Angiogenesis Array (Methodology) Immunohistochemistry
Fixed astrocytomas tumors were embedded in paraffin.
Four-micrometer-thick sections were immunostained on the Benchmark XT automated stainer (Ventana Medical Systems, Tucson, Arizona, USA). Tumor samples were stained with an antibody against CD31 (1:10, clone M0823, DAKO, Hamburg, Germany) located on endothelial cells to identify blood vessels or against p53 (1:50, clone NCL-p53 DO1, Novocastra, Dossenheim, Germany) located in the tumor nuclei.
Antigen–antibody complexes were detected with Ventana’s Enhanced V-Red Detection, which is an alkaline phosphatase that uses naphthol and fast red chromogen. Most approaches to detect MVD, such as the one used in this work, are based on the protocol of Weidner et al. (1991, 1992). Briefly, after immunohistochemical staining of endothelium or other vascular wall components, areas of high- est vessel density were selected and counted either manual or computer-assisted. Consequently, statisti- cal analysis was performed to see if MVD correlates with overall survival, disease-free survival or other measures.
We evaluated MVD in astrocytoma with a modern automatic image analyses algorithm provided by S.CO (S.CO LifeScience Company, Garching, Germany).
In detail: areas of highest neovascularization were selected by scanning CD31 stained tumor sections at low power (25×and 40×) and identifying the areas of infiltrating tumor with the highest number of microves- sel. Vessels belonging the dura mater or the arachnoida were not considered in the vessel counts. However these vessels served as internal controls in assess- ing the quality of the CD31 staining. After the area of highest neovascularization was identified a picture
with 200×magnification was collected and sent to the analyses program.
The degree of vascularization was quantified automatically and objectively with the S.CO Image Analysis System (S.CO LifeScience Company, Garching, Germany), which bases on the Cognition Network TechnologyR by Definiens AG. In detail:
a threshold based approach identifies all red stained objects in the picture and automatically detects a vascular candidate using the criteria colour. Then an object oriented method is applied to eliminate those objects that show only a diffuse and faint red staining compared to the surrounding. The remaining objects are classified into three categories using the criteria object size: small objects = single immunopositive cells (yellow), medium size objects = capillaries (pink), large objects = arteries and veins (red) (Fig.16.1).
The number and area of immunohistochemical pos- itive vessel structures were given as output values separate for each image.
The evaluation for p53 followed a much easier approach and was done manually.
For p53 immunohistochemistry two different distri- bution patterns of stained nuclei were found: Pattern A showed a widespread strong positivity of tumor cell nuclei involving either the entire tissue section or segments of the tumor sample. In pattern B, only sin- gle tumor cells scattered throughout the tumor sample showed an only weak but positive reaction. Only cases of pattern A were considered positive for immunohis- tochemistry.
P53 Mutation Analyses
Characterization of p53 mutations within the tumor tis- sues was done by PCR amplification of genomic DNA extracted from the indicated tumor tissues. Genomic DNA was extracted using a genomic DNA Extraction Kit (Qiagen, Hilden, Germany). Primer pairs were generated to span each exon of the p53 gene, as well as flanking intronic sequence, and PCR products were purified using ExoSAP-ITR PCR purification kit. PCR products were subsequently sequenced on an ABI-Prism 3100 Genetic Analyzer and analysed for mutations.
