Division of Chromatin Networks, German Cancer Research Center (DKFZ) and Bioquant, Heidelberg, Germany.
Hopp Children's Cancer Center (KiTZ), Heidelberg, Germany.
Nucleic Acids Res. 2022 Jun 24;50(11):e61. doi: 10.1093/nar/gkac113.
Alternative lengthening of telomeres (ALT) occurs in ∼10% of cancer entities. However, little is known about the heterogeneity of ALT activity since robust ALT detection assays with high-throughput in situ readouts are lacking. Here, we introduce ALT-FISH, a method to quantitate ALT activity in single cells from the accumulation of single-stranded telomeric DNA and RNA. It involves a one-step fluorescent in situ hybridization approach followed by fluorescence microscopy imaging. Our method reliably identified ALT in cancer cell lines from different tumor entities and was validated in three established models of ALT induction and suppression. Furthermore, we successfully applied ALT-FISH to spatially resolve ALT activity in primary tissue sections from leiomyosarcoma and neuroblastoma tumors. Thus, our assay provides insights into the heterogeneity of ALT tumors and is suited for high-throughput applications, which will facilitate screening for ALT-specific drugs.
端粒的替代性延长 (ALT) 发生在约 10%的癌症实体中。然而，由于缺乏具有高通量原位读数的稳健 ALT 检测试剂盒，因此对 ALT 活性的异质性知之甚少。在这里，我们引入 ALT-FISH，这是一种通过积累单链端粒 DNA 和 RNA 来定量单个细胞中 ALT 活性的方法。它涉及一步荧光原位杂交方法，然后进行荧光显微镜成像。我们的方法可靠地鉴定了来自不同肿瘤实体的癌细胞系中的 ALT，并在三个已建立的 ALT 诱导和抑制模型中得到验证。此外，我们成功地将 ALT-FISH 应用于从平滑肌肉瘤和神经母细胞瘤肿瘤的原发性组织切片中空间解析 ALT 活性。因此，我们的测定法提供了对 ALT 肿瘤异质性的深入了解，适用于高通量应用，这将有助于筛选 ALT 特异性药物。
