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

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+ 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 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.

NF-κB and Enhancer-binding CREB Protein Scaffolded by CREB-binding Protein (CBP)/p300 Proteins Regulate CD59 Protein Expression to Protect Cells from Complement Attack

“…Furthermore, chronic inflammation, especially when induced by pathogens, has been implicated in tumorigenesis, where many proinflammatory and pathogenic virulence factors are produced in local tumor sites (36). These factors, such as TNF-␣, IL-1␤, IL-6, IL-10, and LPS, in turn up-regulate CD59 expression, as demonstrated here and previously (37,38), thereby allowing nascent tumor cells or cancer stem cells to escape from complement surveillance (39,40). Moreover, CD59 also has diverse complement-independent roles, including anti-apoptotic (41) and tumor angiogenic functions (42).…”

Production of RNA for Transcriptomic Analysis from Mouse Spinal Cord Motor Neuron Cell Bodies by Laser Capture Microdissection

“…To date, proteomics analyses of a few thousand cells will reach down through only more abundant species. For example, we have identified <1,000 of the most abundant proteins from 3,000-4,000 motor neuron cell bodies (not shown), and Zhu and coworkers have recently reported identifying 2,665 proteins from ~15,000 tumor cells 1 . With further developments of mass spectrometry, however, it is likely that such analyses will extend to far greater depth.…”

Proteomics Characterization of Primary Human Oral Epithelial Cells Using a Novel Culture Technique for Use in Tissue Regeneration

“…In order to obtain detailed molecular information and infer significant signaling pathways from the proteome profiling results, differentially expressed proteins identified between sample ePUKs small vs. monolayer small were uploaded into the pathway analysis tool IPA (Ingenuity Systems, Redwood City, CA, USA) as previously described [8]. The uploaded Excel file contains the relevant proteins with their fold change and corresponding primary accession number (Supplemental Tables 1-5).…”