Fig. 16.1 Example of an image used to estimate the MVD as a function of p53 mutation status. For both images origi- nal magnification was 200×. (a) Area with the highest den- sity of microvessels in a diffuse low-grade astrocytoma (WHO Grade II). Vessels stained with an antibody against CD31 using fast red chromogen. (b) Same image after applying the vessel quantification algorithm. This procedure identifies all red stained objects in the picture and eliminates background. The remain- ing objects are classified into three categories using the criteria
“object size”: small objects = single immunopositive cells (yellow), medium size objects = capillaries (pink), large objects=arteries and veins (red)
Human Angiogenesis Array
The Human glioblastoma cell line LN229 (p53 mutant CCT (Pro) → CTT (Leu) mutation at codon 98) was used for evaluating the background mecha- nisms for the p53 dependent angiogenesis. We trans- fected LN229 with p53-wild-type vector and Fugene Transfection reagent (Roche Deutschland Holding GmbH, Mannheim, Germany). As a negative control
and to substract transfection associated effects, LN229 was also transfected with an empty vector (Clontech, USA; Mountain View, CA). For analyzing the expres- sion profiles of angiogenesis-related proteins we used the Human Angiogenesis Array Kit (R&D Systems, Ltd., Abingdon, United Kingdom). This array consists of 55 on a nitrocellulose membrane spotted antibodies dedicated to proteins related with angiogenesis. Cell samples (1 × 107 cells from LN229 p53 WT- and empty vector-transfected) were harvested and 300μg of protein was mixed with 15μl of biotinylated detec- tion antibodies. After pre-treatment, the cocktail was incubated overnight at 4◦C on a rocking platform.
Following a wash step to remove unbound mate- rial, streptavidin–horseradish and chemiluminescent detection reagents were added sequentially. The light signal intensity correlates with the amount of con- verted substrate. All experiments were performed in three triplicates. The data on developed X-ray film were quantified by scanning on a transmission-mode scanner and subsequently analyzed by using ImageJ analysis software (http://rsbweb.nih.gov/ij/).
Statistics
Statistical analyses were used to explore the correlation between the presence or absence of p53 mutations and p53 protein expression. Microvessel densities were compared in non-mutated versus mutated tumors and in p53 IHC positive and negative tumors. Therefore, a Two-tailed Students’t-test was used by comparing the MVD between different groups. A value of p < 0.05 was considered significant.
Immunohistochemical Detected P53 Protein Does Not Correlate with Microvessel Density
In a series of 23 diffuse astrocytomas (WHO grade II) we found that 9/23 (39.13%) of the astrocytomas were positive for p53 IHC.
We found no correlation between a genetically detectable p53 mutations and a nuclear p53 protein accumulation. We found also no differences in MVD
between p53 IHC positive and p53 negative astrocy- tomas. We detected a mean MVD of 5.30% for the p53 IHC positive cases and a mean MVD of 5% for the p53 IHC negative cases, respectively.
Astrocytomas with P53 Mutation Have a Higher Number of Vessels
We identified p53 mutations in 11/23 (47%) of the astrocytomas, in these tumors we could observe a sig- nificant higher MVD compared to astrocytomas with a wild-type p53 genotype. MVD for p53 mutated low-grade astrocytomas was 8.30% (±1.5) and 3.6%
(±1.2) for p53 wild-type low-grade astrocytomas, respectively. Additionally the absolute vessel number was significantly increased in p53 mutated astrocy- tomas (60±4) compared to p53 wild-type low-grade astrocytomas (38±5).
Thrombospondin-1, Coagulation Factor III and Serpin E1 are Upregulated Under P53 (Human Angiogenesis Array Data)
We transfected LN229 glioma cells that harboured a TP53 mutation with either an empty plasmid (pcCMV- empty) or with a plasmid that encodes the p53 wild-type protein (pcCMV-p53 wild-type) to eluci- date the molecular mechanisms that potentially lead to a higher MVD in p53 mutated astrocytomas The overexpression of p53 after transfection could be confirmed by western blot. The performed Human Angiogenesis Array Kit detected protein expression differences for the LN229 p53 transfected vs. pcCMV- empty in the following proteins: 5.31-fold increase of thrombospondin-1, a 4.17-fold increase of coagulation factor III, a 1.76-fold increase of serpin E1 (PAI-1) and a 0.39-fold decrease of MMP-9.
Discussion
Glioma is by far the most frequently examined brain neoplasm in terms of angiogenesis. Already in 1989 by Schiffer et al., an increased vascularity could be linked
to increased glioma growth (Schiffer et al., 1989).
