamic Therapy is a novel approach for a more tumor selective treatment. Photodynamic Therapy (PDT) that makes use of a nontoxic photosensitizer (PS), which, upon activation with light of a specific wavelength in the presence of oxygen, generates oxygen radicals that elicit a cytotoxic response(1). Despite its approval almost twenty years ago by the FDA, PDT is nowadays only used to treat a limited number of cancer types (skin, bladder) and nononcological diseases (psoriasis, actinic keratosis)(2). The major advantage of the use of PDT is the ability to perform a local treatment, which prevents systemic side effects. Moreover, it allows the treatment of tumors at delicate sites (e.g. around nerves or blood vessels). Here, an intraoperative application of PDT is considered in osteosarcoma (OS), a tumor of the bone, to target primary tumor satellites left behind in tumor surrounding tissue after surgical tumor resection. The treatment aims at decreasing the number of recurrences and at reducing the risk for (postoperative) metastasis. In the present study, we present in vitro PDT procedures to establish the optimal PDT settings for effective treatment of widely used OS cell lines that are used to reproduce the human disease in well established intratibial OS mouse models. The uptake of the PS mTHPC was examined with a spectrophotometer and phototoxicity was provoked with laser light excitation of mTHPC at 652 nm to induce cell death assessed with a WST-1 assay and by the counting of surviving cells. The established techniques enable us to define the optimal PDT settings for future studies in animal models. They are an easy and quick tool for the evaluation of the efficacy of PDT in vitro before an application in vivo.

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.

Evaluation of F8-TNF-α in Models of Early and Progressive Metastatic Osteosarcoma

The targeted delivery of tumor necrosis factor-α (TNF-α) with antibodies specific to splice isoforms of fibronectin [e.g., F8-TNF, specific to the extra-domain A (EDA) domain of fibronectin] has already shown efficacy against experimental sarcomas but has not yet been investigated in orthotopic sarcomas. Here, we investigated F8-TNF in a syngeneic K7 M2–derived orthotopic model of osteosarcoma as a treatment against pulmonary metastases, the most frequent cause of osteosarcoma-related death. Immunofluorescence on human osteosarcoma tissue confirmed the presence of EDA in primary tumors (PTs) as well as metastases. In mice, the efficacy of F8-TNF against PTs and early pulmonary metastases was evaluated. Intratibial PT growth was not affected by F8-TNF, yet early micrometastases were reduced possibly due to an F8-TNF–dependent attraction of pulmonary CD4+, CD8+, and natural killer cells. Furthermore, immunofluorescence revealed stronger expression of EDA in early pulmonary metastases compared with PT tissue. To study progressing pulmonary metastases, a hind limb amputation model was established, and the efficacy of F8-TNF, alone or combined with doxorubicin, was investigated. Despite the presence of EDA in metastases, no inhibition of progressive metastatic growth was detected. No significant differences in numbers of CD4+ or CD8+ cells or F4/80+ and Ly6G+ myeloid-derived cells were observed, although a strong association between metastatic growth and presence of pulmonary Ly6G+ myeloid-derived cells was detected. In summary, these findings demonstrate the potential of F8-TNF in activating the immune system and reducing early metastatic growth yet suggest a lack of efficacy of F8-TNF alone or combined with doxorubicin against progressing osteosarcoma metastases.