Li Jia-Qing, Xu Baogang J, Shakhtour Bashar, Deane Natasha, Merchant Nipun, Heslin Martin J, Washington Kay, Coffey Robert J, Beauchamp R Daniel, Shyr Yu, Billheimer Dean
Department of Surgery, Vanderbilt University Medical Center, Nashville, TN 37232-6848, USA.
Int J Oncol. 2007 Jul;31(1):103-11.
Knowledge of intrinsic tumor heterogeneity is vital for understanding of tumor progression mechanisms as well as for providing efficient treatments. In situ proteomic profiling of tumors is a powerful technology with potential to enhance our understanding of tumor biology, but sources of variability due to patient and tumor heterogeneity are poorly understood and are the topic of this investigation. Clarification of variability within case and between cases is also important for designing future studies. Direct protein profiling by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a sensitive and powerful technology for obtaining hundreds of protein expression peaks from a thin tissue section. By combining robotic microspotting and laser capture microdissection with MALDI MS, we acquired multiple spectra per case to evaluate inter- and intra-case variability in human colorectal tumor and murine cecal carcinoma. We detected 256 peaks from 164 samples of 111 patients, which consisted of 55 normal colorectal mucosal samples, 24 adenomas, 71 primary carcinomas, and 14 hepatic metastases. In addition, we detected 291 peptide/protein peaks from 34 orthotopically transplanted murine cecal carcinomas and 14 hepatic metastases. In human colorectal samples, we observed that proteomic profiling in adenomas was more homogeneous across patients than in normal mucosa specimens (p=0.0008), but primary carcinoma exhibited greater heterogeneity than normal mucosa and adenomas (both p<0.0001). Murine cecal carcinomas were homogeneous within and between carcinomas, while their hepatic metastases tended toward greater intra-tumor differences (p<0.0001). Inter- and intra-case variability was approximately equal for many protein peaks. Acquiring up to 5 subsamples per case could reduce the total number of cases required, but further reduction from additional subsampling was modest unless intra-case variability comprises a greater proportion of total variation (e.g. >70%). In summary, this study characterizes intra- and inter-case variability of high-throughput protein expression in colorectal tumors, and provides guidance for the sample numbers required for in situ proteomic studies.
了解肿瘤内在异质性对于理解肿瘤进展机制以及提供有效的治疗方法至关重要。肿瘤原位蛋白质组分析是一项强大的技术，有潜力增进我们对肿瘤生物学的理解，但由于患者和肿瘤异质性导致的变异性来源却知之甚少，这也是本研究的主题。明确病例内和病例间的变异性对于设计未来研究也很重要。通过基质辅助激光解吸/电离飞行时间质谱（MALDI-TOF MS）进行直接蛋白质分析是一种灵敏且强大的技术，可从薄组织切片中获取数百个蛋白质表达峰。通过将机器人微点样和激光捕获显微切割与MALDI MS相结合，我们为每个病例获取了多个光谱，以评估人类结直肠癌和小鼠盲肠癌病例内和病例间的变异性。我们从111例患者的164个样本中检测到256个峰，其中包括55个正常结直肠黏膜样本、24个腺瘤、71个原发性癌和14个肝转移瘤。此外，我们从34个原位移植的小鼠盲肠癌和14个肝转移瘤中检测到291个肽/蛋白质峰。在人类结直肠样本中，我们观察到腺瘤中的蛋白质组分析在患者之间比正常黏膜样本更具同质性（p = 0.0008），但原发性癌比正常黏膜和腺瘤表现出更大的异质性（两者p < 0.0001）。小鼠盲肠癌在癌内和癌间是同质的，而它们的肝转移瘤倾向于肿瘤内差异更大（p < 0.0001）。许多蛋白质峰的病例间和病例内变异性大致相等。每个病例获取多达5个亚样本可以减少所需的病例总数，但除非病例内变异性在总变异中占更大比例（例如>70%），否则额外亚采样的进一步减少幅度不大。总之，本研究描述了结直肠癌中高通量蛋白质表达的病例内和病例间变异性，并为原位蛋白质组研究所需的样本数量提供了指导。