Beside that, this work is especially interesting because the authors counted the number of vessels per square mm, the number of nuclei per vessel, and the inter- nal and external diameters, on a conventional histo- chemical staining (Luxol Fast Blue PAS Hematoxylin) thereby introducing a similar approach as done in our work.
Using immunohistochemistry for quantifying ves- sels in gliomas was then done by Leon et al. (1996). In 93 adult astrocytomas, using FVIII-immunostaining, a negative association between increasing microvessel numbers (counting vessels in a 200×magnification) and disease-free survival by could be demonstrated.
Also a qualitative approach by measuring MVD on a scale from 1 to 4 correlated with shortened survival (Leon et al.,1996).
In a large retrospective study about low-grade astro- cytomas, Abdulrauf et al. (1998) could identify that patients with more than seven microvessels per 400× field had a shorter survival (mean, 3.8 years) for progression to high grade astrocytoma. They con- cluded that diffuse astrocytomas are therefore very heterogenous and some tumors were more dependent on neovascularization then others.
In this context the experiments of Wesseling et al.
can be interpretated. They examined a mixed group of astrocytoma (including glioblastomas, astrocytomas and anaplastic astrocytomas) and found a wide range of vessel heterogeneity within each group. They iden- tified areas without increased numbers of microvessel in every subgroup, suggesting that neovascularization may not be a requisite for at least some gliomas (Wesseling et al.,1998). So, to summarize the results it appears that a subgroup of gliomas is more depending on neovascularization than others. This clearly offers the theoretical background for our study because one of the major discriminating factors within astrocy- tomas is the mutation of the p53 gene. The mutation frequency for p53 in low grade astrocytoma is approx.
60% and mutations frequency does not significantly increase during malignant progression to secondary glioblastomas indicating that this genetic change is an early event (Okamoto et al.,2004; Reifernberger et al., 1996; Watanabe et al.,1997).
Some studies found a shorter progression inter- val from low-grade astrocytomas to glioblastoma in
tumors harboring a p53 mutation (Stander et al.,2004;
Watanabe et al.,1997).
In our study we looked for the association of p53 mutation with MVD. We observed that p53 mutated low-grade astrocytomas exhibited a significant higher vascular density and absolute number of vessels com- pared to p53 wild-type astrocytomas. This underlines the important role of p53 in terms of regulation of angiogenesis but it also raises the question of the molecular background involved in that process. Beside the numerous functions already demonstrated for p53, recent papers suggest that the normal p53 protein stim- ulates the expression of genes that prevent the process of angiogenesis (el-Deiry1998; Teodoro et al., 2007;
Vogelstein et al., 2000). Especially in a subsequent, work done by Teodora et al. an activation of the gene encoding α(II) collagen prolyl-4-hydroxylase, which is necessary for the release of the anti-angiogenated collagen-derived peptides, such as endostatin and tum- statin, could be linked to p53 (Teodoro et al., 2006).
They used H1299, a non-small cell lung carcinoma cell line; in glioblastoma cell lines however this mech- anism could not be proven effective (Berger et al., 2010). At least from our data we claim that low grade astrocytomas, in which the angiogenic switch has not occurred yet, have higher likelihood to recruit new blood vessels if p53 is inactivated by mutation. This step seems to provide a critical growth advantage in tumor development.
As a side note, we saw no correlation between p53 protein expressions detected by IHC and MVD. The cause of this is to be found in technical issues. IHC can fail to detect certain p53 mutant protein (Anker et al.,1993; Kupryjanczyk et al.,1993). Although only mutated p53 protein is accumulating in the nucleus of the cell, IHC can still not be used to detect all different types of mutated p53 proteins. The bind- ing ability of the p53 antibody is dependent on the secondary structure of the protein and therefore dif- ferent for each mutation. Hence, it is not surprising that a correlation between p53 IHC and MVD was not found.
To analyze the important molecular background between the increased MVD and a p53 mutation we used an array specially designed for analyzing angiogenesis-related proteins (Human Angiogenesis Array Kit). The investigation was done with a
glioblastoma cell line, LN229 (mutated for p53) due to the lack of low-grade astrocytoma cell lines.
