IBM Research Zürich, Säumerstrasse 4, CH-8803 Rüschlikon, Switzerland.
University Hospital Zurich, Department of Pathology and Molecular Pathology, Schmelzbergstr. 12, CH-8091 Zurich, Switzerland.
Nucleic Acids Res. 2020 Feb 20;48(3):e17. doi: 10.1093/nar/gkz1151.
Multiplexed RNA in situ hybridization for the analysis of gene expression patterns plays an important role in investigating development and disease. Here, we present a method for multiplexed RNA-ISH to detect spatial tumor heterogeneity in tissue sections. We made use of a microfluidic chip to deliver ISH-probes locally to regions of a few hundred micrometers over time periods of tens of minutes. This spatial multiplexing method can be combined with ISH-approaches based on signal amplification, with bright field detection and with the commonly used format of formalin-fixed paraffin-embedded tissue sections. By using this method, we analyzed the expression of HER2 with internal positive and negative controls (ActB, dapB) as well as predictive biomarker panels (ER, PgR, HER2) in a spatially multiplexed manner on single mammary carcinoma sections. We further demonstrated the applicability of the technique for subtype differentiation in breast cancer. Local analysis of HER2 revealed medium to high spatial heterogeneity of gene expression (Cohen effect size r = 0.4) in equivocally tested tumor tissues. Thereby, we exemplify the importance of using such a complementary approach for the analysis of spatial heterogeneity, in particular for equivocally tested tumor samples. As the method is compatible with a range of ISH approaches and tissue samples, it has the potential to find broad applicability in the context of molecular analysis of human diseases.
用于分析基因表达模式的多重 RNA 原位杂交在研究发育和疾病方面发挥着重要作用。在这里，我们提出了一种用于检测组织切片中空间肿瘤异质性的多重 RNA-ISH 方法。我们利用微流控芯片将 ISH 探针局部递送至几百微米大小的区域，持续数十分钟。这种空间多重化方法可以与基于信号放大的 ISH 方法、明场检测以及常用的福尔马林固定石蜡包埋组织切片格式相结合。使用这种方法，我们以空间多重化的方式在单个乳腺肿瘤切片上分析了 HER2 的表达情况，包括内部阳性和阴性对照（ActB、dapB）以及预测生物标志物（ER、PgR、HER2）。我们进一步证明了该技术在乳腺癌亚型分化中的适用性。HER2 的局部分析显示，在存在争议的肿瘤组织中，基因表达具有中等到高度的空间异质性（Cohen 效应大小 r = 0.4）。因此，我们举例说明了使用这种互补方法分析空间异质性的重要性，特别是对于存在争议的肿瘤样本。由于该方法与多种 ISH 方法和组织样本兼容，因此它有可能在人类疾病的分子分析中得到广泛应用。
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