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Does Vasculogenic Mimicry Exist in Astrocytoma?

Wei-Ying Yue and Zhong-Ping Chen

Department of Neurosurgery/Neuro-oncology, Cancer Center, Sun Yat-Sen University, Guangzhou, P.R. China

Correspondence to: Zhong-Ping Chen, Department of Neurosurgery/Neuro-oncology, Cancer Center, Sun Yat-Sen University, Guangzhou, P.R. China 510060. E-mail: chenzp{at}gzsums.edu.cn


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 Literature Cited
 
Vasculogenic mimicry (VM) has been observed in melanoma and in some nonmelanoma tumor types. It is unknown whether a similar VM phenomenon exists in astrocytoma. The present study was to examine 45 astrocytomas (including World Health Organization grade II 15 cases, grade III 15 cases, and grade IV 15 cases) by CD34 endothelial marker periodic acid–Schiff (PAS) dual staining to see if VM existing in these tumors. The results demonstrated that endothelium-lined vessels dominated the tumor microvasculature and stained positively for PAS, laminin, and endothelial marker. PAS-positive pattern of VM was found in two grade IV astrocytomas. Channels stained positively for PAS, laminin, and negatively for CD34 of the VM entrapped in the tumor tissue. Erythrocytes could be observed in some of these channels. In these networks of PAS-positive pattern, spots of weak reaction for CD34 were observed, suggesting the incorporation of VM channel and normal vessel. Furthermore, in astrocytoma, especially glioblastoma, focus of anaplastic tumor cells appeared with CD34 expression, whereas some tumor cells lost glial fibrillary acid protein expression. It is assumed that genetically deregulated tumor cells in astrocytoma could lose the astrocyte-specific protein and express inappropriate markers not expected in cells of astrocyte lineage. The present results suggest that VM phenomenon exists in some malignant astrocytoma. (J Histochem Cytochem 53:997–1002, 2005)

Key Words: astrocytoma • vasculogenic mimicry • periodic acid–Schiff staining • PAS-positive pattern • immunohistochemistry


    Introduction
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
MICROCIRCULATION PLAYS a central role in the tumor growth and hematogenous dissemination. It is a well-accepted paradigm that tumors recruit new blood vessels from the existing circulation (Risau 1997Go)—angiogenesis, true vasculogenesis results in the de novo formation of endothelial cell-lined vessels—induced by factors secreted by the tumor cells, as Folkman (Folkman 1971Go; Hanahan and Folkman 1996Go) has emphasized, or by surrounding stromal cells (Fukumura et al. 1998Go). However, Maniotis et al. (1999)Go described a novel process by which tumors develop a highly patterned microcirculation that is independent of angiogenesis: in aggressive primary and metastatic melanomas, the tumor cells generate nonendothelial cell-lined microcirculatory channels composed of extracellular matrix and lined externally by tumor cells. They named the process "vasculogenic mimicry" (VM). Folberg et al. (2000)Go used periodic acid–Schiff (PAS) staining to show VM network and identified seven morphological patterns of PAS-positive channels in tissue sections of uveal melanomas: straight channels, arrangements of parallel straight channels, straight channels that cross-link, arcs (incompletely closed loops), arcs with branching, closed loops, and networks (networks were defined arbitrarily as at least three back-to-back closed PAS-positive loops). The VM has also been observed in nonmelanoma tumor types, including carcinomas of the breast, prostate, ovary and lung, synoviosarcoma, rhabdomyosarcoma, phaeochromocytoma, and in cytotrophoblasts forming the placenta (Hendrix et al. 2003aGo). Hendrix et al. (2003b)Go showed that the molecular "signature" of aggressive melanoma cells is illustrative of an undifferentiated cell with a gene expression profile that is similar to that of embryonic-like cells. The discovery of VM challenges the prevailing assumption that angiogenesis is the only means by which a tumor acquires a blood supply. Astrocytoma is a diffusely infiltrating tumor and rarely metastasizes to extracranial organs. It is unknown yet whether a similar VM phenomenon exists in this neoplasm. We examined different grade astrocytoma using CD34-PAS dual staining to see if VM exists in this tumor. This article presents our findings.


    Materials and Methods
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 Materials and Methods
 Results
 Discussion
 Literature Cited
 
Histological Classification
All formalin-fixed paraffin astrocytoma samples were drawn from the archives of pathology department, Cancer Center, Sun Yat-Sen University. The hematoxylin and eosin slides were reviewed by neuropathologist. World Health Organization (WHO) 2000 standard histological criteria were used for tumor classification and only astrocytomas were included in this study. The tumors consisted of 15 diffuse astrocytomas (WHO grade II), 15 anaplastic astrocytomas (WHO grade III), and 15 glioblastomas (WHO grade IV). Patients with mixed tumors were excluded from the study.