After digital image analyses of the array we detected four proteins being expressed differentially.
Thrombospondin-1 (TSP-1) was increased in p53 transfected LN229 cells (fivefold increase) compared to empty transfected cells. TSP-1 is a 450-kDa extra- cellular matrix glycoprotein. It has a complex structure and modulates cellular behaviours like motility, adhe- sion, and proliferation. Furthermore TSP-1 acts as a potential key player in angiogenesis. The increase of TSP-1 in p53 wild-type transfected LN229 glioma cells is compatible with the literature showing that decreased expression of TSP-1 is a key step in creating a pro-angiogenetic environment (Iddings et al.,2007).
TSP-1 negatively regulates the growth and migration of endothelial cells both in vitro and in vivo (Hsu et al.,1996; Nor et al., 2000). The loss of p53 led to a deficiency in TSP-1 expression, and the subse- quent inability to shut off angiogenesis. Restoration of p53 expression in these tumors re-established TSP-1 expression (Giuriato et al.,2006). Furthermore Kazuno et al. showed that gliomas lacking expression of the gene for thrombospondin-2, a powerful antiangiogenic molecule with many properties similar to TSP-1, are having quantitatively increased microvessel densities (Kazuno et al.,1999).
In wild-type p53 transfected LN229 cells coagula- tion factor III (CF III) was also increased compared to empty transfected LN229 cells (4.17-fold). CF III is expressed by cells which are normally not exposed to flowing blood such as sub-endothelial cells, e.g.
smooth muscle cells and cells surrounding blood ves- sels. This can change when the blood vessel is dam- aged by for example physical injury or rupture of atherosclerotic plaques. CF III is a cell surface gly- coprotein and enables cells to initiate the blood coag- ulation cascades, and it functions as the high-affinity receptor for the coagulation factor VII. Unlike other cofactors of these protease cascades, which circulate as nonfunctional precursors, this factor is a potent ini- tiator that is fully functional when expressed on cell surfaces. Aside from its role in the coagulation cascade binding of CF III to factor VIIa of the coagulation cas- cade has also been found to start signaling processes inside the cell. The signaling function of CF III/VIIa plays a role in angiogenesis and apoptosis (Belting et al.,2005).
An association of CF III and p53 has not been published to the best of our knowledge.
Plasminogen activator inhibitor type-1 (PAI-1) was 1.76-fold over expressed in wild-type p53 transfected LN229. It has already been reported that p53 also reg- ulate genes responsible for the proteolytic degradation of the extracellular matrix, which is a crucial fea- ture for local invasion and metastasis of neoplastic cells. An important and highly regulated cascade of such proteolytic events involves the plasminogen acti- vator (PA) and inhibitor (PAI) system. So our results are in line with previous reports since it has been demonstrated that wild-type but not mutant p53 specif- ically binds to and activates the promoter of the PAI-1 gene (Kunz et al., 1995). PAI-1 inhibits the serine proteases tissue-type plasminogen activator and urokinase-type (Kunz et al., 1995). Cellular transfor- mation often results in a dramatic increase in the production of the plasminogen activators (PA) and in altered expression of the inhibitor, PAI-1. The balance between PAs and their inhibitors appears to be criti- cal for the invasive phenotype of tumor cells implying that altered expression slightly in favor of plasmino- gen activators contributes to the malignant phenotype (Kunz et al.,1995).
Matrix metalloproteinase-9 (MMP-9) was decreased by a factor of 0.39 in p53 transfected LN229 glioma cells. Our results are in line with data from the current literature showing that reintroduction of wild-type p53 into mutant p53 soft tissue tumor cells decreased MMP-9 mRNA and protein levels (Liu et al.,2006). In glioblastoma cells antisense MMP-9 revealed marked reduction in the invasiveness of the adenoviral infected cells compared with parental and vector controls (Lakka et al.,2003).
