iTRAQ技术是目前比较成熟且常用的定量蛋白质组学技术，在寻找疾病蛋白质biomarkers的研究中经常用到该技术。寻找蛋白质biomarkers的研究经常分为两部分：一为通过差异定量的方法高通量筛选，并利用生物信息学的手段缩小候选验证的范围；二为利用各种验证手段对前期高通量筛选的结果进行验证，筛选出最具有潜力的候选biomarkers，以供更进一步地临床分析。

本篇文献里作者先是采用基于质谱的高通量定量蛋白质组学技术——iTRAQ，筛选出与结肠癌诊断相关的biomarkers，接着分别通过免疫组化、QPCR、多例病例的数据集等研究对筛选的候选蛋白质biomarkers进行验证，最终认为LOXL2是最具有潜力的诊断标志物。

文献来源：LOXL2 is highly expressed in cancer-associated fibroblasts and associates to poor colon cancer survival. Clin Cancer Res. 2015 Jul 23. pii: clincanres.3096.2014.

文章摘要

对结肠癌发展不同阶段的正确区分（尤其是II期和III期），迫切需要寻找到足够多的诊断标志物。肿瘤相关的成纤维细胞（CAFs）是肿瘤微环境的主要参与者，与肿瘤的复发和存活密切相关。作者利用蛋白质组学技术（iTRAQ）分析了从肿瘤病人组织中分离纯化出的结肠癌相关成纤维细胞（CAFs）与正常成纤维细胞（NFs）的蛋白表达差异，发现大量间质相关蛋白可能作为诊断标志物。其中LOXL2和TAGLN表现出很强的诊断价值，LOXL2的高表达与较差的预后直接相关，并能够很好地指针高风险的II期和III期病人。LOXL2可能在临床应用中具有很好的风险评估潜力，有利于判断后续治疗策略及改善最终的预后。

实验过程

收集12对结肠癌病人的肿瘤组织及癌旁组织，将肿瘤相关成纤维细胞（CAFs）与正常成纤维细胞（NFs）进行纯化分离，利用4-Plex iTRAQ对成纤维细胞的细胞上清和胞内蛋白进行差异蛋白质组学分析。差异表达的间质相关蛋白随后进一步利用免疫组化进行验证。预后诊断潜力评价过程中，① 外源基因表达数据集（共包含超过300例病人的数据）被用于进行训练和风险界定，② 70例病例组织的PCR结果以及 ③ 121例病例组织的免疫组化结果被用于进行验证。整合的数据集被用于对II期和III期病人进行风险预测。

iTRAQ workflow used for the proteomic analysis of CAFs and NFs.

CM: Cell Conditioned Medium WCE: Whole Cell Extract

实验结果

iTRAQ分别鉴定到1780个胞内和305个细胞上清蛋白，其中分别有57 和43蛋白具有表达差异，作者选择了其中15个差异表达蛋白进行了WB和PCR验证。体外培养的成纤维细胞经TGF-β刺激后，表现出了相似的蛋白表达谱。

Proteins identified and quantified in both iTRAQ replicates. About 78% of identified proteins and 82% of quantified proteins were coincident in both analyses.

Figure 1. Validation of CAFs deregulated proteins. (A) Whole cellular extracts or concentrated protein samples from conditioned medium of CAFs and NFs were separated on SDS-PAGE gels, transferred to nitrocellulose membranes and probed with the indicated antibodies. Tubulin was used as a loading control. Protein abundance was quantified by densitometry to compare the expression with the iTRAQ ratios. (B) cDNAs synthesized from total RNA obtained from NFs and CAFs were subjected to QRT-PCR using specific primers for the selected genes and 18S rRNA for normalization. BJh-TERT cells were starved in serum-free medium, treated with TGFβ (5 ng/mL) for 24 h and lysed. The extracts were analyzed by QPCR (C) and Western blot (D) using specific primers and the indicated antibodies. Data represent the mean ± SD of three experiments. (*, p < 0.05, **, p < 0.01; ***, p < 0.001).

