In situ visualization of intratumor growth factor signaling: immunohistochemical localization of activated ERK/MAP kinase in glial neoplasms

Abstract

Abnormal growth factor signaling is implicated in the pathogenesis of gliomas. The extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway is a likely target, linking receptor tyrosine kinase activation to downstream serine/threonine phosphorylation events regulating proliferation and differentiation. Signaling within heterogeneous cell populations of gliomas cannot be adequately assessed by traditional biochemical enzyme assays. Immunohistochemical detection of doubly phosphorylated (activated) ERK/MAPK permitted visualization of spatially discrete cellular patterns of ERK/MAPK activation, compared with the relatively uniform expression of total ERK/MAPK protein. The astrocytic tumors, regardless of grade, had the highest overall degree of enzyme activation, whereas oligodendrogliomas had the least. Anaplastic progression in oligodendrogliomas resulted in a larger number of cells with active ERK/MAPK. Within glioblastomas, microvascular hyperplasia and necrosis were associated with ERK/MAPK activation in adjacent tumor cells. In addition to spatial patterns of intratumor paracrine signaling, a possible cell-cycle-associated regulation was detected: mitotic and actively cycling tumor cells showed diminished activation relative to cells in G0. Although ERK/MAPK activation was not restricted to neoplastic glia, consistent patterns of selective activation in tumor cells suggests that sustained activation may contribute to the neoplastic glial phenotype.

Figure 1.

Immunoblot analysis of ERK/MAPK protein expression and phosphorylation in non-neoplastic cortex and astrocytomas. ERK/MAPK protein expression levels (bottom panel; total ERK/MAPK) are similar in non-neoplastic cortex (lanes 1 to 3) and astrocytic tumors of all grades (lanes 4 to 7). In contrast, dually phosphorylated ERK/MAPK (top panel; phospho-MAP kinase) is generally elevated in astrocytic tumors compared with the barely detectable levels in three different normal cortex samples. Although there was great variation in dp-ERK/MAPK levels among tumors, intense dp-ERK/MAPK bands like those in the tumor samples illustrated were never observed in non-neoplastic cortex samples, obtained from lobectomy specimens for intractable epilepsy. Equal quantities of protein were loaded for each specimen.

Figure 2.

Demonstration of dp-ERK/MAPK antibody specificity in immunohistochemistry. dp-ERK/MAPK (phospho MAPK) immunoreactivity is abolished by preadsorbtion with the doubly phosphorylated immunizing peptide (B) but not the unphosphorylated peptide (A). Alkaline phosphatase pretreatment almost completely eliminates phospho-MAPK immunoreactivity (D) compared with buffer-only control (C). Staining with the phospho-insensitive general ERK/MAPK antibody is unaffected by phosphatase treatment (E and F). Immunoperoxidase staining with hematoxylin counterstain; original magnification, ×50.

Figure 3.

Localization of activated ERK/MAPK in non-neoplastic human cortex. dp-ERK/MAPK (pMAPK) immunoreactivity is undetectable in non-neoplastic gray (A) and white (C) matter, with the exception of subpial astrocytes in regions of mild gliosis (corner inset, A) and occasional capillary endothelial cells (arrows, A and C). In contrast, total ERK/MAPK (MAPK) immunoreactivity is uniformly present in gray (B) and white (D) matter, especially in pyramidal cell somata (B), and in the processes of subpial astrocytes (inset, B). Immunoperoxidase staining with hematoxylin counterstain; original magnification, ×100.

Figure 4.