In conclusion our data showed a higher MVD in p53 mutated low-grade astrocytomas and sup- port the hypothesis of a p53-mediated regulation on angiogenesis in diffuse low-grade astrocytomas.
Additionally the differential angiogenesis protein anal- yses for p53 transfected cell lines provides evidence that Thrombospondin-1, Coagulation factor III and Serpin E1 are potentially p53 targets and important key players in regulating angiogenesis in gliomas. For future analyses the influence of the EGFR signaling pathway, e.g. PTEN and EGFR alterations, and IDH-1 mutational status on MVD and angiogenesis might be worthwhile to explore since both pathways are closely linked to metabolism and hypoxia/ischemia.
References
Abdulrauf SI, Edvardsen K, Ho KL, Yang XY, Rock JP, Rosenblum ML (1998) Vascular endothelial growth factor expression and vascular density as prognostic markers of sur- vival in patients with low-grade astrocytoma. J Neurosurg 88(3):513–520
Anker L, Ohgaki H, Ludeke BI, Herrmann HD, Kleihues P, Westphal M (1993) p53 protein accumulation and gene muta- tions in human glioma cell lines. Int J Cancer 55(6):982–987 Belting M, Ahamed J, Ruf W (2005) Signaling of the tis- sue factor coagulation pathway in angiogenesis and cancer.
Arterioscler Thromb Vasc Biol 25(8):1545–1550
Berger B, Capper D, Lemke D, Pfenning PN, Platten M, Weller M, von Deimling A, Wick W, Weiler M (2010) Defective p53 antiangiogenic signaling in glioblastoma. Neuro Oncol 12(9):894–907
Boehme KA, Blattner C (2009) Regulation of p53–insights into a complex process. Crit Rev Biochem Mol Biol 44(6):
367–392
Dameron KM, Volpert OV, Tainsky MA, Bouck N (1994a) Control of angiogenesis in fibroblasts by p53 regulation of thrombospondin-1. Science 265(5178):1582–1584
Dameron KM, Volpert OV, Tainsky MA, Bouck N (1994b) The p53 tumor suppressor gene inhibits angiogenesis by stimu- lating the production of thrombospondin. Cold Spring Harb Symp Quant Biol 59:483–489
Donehower LA, Harvey M, Slagle BL, McArthur MJ, Montgomery CA Jr., Butel JS, Bradley A (1992) Mice defi- cient for p53 are developmentally normal but susceptible to spontaneous tumours. Nature 356(6366):215–221
el-Deiry, W.S. (1998) Regulation of p53 downstream genes.
Semin Cancer Biol 8(5):345–357
Folkman J (1990) What is the evidence that tumors are angio- genesis dependent? J Natl Cancer Inst 82(1):4–6
Giuriato S, Ryeom S, Fan AC, Bachireddy P, Lynch RC, Rioth MJ, van Riggelen J, Kopelman AM, Passegue E, Tang F, Folkman J, Felsher DW (2006) Sustained regres- sion of tumors upon MYC inactivation requires p53 or thrombospondin-1 to reverse the angiogenic switch. Proc Natl Acad Sci USA 103(44):16266–16271
Greenblatt MS, Bennett WP, Hollstein M, Harris CC (1994) Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis. Cancer Res 54(18):4855–4878
Hainaut P, Hollstein M (2000) p53 and human cancer: the first ten thousand mutations. Adv Cancer Res 77:81–137 Hanahan D, Weinberg RA (2000) The hallmarks of cancer. Cell
100(1):57–70
Haupt S, Berger M, Goldberg Z, Haupt Y (2003) Apoptosis – the p53 network. J Cell Sci 116(Pt 20):4077–4085
Hollstein M, Sidransky D, Vogelstein B, Harris CC (1991) p53 mutations in human cancers. Science 253(5015):49–53 Hsu SC, Volpert OV, Steck PA, Mikkelsen T, Polverini PJ,
