Three-dimensional imaging and quantitative analysis in CLARITY processed breast cancer tissues
Three-dimensional imaging and quantitative analysis in CLARITY processed breast cancer tissues
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DOI：
10.1038/s41598-019-41957-w
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发表时间：
2019-04-04
影响因子：
4.6
通讯作者：
Goodman, Laurie J.
中科院分区：
文献类型：
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作者：
Chen, Yi;Shen, Qi;Goodman, Laurie J.
肿瘤微环境在空间上可能是异质的，这使得用基于标准2D组织学的方法充分表征具有挑战性。在这项研究中，我们确定了一个完整的组织处理方法来分析乳腺癌患者活检的可行性。将福尔马林固定的人乳腺癌芯针活检标本包埋、脂质清除和多重免疫染色，以鉴定关键生物标志物（泛细胞角蛋白、Ki 67、CD 3）。然后使用共聚焦显微镜对折射率匹配后的样品进行成像。然后将这些数据集与传统的基于载玻片的FFPE组织学进行定量比较。使用显微镜，组织的大体和细胞形态得到了良好的保存，并且除了纤维化区域之外，还实现了高光学透明度。使用优化的抗体条件实现了各种细胞和核标志物的特异性染色。从组织学数据集手动确定的复合Ki67评分与组织学结果一致。然而，BIQUITY数据集（3D）显示了肿瘤内Ki67表达的变化，这在单个FFPE切片（2D）中不明显。我们进一步证明了存档的FFPE临床标本可以进行自动处理、免疫染色和成像。简而言之，与传统的基于载玻片的组织学相比，经自动化处理的标本可以实现对肿瘤样本的更准确、无偏倚的分析，从而改善肿瘤内异质性的可视化。
The tumor microenvironment can be spatially heterogenous, which makes it challenging to fully characterize with standard 2D histology-based methods. In this study, we determined the feasibility of a CLARITY tissue-processing approach to analyze biopsies from breast cancer patients. Formalin-fixed human breast cancer core-needle biopsy specimens, were embedded, lipid-cleared, and multiplexed immunostained to identify key biomarkers (pan-cytokeratin, Ki67, CD3). Confocal microscopy was then used to image the specimens after refractive index matching. These data sets were then quantitatively compared to conventional slide-based FFPE histology. Using CLARITY, the gross and cellular morphology of the tissues were well preserved, and high optical transparency was achieved, with the exception of fibrotic regions. Specific staining of various cellular and nuclear markers was achieved using optimized antibody conditions. Manually determined composite Ki67 scores from the CLARITY datasets agreed with histology results. However, the CLARITY datasets (3D) revealed variation in the intra-tumoral Ki67 expression that was not evident in individual FFPE sections (2D). We further demonstrated that archived FFPE clinical specimens can be CLARITY-processed, immunostained, and imaged. In short, CLARITY-processed specimens may enable a more accurate, unbiased analysis of tumor samples in comparison to conventional slide-based histology, thus allowing for improved visualization of intra-tumoral heterogeneity.