Immunohistochemical demonstration of ERK/MAPK phosphorylation in astrocytic neoplasms. For each tumor, adjacent sections were immunolabeled with the phospho-specific ERK/MAPK antibody (pMAPK; left panels ) and a phospho-insensitive ERK/MAPK antibody (MAPK; right panels). Elevated nuclear and cytoplasmic dp-ERK/MAPK immunoreactivity, relative to normal quiescent astrocytes, was detected in a subset of tumor cells within a variety of astrocytic neoplasms. In contrast, ERK/MAPK immunoreactivity was more uniformly expressed. A and B: Astrocytoma, WHO grade II. Some diffusely infiltrating astrocytoma cells show increased nuclear and cytoplasmic dp-ERK/MAPK immunoreactivity. Note the absence of labeling in adjacent normal neurons. ERK/MAPK protein is more evenly expressed in tumor cell cytoplasm and in neurons. C and D: Anaplastic astrocytoma, WHO grade III. A subset of gemistocytic tumor cells shows high cytoplasmic and nuclear dp-ERK/MAPK immunoreactivity. E and F: Ependymoma. A subpopulation of highly fibrillar ependymoma cells, especially those forming perivascular pseudorosettes, shows elevated dp-ERK/MAPK immunoreactivity; compare with the relatively uniform expression of ERK/MAPK protein. G and H: Glioblastoma multiforme (astrocytoma, WHO grade IV). Tumor cells near the hyperplastic microvasculature show the most intense nuclear and cytoplasmic pMAPK immunoreactivity. Some fine immunoreactive tumor cell processes appear to be oriented toward the microvasculature (corner inset). Total MAPK immunoreactivity is much more uniform, and not restricted to the perivascular tumor cells. I and J: Glioblastoma with giant cell transformation (astrocytoma, WHO grade IV). Only the large, bizarre, multinucleated tumor cells have detectable dp-ERK/MAPK immunoreactivity; the small cell population is completely negative. Note the absence of dp-ERK/MAPK immunoreactivity in a mitotic tumor cell (left side). In contrast, both small and large tumor cell populations show uniform ERK/MAPK protein immunoreactivity. Also, a mitotic cell is visible that has detectable MAPK immunoreactivity (right side). K to N: Selective dephosphorylation of ERK/MAPK within mitotic and cycling glioblastoma cells. K and L: Immunoreactivity for dp-ERK/MAPK is absent within individual mitotic tumor cells in two additional glioblastoma specimens; adjacent interphase cells show abundant cytoplasmic and nuclear immunoreactivity. M: Evidence that mitotic phosphoepitopes in general are not lost in routinely processed surgical specimens comes from immunolabeling with monoclonal antibody MPM-2, which recognizes a number of mitosis-specific phosphorylated proteins. Strong perichromosomal staining is present in mitotic cells. N: Double-label immunohistochemistry for dp-ERK/MAPK (red-brown) and Ki-67 (dark blue) reveals essentially complete non-co-localization. This suggests that cycling cells (those with nuclear Ki-67 immunolabeling) and those with activated ERK/MAPK (dp-ERK/MAPK immunoreactive) are generally mutually exclusive populations. A to M: Immunoperoxidase staining (DAB) with hematoxylin counterstain. N: Double-label immunoperoxidase with aminoethylcarbazole and Vector SG substrates; no counterstain. Scale bar in A represents 50 μm and applies to A to J and M; bar in N represents 20 μm and applies to K, L, and N.

Figure 5.

ERK/MAPK in glioblastomas, showing increased phosphorylation in tumor cells near hyperplastic microvessels and surrounding zones of tumor necrosis. Glioblastoma cells in contact with or near hyperplastic microvessels show increased dp-ERK/MAPK immunoreactivity relative to those at a distance (A). Total ERK/MAPK immunoreactivity in the same microscopic field is uniform in all tumor cells (B). Increased dp-ERK/MAPK is present in a zone of viable tumor cells surrounding a focus of tumor necrosis (C), whereas total ERK/MAPK immunoreactivity is uniform in the same microscopic field (D). Immunoperoxidase with hematoxylin counterstain. Scale bar in D represents 175 μm.

Figure 6.

Focal ERK/MAPK activation in non-infiltrating (circumscribed) astrocytomas. Immunoreactivity for activated ERK/MAPK is focally prominent in cytoplasm and coarse processes of pilocytic astrocytoma cells (A). A glomeruloid vessel (v) is devoid of the phosphorylated ERK/MAPK epitope. Total ERK/MAPK immunoreactivity is present in all cells in this tumor (B). A pleomorphic xanthoastrocytoma shows focally elevated cytoplasmic dp-ERK/MAPK immunoreactivity and occasional nuclear labeling in tumor cells (C); adjacent non-neoplastic brain (br) is negative. Total ERK/MAPK immunoreactivity is present in all tumor cells as well as non-neoplastic brain (br; D). Original magnification, ×100.

Figure 7.

ERK/MAPK phosphorylation in non-astrocytic brain tumors. A and B: A grade II oligodendroglioma shows generally low dp-ERK/MAPK immunolabeling, with a few immunoreactive nuclei. C and D: In contrast, an anaplastic oligodendroglioma (grade III) shows greatly elevated cytoplasmic and nuclear dp-ERK/MAPK immunoreactivity in a large subpopulation of tumor cells. E and F: A primary CNS lymphoma shows no detectable dp-ERK/MAPK immunoreactivity; note the intense immunolabeling within an entrapped reactive astrocyte. G and H: A metastatic poorly differentiated adenocarcinoma shows detectable but low dp-ERK/MAPK immunoreactivity compared with the intense labeling of surrounding reactive astrocytes. Immunoperoxidase with hematoxylin counterstain. Scale bar in H represents 50 μm.