（以下几段均为通过不同方法对iTRAQ结果中筛选的差异蛋白进行验证）

为了分析这些差异表达蛋白的细胞表达特异性，作者采用了GSE39396 dataset（从6个结肠癌病人分离得到的4种细胞endothelial cells: CD31+, epithelial cells: EPCAM+, inflammatory cells: CD45+ and CAFs: FAP+）进行了初步分析，发现其中60个上调表达蛋白中的28个蛋白，以及40个下调表达蛋白中的24个蛋白，与成纤维细胞的高表基因状态一致。

接下来，作者用QPCR的方法分析了这些蛋白在KM12C、KM12SM、SW480 和SW620上皮结肠癌细胞系中的基因表达，发现其中的10个蛋白特异性地或者过表达于成纤维细胞中。

免疫组化试验进一步证实，ACAN、αSMA、CDH13、DKK3、TAGLN、TGM2和LOXL2均选择性表达于结肠癌间质中，而在上皮或正常的结肠黏膜中不表达或无规律性表达。这些结果说明，筛选出的蛋白具有作为特异性肿瘤间质标志物的价值。

Figure 2. Stromal specificity of CAFs proteins. Differentially-expressed genes in CAFs vs NFs (60 up-regulated (A) and 40 down-regulated (B)) are represented by heatmaps of scaled gene-expression levels according to a dataset for: epithelial cells: EPCAM+, leukocytes: CD45+, fibroblasts: FAP+ and endothelial cells: CD31+ (28). Color scale represents the value of the z-score calculated in the dataset. Red indicates a higher z-score value, whereas blue indicates a lower value. (C) cDNAs synthesized from total RNA from NFs, CAFs and epithelial colon cancer cell lines (KM12SM, KM12C, SW480 and SW620) were subjected to QPCR as in Figure 1. Data represent the mean ± SD of three experiments.

Figure 3. Expression of stromal biomarkers in paired tissues of colon cancer patients. Immunohistochemical staining for ACAN, αSMA, COL14A1, CDH13, DKK3, TAGLN, TGM2 and LOXL2 in normal mucosa, primary colon cancer and liver metastasis. Pictures were taken at x200 magnification.

为了评估这些蛋白是否能够指针疾病的复发风险和预后，作者首先在一个由70例结肠癌病人组成的队列中进行了验证。肿瘤组织中的LOXL2表达水平比正常组织高出44%，LOXL2的高表达与整体存活（P = 0.001 HR: 8.52 (1.90-38.29)）和高复发率（P = 0.001 HR: 5.38 (1.70-17.01)）一致。

多变量统计分析数据集的结果证实，LOXL2的表达水平与较差的预后相关，并可作为一个独立的无疾病存活和整体存活的预后因子（Table 1）。

随后，另外一批121个结肠癌病人的免疫组化结果进一步证实，大约32%的病人表现出较强的LOXL2表达（>50%的阳性标记），间质细胞核的阳性标记与无疾病存活和整体存活有关。将LOXL2和其他标志物进行组合诊断，并不能明显提高诊断能力。

Figure 4. Prognostic value of LOXL2 and TAGLN in colon cancer. Biomarker validation was carried out in two steps: 1. By QPCR in a different cohort of 70 colon cancer patients: (A) overall survival and (B) disease-free. 2. By immunohistochemistry analysis using 121 samples in a tissue microarray slide. Positive expression was associated to nuclear staining of the stromal fibroblasts. (C) Overall survival and (D) disease-free survival. Kaplan-Meier curves were performed using the log-rank test. The Hazard Ratio was based on the Cox model.

为了评价LOXL2对II期和III期病人的风险评估价值，作者采用多个数据集分析了II期病人的复发率与表达水平的关系。结果发现，LOXL2高表达的II期病人的复发周期更短（P value = 0.011 HR: 2.74 (1.22-6.20)），其三年DFS为73.9%，而低表达组为89.6%。高表达LOXL2的47例II期和III期病人的无病期更短。55个II期病人的免疫组化结果显示，其无病期为P= 0.005 HR: 4.35 (1.43-13.25)。LOXL2高表达也与III期病人的整体存活（P = 0.004 HR: 3.40 (1.40-8.24)）有关。

Figure 5. Risk stratification of colon cancer patients at stages II and III using LOXL2 expression. (A) Tumor recurrence-free survival was determined using: GSE17538+GSE31331 datasets of colon patients at stage II, QPCR with a different cohort of 48 patients at stage II-III or immunohistochemistry (IHC) with 55 samples from patients at stage II. (B) Overall survival was determined using GSE17538+GSE12945 datasets of patients at stage III or QPCR with samples from 48 patients at stage II-III. Kaplan-Meier curves were performed using the log-rank test. The Hazard Ratio was based on the Cox model.

结论

通过蛋白质组学的筛选和后续验证，该研究证实LOXL2的表达水平与结肠癌的预后有关，并可通过区分II期和III期阶段为后续的治疗决策提供帮助。

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领域：蛋白/抗体/蛋白质组