Rao S, Chou P, Bouck NP (1996) Inhibition of angiogene- sis in human glioblastomas by chromosome 10 induction of thrombospondin-1. Cancer Res 56(24):5684–5691
Iddings DM, Koda EA, Grewal SS, Parker R, Saha S, Bilchik A (2007) Association of angiogenesis markers with lymph
node metastasis in early colorectal cancer. Arch Surg 142(8):738–744. discussion 744–735
Kazuno M, Tokunaga T, Oshika Y, Tanaka Y, Tsugane R, Kijima H, Yamazaki H, Ueyama Y, Nakamura M (1999) Thrombospondin-2 (TSP2) expression is inversely corre- lated with vascularity in glioma. Eur J Cancer 35(3):
502–506
Krex D, Klink B, Hartmann C, von Deimling A, Pietsch T, Simon M, Sabel M, Steinbach JP, Heese O, Reifenberger G, Weller M, Schackert G (2007) Long-term sur- vival with glioblastoma multiforme. Brain 130(Pt 10):
2596–2606
Kunz C, Pebler S, Otte J, von der Ahe D (1995) Differential regulation of plasminogen activator and inhibitor gene tran- scription by the tumor suppressor p53. Nucleic Acids Res 23(18):3710–3717
Kupryjanczyk J, Thor AD, Beauchamp R, Merritt V, Edgerton SM, Bell DA, Yandell DW (1993) p53 gene mutations and protein accumulation in human ovarian cancer. Proc Natl Acad Sci USA 90(11):4961–4965
Lakka SS, Gondi CS, Yanamandra N, Dinh DH, Olivero WC, Gujrati M, Rao JS (2003) Synergistic down-regulation of urokinase plasminogen activator receptor and matrix metalloproteinase-9 in SNB19 glioblastoma cells efficiently inhibits glioma cell invasion, angiogenesis, and tumor growth. Cancer Res 63(10):2454–2461
Leon SP, Folkerth RD, Black PM (1996) Microvessel density is a prognostic indicator for patients with astroglial brain tumors.
Cancer 77(2):362–372
Liu J, Zhan M, Hannay JA, Das P, Bolshakov SV, Kotilingam D, Yu D, Lazar AF, Pollock RE, Lev D (2006) Wild- type p53 inhibits nuclear factor-kappaB-induced matrix metalloproteinase-9 promoter activation: implications for soft tissue sarcoma growth and metastasis. Mol Cancer Res 4(11):803–810
Louis DN, Ohgaki H, Wiestler OD, Cavenee WK, Burger PC, Jouvet A, Scheithauer BW, Kleihues P (2007) The 2007 WHO classification of tumours of the central nervous system.
Acta Neuropathol 114(2):97–109
Lutzker SG, Levine AJ (1996) A functionally inactive p53 protein in teratocarcinoma cells is activated by either DNA damage or cellular differentiation. Nat Med 2(7):
804–810
Nor JE, Mitra RS, Sutorik MM, Mooney DJ, Castle VP, Polverini PJ (2000) Thrombospondin-1 induces endothelial cell apoptosis and inhibits angiogenesis by activating the caspase death pathway. J Vasc Res 37(3):209–218
Okamoto Y, Di Patre PL, Burkhard C, Horstmann S, Jourde B, Fahey M, Schuler D, Probst-Hensch NM, Yasargil MG, Yonekawa Y, Lutolf UM, Kleihues P, Ohgaki H (2004) Population-based study on incidence, survival rates, and genetic alterations of low-grade diffuse astrocytomas and oligodendrogliomas. Acta Neuropathol 108(1):49–56 Reifenberger J, Ring GU, Gies U, Cobbers L, Oberstrass J, An
HX, Niederacher D, Wechsler W, Reifenberger G (1996) Analysis of p53 mutation and epidermal growth factor receptor amplification in recurrent gliomas with malignant progression. J Neuropathol Exp Neurol 55(7):822–831 Schiffer D, Chio A, Giordana MT, Mauro A, Migheli A, Vigliani
MC (1989) The vascular response to tumor infiltration in