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.

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

BACKGROUND: Osteosarcoma 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. METHODS: We 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). RESULTS: First, an effective concentration of 4 mg/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. CONCLUSIONS: Our 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.

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

BACKGROUND: Primary 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. METHODS: We 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. RESULTS: No significant associations between biomarker expression in BXs and patient survival or chemotherapy response were detected. In univariate analysis, positive immunohistochemistry of P53 (P = 0.008) and P16 (P16; P = 0.033) 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 (P = 0.003; HR = 0.067) while absence of P16 was associated with good chemotherapy response (P = 0.004; OR = 74.076). Presence of PTEN on tumor RXs was significantly associated with an improved survival prognosis (P = 0.022). CONCLUSIONS: Positive 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.