PURPOSE Osteosarcoma and Ewing's sarcoma are the most common bone tumors in children and adolescents. Despite intensive chemotherapy, patients with advanced disease have a poor prognosis,... Show morePURPOSE Osteosarcoma and Ewing's sarcoma are the most common bone tumors in children and adolescents. Despite intensive chemotherapy, patients with advanced disease have a poor prognosis, illustrating the need for alternative therapies. Sarcoma cells are susceptible to the cytolytic activity of resting natural killer (NK) cells which can be improved by interleukin (IL)-15 stimulation. In this study, we explored whether the cytolytic function of resting NK cells can be augmented and specifically directed toward sarcoma cells by antibody-dependent cellular cytotoxicity (ADCC). EXPERIMENTAL DESIGN Epidermal growth factor receptor (EGFR) expression was examined on osteosarcoma and Ewing's sarcoma cell lines by flow cytometry and in osteosarcoma biopsy and resection specimens by immunohistochemistry. Cetuximab-mediated ADCC by NK cells from osteosarcoma patients and healthy controls was measured with 4-hour (51)Cr release assays. RESULTS EGFR surface expression was shown on chemotherapy-sensitive and chemotherapy-resistant osteosarcoma cells (12/12), most primary osteosarcoma cultures (4/5), and few Ewing's sarcoma cell lines (2/7). In the presence of cetuximab, the cytolytic activity of resting NK cells against all EGFR-expressing sarcoma cells was substantially increased and comparable with that of IL-15-activated NK cells. Surface EGFR expression on primary osteosarcoma cultures correlated with EGFR expression in the original tumor. The cytolytic activity of osteosarcoma patient-derived NK cells against autologous tumor cells was as efficient as that of NK cells from healthy donors. CONCLUSION Our data show that the cytolytic potential of resting NK cells can be potentiated and directed toward osteosarcoma cells with cetuximab. Therefore, cetuximab-mediated immunotherapy may be considered a novel treatment modality in the management of advanced osteosarcoma. Show less
High-grade osteosarcoma occurs predominantly in adolescents and young adults and has an overall survival rate of about 60%, despite chemotherapy and surgery. Therefore, novel treatment modalities... Show moreHigh-grade osteosarcoma occurs predominantly in adolescents and young adults and has an overall survival rate of about 60%, despite chemotherapy and surgery. Therefore, novel treatment modalities are needed to prevent or treat recurrent disease. Natural killer (NK) cells are lymphocytes with cytotoxic activity toward virus-infected or malignant cells. We explored the feasibility of autologous and allogeneic NK cell-mediated therapies for chemotherapy-resistant and chemotherapy-sensitive high-grade osteosarcoma. The expression by osteosarcoma cells of ligands for activating NK cell receptors was studied in vitro and in vivo, and their contribution to NK cell-mediated cytolysis was studied by specific antibody blockade. Chromium release cytotoxicity assays revealed chemotherapy-sensitive and chemotherapy-resistant osteosarcoma cell lines and osteosarcoma primary cultures to be sensitive to NK cell-mediated cytolysis. Cytolytic activity was strongly enhanced by IL-15 activation and was dependent on DNAM-1 and NKG2D pathways. Autologous and allogeneic activated NK cells lysed osteosarcoma primary cultures equally well. Osteosarcoma patient-derived NK cells were functionally and phenotypically unimpaired. In conclusion, osteosarcoma cells, including chemoresistant variants, are highly susceptible to lysis by IL-15-induced NK cells from both allogeneic and autologous origin. Our data support the exploitation of NK cells or NK cell-activating agents in patients with high-grade osteosarcoma. Show less
High-grade osteosarcoma occurs predominantly in adolescents and young adults and has an overall survival rate of about 60%, despite chemotherapy and surgery. Therefore, novel treatment modalities... Show moreHigh-grade osteosarcoma occurs predominantly in adolescents and young adults and has an overall survival rate of about 60%, despite chemotherapy and surgery. Therefore, novel treatment modalities are needed to prevent or treat recurrent disease. Natural killer (NK) cells are lymphocytes with cytotoxic activity toward virus-infected or malignant cells. We explored the feasibility of autologous and allogeneic NK cell-mediated therapies for chemotherapy-resistant and chemotherapy-sensitive high-grade osteosarcoma. The expression by osteosarcoma cells of ligands for activating NK cell receptors was studied in vitro and in vivo, and their contribution to NK cell-mediated cytolysis was studied by specific antibody blockade. Chromium