Microscopy analysis of tumour samples is commonly performed on fixed, thinly sectioned and protein-labelled tissues. However, these examinations do not reveal the intricate three-dimensional structures of tumours, nor enable the detection of aberrant transcripts. Here, we report a method, which we name DIIFCO (for diagnosing in situ immunofluorescence-labelled cleared oncosamples), for the multimodal volumetric imaging of RNAs and proteins in intact tumour volumes and organoids. We used DIIFCO to spatially profile the expression of diverse coding RNAs and non-coding RNAs at the single-cell resolution in a variety of cancer tissues. Quantitative single-cell analysis revealed spatial niches of cancer stem-like cells, and showed that the niches were present at a higher density in triple-negative breast cancer tissue. The improved molecular phenotyping and histopathological diagnosis of cancers may lead to new insights into the biology of tumours of patients.

肿瘤样本的显微镜分析通常在固定的、薄切片和蛋白质标记的组织上进行。然而，这些检查并未揭示肿瘤复杂的三维结构，也无法检测异常转录本。在这里，我们报告了一种方法，我们将其命名为 DIIFCO（用于诊断原位免疫荧光标记的清除肿瘤样本），用于对完整肿瘤体积和类器官中的 RNA 和蛋白质进行多模式体积成像。我们使用 DIIFCO 以单细胞分辨率在各种癌组织中对不同编码 RNA 和非编码 RNA 的表达进行空间分析。定量单细胞分析揭示了癌症干细胞样细胞的空间生态位，并表明这些生态位在三阴性乳腺癌组织中的密度更高。