Targeted tissue proteomic analysis of human astrocytomas

Abstract

Complicating proteomic analysis of whole tissues is the obvious problem of cell heterogeneity in tissues, which often results in misleading or confusing molecular findings. Thus, the coupling of tissue microdissection for tumor cell enrichment with capillary isotachophoresis-based selective analyte concentration not only serves as a synergistic strategy to characterize low abundance proteins, but it can also be employed to conduct comparative proteomic studies of human astrocytomas. A set of fresh frozen brain biopsies were selectively microdissected to provide an enriched, high quality, and reproducible sample of tumor cells. Despite sharing many common proteins, there are significant differences in the protein expression level among different grades of astrocytomas. A large number of proteins, such as plasma membrane proteins EGFR and Erbb2, are up-regulated in glioblastoma. Besides facilitating the prioritization of follow-on biomarker selection and validation, comparative proteomics involving measurements in changes of pathways are expected to reveal the molecular relationships among different pathological grades of gliomas and potential molecular mechanisms that drive gliomagenesis.

Figure 1

Demonstration of selective tissue microdissection of a relatively pure population of tumor cells. Left, a hypercellular focus of GBM cells, abutting the acellular space to the left of the slide, surrounded on the right by white matter (brain) infiltrated by tumor cells, but at a substantially lower cellular density; middle, the tumor cells selectively removed by LCM; and, right, the tumor cells isolated on the LCM cap.

Figure 2

The Venn diagrams comparing (A) total proteins and (B) membrane proteins identified from grade II astrocytoma (black), grade III anaplastic astrocytoma (blue), and grade IV GBM (red).

Figure 3

Pearson correlation coefficient plot of two proteomic runs analyzing adjacent GBM sections of the same tissue specimen.

Figure 4

Volcano plots of (a) grade IV vs. grade III and (b) grade IV vs. grade II.

Figure 5

Unsupervised hierarchical clustering of forty proteins with the most significant t-test values resulting from the comparison of grade III and grade IV astrocytomas.

Figure 6

Top functional pathways constructed from the up-regulated GBM proteome dataset using the Ingenuity Pathway Analysis™.

Figure 7

An up-regulated GBM protein network containing EGFR and Erbb2.

Figure 8

IQGAP1 expression relative to correlation with astrocytoma grade.

Figure 9

Scoring of TMA with IHC staining of IQGAP1. “Intensity” refers to staining intensity using IHC; “percentage” refers to the percentage of tumor cells within each TMA that were positive by IHC; and “grade” refers to WHO grade I-IV gliomas. IHC results validate MS-based quantitative proteomic profiling of microdissected astrocytoma tissues.