In vivo model of hypoxia in Ewing Sarcoma

描述（申请人提供）：尤文氏肉瘤家族肿瘤（Ewing’s sarcoma family of tumor， ESFT）是一组高度侵袭性的恶性肿瘤，其治疗仍是临床未解决的难题。转移的存在是ESFT唯一最强大的不良预后因素，局限性和转移性疾病患者的无事件生存率分别为72%和27%。最不利的预后与骨转移的存在有关。因此，转移性ESFT的治疗和预防疾病传播是ESFT临床管理的关键问题。然而，尽管如此，控制ESFT转移形成的机制尚不清楚。临床和实验数据表明，缺氧与ESFT的进展有关。在ESFT患者中，肿瘤组织中存在未灌注区域与不利的转移模式（多发性转移伴骨累及）相关，而缺乏此类区域则预后较好。在细胞培养中，缺氧已被证明可以上调EWS-FLI1（一种触发ESFT发育的异常转录因子），并将其转录活性向pro方向转移

DESCRIPTION (provided by applicant): Ewing's sarcoma family of tumors (ESFT) is a group of highly aggressive malignancies, the treatment of which remains an unsolved clinical problem. The presence of metastases is the single most powerful adverse prognostic factor for ESFT, with event-free survival at 72% and 27% for patients with localized and metastatic disease, respectively. The most unfavorable prognosis is associated with the presence of bone metastases. Thus, the treatment of metastatic ESFT and preventing disease dissemination is a crucial problem in ESFT clinical management. Despite that, however, the mechanisms governing the metastases formation in ESFT are poorly understood. Hypoxia has been implicated in ESFT progression by clinical and experimental data. In ESFT patients, the presence of unperfused areas within tumor tissue associates with an unfavorable metastatic pattern (multiple metastases with bone involvement), while the lack of such areas with better prognosis. In cell culture, hypoxia has been shown to up-regulate EWS-FLI1, an aberrant transcription factor triggering ESFT development, and shift its transcriptional activity toward pro metastatic and pro-survival genes. On functional levels, low oxygen augments ESFT cell invasiveness and tumorigenic potential. We have also shown hypoxia-induced enrichment in ESFT cells with a high activity of aldehyde dehydrogenase (ALDH), characterized as cancer stem cells, as well as identified neuropeptide Y (NPY), an EWS-FLI1 target gene, as a candidate hypoxia-induced pro-metastatic factor in ESFT. NPY has also been implicated in the regulation of bone homeostasis, suggesting its potential role in bone invasion. Based on these data, we hypothesize that hypoxia in primary tumor promotes ESFT metastases formation and changes their pattern by favoring bone invasion. To provide a direct proof for this notion, we propose developing a novel animal model by performing femoral artery ligation (FAL) in mice bearing orthotopic ESFT xenografts in their gastrocnemius muscles, followed by excision of the primary tumor and long-term monitoring of metastases formation. This approach will allow us to create transient hypoxia in growing ESFT tumor and test its effect on disease dissemination. The aims of our study are: 1) to establish a model of hypoxia in ESFT primary tumor by testing the impact of FAL on the oxygen level within tumor tissue; 2) to determine the effect of hypoxia on metastases formation by comparing number, sizes, latency and localization of metastases in animals with or without hypoxic challenge. Our study will provide direct proof for the role of hypoxia in ESFT metastases formation and disease progression. It will also establish a model that allows for testing factors already implicated in hypoxia-induced metastases formation, such as NPY, and identifying novel molecules involved in this process. This, in turn, will significantl improve our understanding of the metastatic processes in ESFT and address one of the most critical, yet understudied problems in its biology. In the future, this may result in the development of novel therapeutic strategies and prognostic factors, directly benefitting ESFT patients.