Target Proteomic Profiling of Frozen Pancreatic CD24+
Adenocarcinoma Tissues by Immuno-Laser Capture Microdissection and
Nano-LC–MS/MS
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Abstract
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<sup>+</sup> adenocarcinoma cells from early stage tumors using iLCM and
LC–MS/MS and a comparison with CD24<sup>–</sup> 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<sup>+</sup> versus CD24<sup>–</sup> 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