Epithelium percentage estimation facilitates epithelial quantitative protein measurement in tissue specimens
Epithelium percentage estimation facilitates epithelial quantitative protein measurement in tissue specimens
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DOI：
10.1186/1559-0275-10-18
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发表时间：
2013-01-01
影响因子：
3.8
通讯作者：
Zhang, Hui
中科院分区：
文献类型：
--
作者：
Chen, Jing;Eshghi, Shadi Toghi;Zhang, Hui
背景：用于蛋白质定量测量的高通量工具的快速发展已经证明了鉴定与癌症相关的蛋白质的潜力。然而，肿瘤组织标本的定量结果往往因组织异质性而混淆，例如，肿瘤区域通常具有明显较高的上皮含量而较低的间质含量。目的：因此有必要开发一种工具，以方便解释组织标本中蛋白质测量的结果。方法：采用免疫组化染色（IHC）测定染色强度，确定正常和肿瘤前列腺组织中上皮细胞粘附分子（Epithelial cell adhesion molecule, EpCAM）和组织蛋白酶L （cathepsin L， CTSL）的表达。用ELISA法检测OCT包埋冷冻前列腺组织蛋白提取物中这些蛋白的表达。为了消除组织异质性对ELISA测定的上皮蛋白定量的影响，我们开发了一种基于颜色的分割方法，利用来自同一前列腺组织的H&E组织学切片估计上皮含量，并使用估计的上皮百分比对ELISA结果进行归一化。病理学家也对同一载玻片的上皮内容进行了估计，并用于标准化ELISA结果。将基于计算机的结果与病理学家的读数进行比较。结果：我们发现EpCAM和CTSL水平均受组织标本中上皮含量的影响很大。在不调整上皮百分比的情况下，肿瘤组织中EpCAM和CTSL水平均明显高于正常组织，p值小于0.001。然而，在上皮百分比归一化后，ELISA测量EpCAM和CTSL与免疫组化染色结果一致，显示只有EpCAM表达显著增加，而CTSL在癌组织中的表达无差异。这些结果是通过计算机估计和病理估计的上皮百分比归一化得到的。结论：我们的结果表明，使用我们基于颜色的分割方法估计组织上皮百分比与病理学家估计的组织上皮百分比有很好的相关性。基于颜色分割估计的上皮内容物可用于肿瘤蛋白的免疫分析或临床蛋白质组学分析。用于上皮估计的代码以及估计上皮内容的显微照片可在线获得。
Background: The rapid advancement of high-throughput tools for quantitative measurement of proteins has demonstrated the potential for the identification of proteins associated with cancer. However, the quantitative results on cancer tissue specimens are usually confounded by tissue heterogeneity, e.g. regions with cancer usually have significantly higher epithelium content yet lower stromal content.Objective: It is therefore necessary to develop a tool to facilitate the interpretation of the results of protein measurements in tissue specimens.Methods: Epithelial cell adhesion molecule (EpCAM) and cathepsin L (CTSL) are two epithelial proteins whose expressions in normal and tumorous prostate tissues were confirmed by measuring staining intensity with immunohistochemical staining (IHC). The expressions of these proteins were measured by ELISA in protein extracts from OCT embedded frozen prostate tissues. To eliminate the influence of tissue heterogeneity on epithelial protein quantification measured by ELISA, a color-based segmentation method was developed in-house for estimation of epithelium content using H&E histology slides from the same prostate tissues and the estimated epithelium percentage was used to normalize the ELISA results. The epithelium contents of the same slides were also estimated by a pathologist and used to normalize the ELISA results. The computer based results were compared with the pathologist's reading.Results: We found that both EpCAM and CTSL levels, measured by ELISA assays itself, were greatly affected by epithelium content in the tissue specimens. Without adjusting for epithelium percentage, both EpCAM and CTSL levels appeared significantly higher in tumor tissues than normal tissues with a p value less than 0.001. However, after normalization by the epithelium percentage, ELISA measurements of both EpCAM and CTSL were in agreement with IHC staining results, showing a significant increase only in EpCAM with no difference in CTSL expression in cancer tissues. These results were obtained with normalization by both the computer estimated and pathologist estimated epithelium percentage.Conclusions: Our results show that estimation of tissue epithelium percentage using our color-based segmentation method correlates well with pathologists' estimation of tissue epithelium percentages. The epithelium contents estimated by color-based segmentation may be useful in immuno-based analysis or clinical proteomic analysis of tumor proteins. The codes used for epithelium estimation as well as the micrographs with estimated epithelium content are available online.