CD34-PAS Dual Staining
The PAS staining method used by Maniotis group to identify the matrix-associated vascular channels of uveal melanomas (Maniotis et al. 1999Go): tissues were stained with PAS, omitting hematoxylin counterstaining to reduce visual noise; black and white photography with a green filter further highlighted the PAS-positive patterns.

Because the endothelium-lined vessels also show positive reaction to PAS staining, which could be confused with VM, to distinguish between the PAS-positive network of endothelium-lined vessels and VM, the tissue slides were immunohistochemically processed with CD34 before the PAS staining to identify endotheliums. Hematoxylin counterstaining was restored to get clear histology discrimination. The VM PAS-positive pattern network was evaluated by the criteria of Folberg et al. (2000)Go.

We have compared the utility of F-VIII, CD34, and CD31 in determining endothelium in astrocytoma and found that CD34 results in a more distinct labeling and easier determination of endothelium than do other markers. The comparison result in astrocytoma agrees with document in melanoma (Chen et al. 2002Go). Thus we selected CD34 in the present study as the endothelium marker.

For the CD34-PAS dual staining steps, paraffin sections were cut at 4–5 µm. Slides were deparaffinized using xylene and absolute ethanol, rinsed in distilled water, exposed to 3% H2O2 for 10 min at 37C, then put the slides in EDTA antigen-unmasking solution. The antigen-unmasking solution and slides were heated in an oven at 90C for 15 min. Slides were cooled to room temperature in unmasking solution. The sections were rinsed with PBS and blocked subsequently with 10% normal horse serum in PBS. Sections were stained with the CD34 primary antibody for 30 min, at 36.5C, then incubated for 20 min at 36.5C with biotinylated anti-mouse immunoglobulins in PBS. This was followed by incubation with streptavidin conjugated to horseradish peroxidase in PBS for 20 min, then by a brief rinse in PBS. Sections were exposed to diaminobenzidine tetrahydrochloride chromogen for up to 5 min, and rinsed in distilled water (all the reagents were bought from Zymed, South San Francisco, CA). To highlight the matrix-associated vascular channels of astrocytoma, after CD34 immunohistochemical staining, tissues were stained with PAS, counterstained with Mayer's hematoxylin for 1 min, and cover slipped with a permanent mounting medium. These sections were viewed under light microscopy to detect CD34 and PAS signals. Adult liver tissue was used as positive control and stained appropriately.

Vimentin-PAS Dual Staining
The composition of glioblastoma is heterogeneous, poorly differentiated tumor cells show vimentin expression. We have used Vimentin-PAS double staining to clarify if the channel is lined by tumor cells. Processed in EDTA antigen unmasking solution was not needed, the other step was the same as in CD34-PAS dual staining.

Immunohistochemistry
Glial fibrillary acid protein (GFAP), an astrocyte-specific protein, was used to confirm the tumor with PAS-positive pattern as astrocytoma. Hendrix et al. (2003a)Go reported the PAS-positive networks in melanoma were rich in laminin; therefore, we selected this antibody to identify the similarity between the VM PAS-positive networks in astrocytoma and melanoma.

Tissue sections were digested with trypsin in PBS at 1 mg/ml, for 10 min at 37C for antigen unmasking. The sections were covered with laminin primary antibody diluted at 1:50 and incubated overnight at 4C in NeoMarkers' monoclonal antibody, clone number LAM-89. Sections for GFAP (Zymed, monoclonal antibody, clone number ZCG29) staining was not digested with trypsin. The other steps were performed according to CD34 immunohistochemistry staining description. All sections were then counterstained with Mayer's hematoxylin, dehydrated, and cover slipped. Adult liver tissue was used as positive control and stained appropriately.

PAS-positive Pattern Evaluation
The astrocytoma slides processed with CD34-PAS dual staining were observed under light microscope. PAS-positive patterns were evaluated according to the standard of Folberg et al. (2000)Go. To obtain a clear discrimination, the green filter was not used in the microscope light pathway. Magnification 100x microscope was used to screen the PAS-positive pattern in the whole slide of 45 cases. The high-power (400x) microscope was used to discriminate the CD34-negative PAS-positive channel.


