In vivo model of hypoxia in Ewing Sarcoma

描述（由申请人提供）：尤文肉瘤家族肿瘤（ESFT）是一组高度侵袭性的恶性肿瘤，其治疗仍然是一个未解决的临床问题。 转移瘤的存在是ESFT最有力的不良预后因素，局部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，治疗转移性ESFT和防止疾病传播是ESFT临床管理的关键问题。 然而，尽管如此，对ESFT转移形成的机制仍知之甚少。 临床和实验数据表明，缺氧与ESFT进展有关。 在ESFT患者中，肿瘤组织内存在未灌注区域与不利的转移模式（多发性转移伴骨受累）相关，而缺乏此类区域则预后较好。 在细胞培养中，缺氧已被证明上调EWS-FLI 1，一种触发ESFT发展的异常转录因子，并将其转录活性转向促ESFT发生。 转移和促存活基因。 在功能水平上，低氧增强了ESFT细胞的侵袭力和致瘤潜力。 我们还显示了在具有高活性醛脱氢酶（ALDH）的ESFT细胞中缺氧诱导的富集，其特征在于癌症干细胞，以及鉴定的神经肽Y（NPY），EWS-FLI 1靶基因，作为ESFT中缺氧诱导的促转移因子的候选物。 神经肽Y还参与骨稳态的调节，提示其在骨侵袭中的潜在作用。 基于这些数据，我们假设原发肿瘤中的缺氧促进了ESFT转移的形成，并通过有利于骨侵袭来改变其模式。 为了直接证明这一观点，我们建议开发一种新的动物模型，通过在腓肠肌中原位ESFT异种移植的小鼠中进行股动脉结扎（FAL），然后切除原发性肿瘤并长期监测转移形成。 这种方法将使我们能够在生长的ESFT肿瘤中创造短暂的缺氧，并测试其对疾病传播的影响。 我们研究的目的是：1）通过测试FAL对肿瘤组织内氧水平的影响来建立ESFT原发性肿瘤中的缺氧模型; 2）通过比较有或没有缺氧攻击的动物中转移瘤的数量、大小、潜伏期和定位来确定缺氧对转移瘤形成的影响。 我们的研究将为缺氧在ESFT转移形成和疾病进展中的作用提供直接证据。 它还将建立一个模型，允许测试已经参与缺氧诱导的转移形成的因素，如NPY，并确定参与这一过程的新分子。 反过来，这将显著提高我们对ESFT转移过程的理解，并解决其生物学中最关键但研究不足的问题之一。 在未来，这可能会导致新的治疗策略和预后因素的发展，直接受益ESFT患者。

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.