Smith M J, Culhane A C, Donovan M, Coffey J C, Barry B D, Kelly M A, Higgins D G, Wang J H, Kirwan W O, Cotter T G, Redmond H P
Department of Academic Surgery, University College Cork, Cork, Ireland.
Br J Cancer. 2009 May 5;100(9):1452-64. doi: 10.1038/sj.bjc.6604931.
Tumour stroma gene expression in biopsy specimens may obscure the expression of tumour parenchyma, hampering the predictive power of microarrays. We aimed to assess the utility of fluorescence-activated cell sorting (FACS) for generating cell populations for gene expression analysis and to compare the gene expression of FACS-purified tumour parenchyma to that of whole tumour biopsies. Single cell suspensions were generated from colorectal tumour biopsies and tumour parenchyma was separated using FACS. Fluorescence-activated cell sorting allowed reliable estimation and purification of cell populations, generating parenchymal purity above 90%. RNA from FACS-purified and corresponding whole tumour biopsies was hybridised to Affymetrix oligonucleotide microarrays. Whole tumour and parenchymal samples demonstrated differential gene expression, with 289 genes significantly overexpressed in the whole tumour, many of which were consistent with stromal gene expression (e.g., COL6A3, COL1A2, POSTN, TIMP2). Genes characteristic of colorectal carcinoma were overexpressed in the FACS-purified cells (e.g., HOX2D and RHOB). We found FACS to be a robust method for generating samples for gene expression analysis, allowing simultaneous assessment of parenchymal and stromal compartments. Gross stromal contamination may affect the interpretation of cancer gene expression microarray experiments, with implications for hypotheses generation and the stability of expression signatures used for predicting clinical outcomes.
活检标本中的肿瘤基质基因表达可能会掩盖肿瘤实质的表达，从而削弱微阵列的预测能力。我们旨在评估荧光激活细胞分选（FACS）在生成用于基因表达分析的细胞群体方面的效用，并比较FACS纯化的肿瘤实质与整个肿瘤活检组织的基因表达。从结直肠癌活检组织中制备单细胞悬液，并使用FACS分离肿瘤实质。荧光激活细胞分选能够可靠地估计和纯化细胞群体，使实质纯度超过90%。将FACS纯化的样本和相应的整个肿瘤活检组织的RNA与Affymetrix寡核苷酸微阵列进行杂交。整个肿瘤样本和实质样本表现出不同的基因表达，289个基因在整个肿瘤中显著过表达，其中许多与基质基因表达一致（例如，COL6A3、COL1A2、POSTN、TIMP2）。结直肠癌特征性基因在FACS纯化的细胞中过表达（例如，HOX2D和RHOB）。我们发现FACS是一种用于生成基因表达分析样本的可靠方法，能够同时评估实质和基质成分。大量的基质污染可能会影响癌症基因表达微阵列实验的解读，这对假设生成以及用于预测临床结果的表达特征的稳定性具有重要意义。
