Target Proteomic Profiling of Frozen Pancreatic CD24+ Adenocarcinoma Tissues by Immuno-Laser Capture Microdissection and Nano-LC–MS/MS

Cellular
heterogeneity of solid tumors represents a common problem
in mass spectrometry (MS)-based analysis of tissue specimens. Combining
immuno-laser capture microdissection (iLCM) and mass spectrometry
(MS) provides a means to study proteins that are specific for pure
cell subpopulations in complex tissues. CD24, as a cell surface marker
for detecting pancreatic cancer stem cells (CSCs), is directly correlated
with the development and metastasis of pancreatic cancer. Herein,
we describe an in-depth proteomic profiling of frozen pancreatic CD24+ adenocarcinoma cells from early stage tumors using iLCM and
LC–MS/MS and a comparison with CD24– cells
dissected from patient-matched adjacent normal tissues. Approximately
40 nL of tissue was procured from each specimen and subjected to tandem
MS analysis in triplicate. A total of 2665 proteins were identified,
with 375 proteins in common that were significantly differentially
expressed in CD24+ versus CD24– cells
by at least a 2-fold change. The major groups of the differentially
overexpressed proteins are involved in promoting tumor cell migration
and invasion, immune escape, and tumor progression. Three selected
candidates relevant to mediating immune escape, CD59, CD70, and CD74,
and a tumor promoter, TGFBI, were further validated by immunohistochemistry
analysis on tissue microarrays. These proteins showed significantly
increased expression in a large group of clinical pancreatic adenocarcinomas
but were negative in all normal pancreas samples. The significant
coexpression of these proteins with CD24 suggests that they may play
important roles in the progression of pancreatic cancer and could
serve as promising prognosis markers and novel therapeutic targets
for this deadly disease.

实体瘤的细胞异质性是基于质谱（MS）的组织标本分析中普遍存在的共性难题。将免疫激光捕获显微切割（iLCM）与质谱（MS）技术联用，可为研究复杂组织中纯细胞亚群的特异性蛋白提供可行手段。CD24作为一种用于检测胰腺癌干细胞（CSCs）的细胞表面标志物，与胰腺癌的发生、发展及转移密切相关。本研究采用免疫激光捕获显微切割（iLCM）结合液相色谱-串联质谱（LC-MS/MS）技术，对早期肿瘤来源的冰冻CD24阳性（CD24+）胰腺腺癌细胞进行了深度蛋白质组学分析，并与患者匹配的癌旁正常组织中分离得到的CD24阴性（CD24-）细胞进行对照比较。每份标本获取约40纳升（nL）组织样本，随后进行三次重复的串联质谱分析。本次研究共鉴定出2665种蛋白质，其中375种蛋白质在CD24+与CD24-细胞中存在至少2倍的显著差异表达。差异高表达蛋白的主要功能类别涵盖促进肿瘤细胞迁移侵袭、免疫逃逸以及肿瘤进展。研究选取了3种与免疫逃逸调控相关的候选蛋白CD59、CD70、CD74，以及肿瘤促进因子TGFBI，通过组织微阵列（tissue microarrays）的免疫组化（immunohistochemistry）分析进行了验证。结果显示，这些蛋白在大量临床胰腺腺癌组织中表达水平显著升高，而在所有正常胰腺样本中均呈阴性表达。这些蛋白与CD24存在显著共表达现象，提示它们可能在胰腺癌进展过程中发挥重要作用，有望成为该恶性疾病的潜在预后标志物及新型治疗靶点。