大多数癌症死亡是由实体瘤引起的，其中四种最常见的癌症（乳腺癌、肺癌、结直肠癌和前列腺癌）占所有病例的 60% 以上 (1)。由可变癌症微环境（例如缺氧）驱动的肿瘤细胞异质性是治疗结果的关键决定因素。我们开发了一种新颖的培养方案，称为长期缺氧（LTHY）时间过程，以概括体内严重缺氧的逐渐发展，以模拟原发性肿瘤中观察到的条件。接受 LTHY 处理的细胞经历了基于 miRNA 和 mRNA 特征的非典型上皮间质转化 (EMT)，并表现出类似 EMT 的形态变化。与此同时，我们报告了一种新型截短型 WT1 转录因子 (tWt1) 的产生，这是一种非规范的 EMT 驱动程序，其表达由尚未描述的内含子启动子通过缺氧反应元件 (HRE) 驱动。我们进一步证明 tWt1 从内含子衍生的起始密码子开始翻译，保留正确的亚细胞定位和 DNA 结合。类似的 tWt1 也在 LTHY 培养的人类癌细胞系以及原发性癌症中表达，并预测患者的长期生存。我们的研究不仅证明了更好地模拟原发性癌症中观察到的培养条件的重要性，特别是在缺氧方面，而且还确定了一种新的 WT1 亚型，它与卵巢癌长期生存率较差相关。© 2024。作者（s）。

The majority of cancer deaths are caused by solid tumors, where the four most prevalent cancers (breast, lung, colorectal and prostate) account for more than 60% of all cases (1). Tumor cell heterogeneity driven by variable cancer microenvironments, such as hypoxia, is a key determinant of therapeutic outcome. We developed a novel culture protocol, termed the Long-Term Hypoxia (LTHY) time course, to recapitulate the gradual development of severe hypoxia seen in vivo to mimic conditions observed in primary tumors. Cells subjected to LTHY underwent a non-canonical epithelial to mesenchymal transition (EMT) based on miRNA and mRNA signatures as well as displayed EMT-like morphological changes. Concomitant to this, we report production of a novel truncated isoform of WT1 transcription factor (tWt1), a non-canonical EMT driver, with expression driven by a yet undescribed intronic promoter through hypoxia-responsive elements (HREs). We further demonstrated that tWt1 initiates translation from an intron-derived start codon, retains proper subcellular localization and DNA binding. A similar tWt1 is also expressed in LTHY-cultured human cancer cell lines as well as primary cancers and predicts long-term patient survival. Our study not only demonstrates the importance of culture conditions that better mimic those observed in primary cancers, especially with regards to hypoxia, but also identifies a novel isoform of WT1 which correlates with poor long-term survival in ovarian cancer.© 2024. The Author(s).