Center for Applied Proteomics and Molecular Medicine, George Mason University, Manassas, VA, USA.
Lab Invest. 2010 May;90(5):787-96. doi: 10.1038/labinvest.2010.47. Epub 2010 Mar 1.
Tissues are complex structures composed of different cell types, each of which present specific functions and characteristics. To better understand and measure the effect of tumor cell enrichment on protein pathway profiling and drug target activation measurements, the signaling activation portraits of laser capture microdissected (LCM) cancer epithelium and tumor stroma were compared with patient-matched whole-tissue specimens from 53 primary colorectal cancer samples. Microdissected material and whole-tissue lysate from contiguous cryostat sections were subjected to reverse-phase protein microarray analysis to determine the level of phopshorylation and expression of 75 different proteins known to be involved in cancer progression. The results revealed distinct differences in the protein activation portraits of cancer epithelium and stroma. Moreover, we found that the signaling activation profiles of the undissected whole-tissue specimens are profoundly different from the matched LCM material. Attempts to rescale the undissected pathway information based on percent endogenous tumor epithelium content were unsuccessful in recapitulating the LCM tumor epithelial signatures. Analysis of epidermal growth factor receptor phosphorylation and COX2 expression in these same sample sets revealed wholesale differences in the rank ordering of patient determination when LCM was compared with undissected samples. On the basis of these data, we conclude that accurate protein pathway activation status, which is under evaluation as a basis for patient selection and stratification for personalized therapy, must include upfront cellular-enrichment techniques such as LCM to generate accurate drug target activation status.
组织是由不同类型的细胞组成的复杂结构，每个细胞都具有特定的功能和特征。为了更好地理解和衡量肿瘤细胞富集对蛋白质通路分析和药物靶点激活测量的影响，对 53 例原发性结直肠癌样本中 53 例患者匹配的全组织标本的激光捕获显微切割（LCM）癌上皮和肿瘤基质的信号激活特征进行了比较。对连续冷冻切片的微切割材料和全组织裂解物进行反相蛋白微阵列分析，以确定 75 种不同的磷酸化水平和已知参与癌症进展的蛋白质的表达。结果显示，癌上皮和基质的蛋白质激活特征存在明显差异。此外，我们发现未切割的全组织标本的信号激活谱与匹配的 LCM 材料有很大的不同。尝试根据内源性肿瘤上皮细胞含量的百分比重新调整未切割的通路信息，未能成功重现 LCM 肿瘤上皮特征。对这些相同样本集中表皮生长因子受体磷酸化和 COX2 表达的分析表明，当与未切割样本相比时，LCM 比较时患者确定的排序存在整体差异。基于这些数据，我们得出结论，作为患者选择和分层进行个体化治疗的基础，准确的蛋白质通路激活状态必须包括细胞富集技术，如 LCM，以生成准确的药物靶点激活状态。