release cytotoxicity assays revealed chemotherapy-sensitive and chemotherapy-resistant osteosarcoma cell lines and osteosarcoma primary cultures to be sensitive to NK cell-mediated cytolysis. Cytolytic activity was strongly enhanced by IL-15 activation and was dependent on DNAM-1 and NKG2D pathways. Autologous and allogeneic activated NK cells lysed osteosarcoma primary cultures equally well. Osteosarcoma patient-derived NK cells were functionally and phenotypically unimpaired. In conclusion, osteosarcoma cells, including chemoresistant variants, are highly susceptible to lysis by IL-15-induced NK cells from both allogeneic and autologous origin. Our data support the exploitation of NK cells or NK cell-activating agents in patients with high-grade osteosarcoma. Show less
Purpose: High-grade osteosarcoma is a malignant primary bone tumor with a peak incidence in adolescence. Overall survival (OS) of patients with resectable metastatic disease is approximately 20%.... Show morePurpose: High-grade osteosarcoma is a malignant primary bone tumor with a peak incidence in adolescence. Overall survival (OS) of patients with resectable metastatic disease is approximately 20%. The exact mechanisms of development of metastases in osteosarcoma remain unclear. Most studies focus on tumor cells, but it is increasingly evident that stroma plays an important role in tumorigenesis and metastasis. We investigated the development of metastasis by studying tumor cells and their stromal context. Experimental Design: To identify gene signatures playing a role in metastasis, we carried out genome-wide gene expression profiling on prechemotherapy biopsies of patients who did (n = 34) and patients who did not (n = 19) develop metastases within 5 years. Immunohistochemistry (IHC) was performed on pretreatment biopsies from 2 additional cohorts (n = 63 and n = 16) and corresponding postchemotherapy resections and metastases. Results: A total of 118/132 differentially expressed genes were upregulated in patients without metastases. Remarkably, almost half of these upregulated genes had immunological functions, particularly related to macrophages. Macrophage-associated genes were expressed by infiltrating cells and not by osteosarcoma cells. Tumor-associated macrophages (TAM) were quantified with IHC and associated with significantly better overall survival (OS) in the additional patient cohorts. Osteosarcoma samples contained both M1-(CD14/HLA-DR alpha positive) and M2-type TAMs (CD14/CD163 positive and association with angiogenesis). Conclusions: In contrast to most other tumor types, TAMs are associated with reduced metastasis and improved survival in high-grade osteosarcoma. This study provides a biological rationale for the adjuvant treatment of high-grade osteosarcoma patients with macrophage activating agents, such as muramyl tripeptide. Clin Cancer Res; 17(8); 2110-9. (C) 2011 AACR. Show less
Usage of cancer cell lines has repeatedly generated conflicting results provoked by differences among subdones or contamination with mycoplasm or other immortal mammalian cells. To overcome these... Show moreUsage of cancer cell lines has repeatedly generated conflicting results provoked by differences among subdones or contamination with mycoplasm or other immortal mammalian cells. To overcome these limitations, we decided within the EuroBoNeT consortium to characterize a common set of cell lines including osteosarcomas (OS), Ewing sarcomas (ES), and chondrosarcomas (CS). DNA fingerprinting was used to guarantee the identity of all of the cell lines and to distinguish subdones of osteosarcoma cell line HOS. Screening for homozygous loss of 38 tumor suppressor genes by MLPA revealed deletion of CDKN2A as the most common event (15/36), strictly associated with absence of the CDKN2A (p 16) protein. Ten cell lines showed missense mutations of the TPS3 gene while another set of nine cell lines showed mutations resulting in truncation of the TP53 protein. Cells harboring missense mutations expressed high levels of nuclear TP53, while cell lines with nonsense mutations showed weak/absent staining for TP53. TP53(wt) cell lines usually expressed the protein in 2-10% of the cells. However, seven TP53(wt) osteosarcomas were negative for both mRNA and protein expression. Our analyses shed light on the correlation between immunohistochemical and genetic data for CDKN2A and TP53, and confirm the importance of these signaling pathways. The characterization of a substantial number of cell lines represents an important step to supply research groups with proven models for further advanced studies on tumor biology and may help to make results from different laboratories more comparable. (C) 2009 Wiley-Liss, Inc. Show less