    Results
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 Materials and Methods
 Results
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 Literature Cited
 
CD34-PAS dual staining revealed that most of the intratumor vessels showed positive reaction for CD34 on the lumen internal surface and PAS positive reaction on vessel wall (Figure 1A) . In some astrocytoma, the PAS-positive wall of these vessels formed PAS-positive networks. However, it is easily distinguished from VM PAS-positive pattern under a light microscope: all of the vessels showed positive reaction for CD34 on the lumen internal surface.



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Figure 1

Light microscopic observation of astrocytoma. (A) Grade IV astrocytoma in mature vessel CD34 staining (buffy) could be seen in the internal lumen of vessels, periodic acid–Schiff (PAS) staining (pin) located at vessel wall (black arrow). (B) PAS-positive arcs (incomplete loops) are identified at the center of the micrograph, and a cluster of back-to-back complete loops is identified at the top right. Networks with at least three back-to-back loops could be seen. (C) Magnification of focus PAS-positive patterned network in (B). Focus of CD34 weak reaction (buffy) could be seen at the PAS-positive patterned networks (white arrow), PAS-positive CD34-negative channel was shown in the center of the picture, erythrocyte shadows could be seen in the channels (black arrow). (D) One of the PAS-positive patterned networks: parallel straight channels cross-link. (E) Vimentin-positive tumor cells lined out of the channel in the Vimentin-PAS dual staining slide (white arrow). Erythrocyte shadows could be seen in the channels, whereas endothelium could not be identified in the internal lumen surface. (F) Stained conventionally by hematoxylin and eosin. Erythrocyte could be seen in the channels (white arrow), endothelium could not be seen in the internal lumen surface. (G) Laminin in the basal lamina of blood vessels, patterned networks, and loops, with a reticular meshwork formation in the latter (black arrow). This picture was taken from the adjacent section of (F) and stained with laminin. (H) Glial fibrillary acid protein (GFAP) expression in the adjacent section of slide with PAS-positive pattern confirmed this tumor as astrocytoma. In this slide, some tumor cells lost the expression of GFAP (black arrow). (A–D) Dual staining (CD34-PAS-hematoxylin), (E) Dual staining (Vimentin-PAS-hematoxylin). (F) Hematoxylin and eosin. (G, H) Immunohistochemistry. Magnifications: A,C,E–H = x400; B,D = x200.

 
The CD34-negative PAS-positive channel was found in the VM PAS-positive pattern of two WHO grade IV astrocytomas. The VM PAS-positive pattern located in viable area of the tumor, far from necrosis, not within highly cellular areas. About one CD34-negative PAS-positive channel per high power field (400x) was found in the positive case.

CD34-PAS dual staining demonstrated the formation of complex networks and closed loops within the tumor, each loop surrounding smaller lobules or nests of astrocytoma tumor cells (Figure 1B). Arcs (incomplete loops) and a cluster of back-to-back complete loops was identified at the center of the micrograph (Figure 1B). Parallel straight channels with cross-links have also been found (Figure 1D). Tumors consisted of solid sheets of astrocytoma cells disposed in a predominant lobular growth pattern (Figures 1B and 1C). In networks of PAS-positive patterns, spots of weak reaction for CD34 were observed (Figures 1B and 1C). CD34 positive vessels were embedded in some area of the PAS positive pattern (Figures 1B and 1C). Erythrocyte shadows could be seen in some PAS-positive CD34-negative channels (Figure 1C). Vimentin-positive tumor cells lined out of the channel could be found in the Vimentin-PAS dual staining slide. Erythrocyte shadows could be seen in the Vimentin-PAS dual staining channels, whereas endothelium could not be identified in the internal lumen surface (Figure 1E). Stained conventionally by hematoxylin and eosin, erythrocyte could be seen in the channels with tumor cells lined out of the channel, whereas endothelium could not be identified in the internal lumen surface (Figure 1F). Laminin regularly stained the basal lamina of the normal vessels, intratumor blood vessels, and areas containing PAS-positive networks and loops. In the latter, laminin positivity produced a reticular meshwork pattern (Figure 1G). The tumor with PAS-positive pattern was confirmed by GFAP staining as astrocytoma (Figure 1H).

Interestingly, some astrocytoma cells showed CD34 expression (Figure 2A) , whereas some lost GFAP expression (Figure 1H). The case with CD34 expression on tumor cells was confirmed by GFAP staining as astrocytoma (Figure 2B).



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Figure 2

(A) CD34 periodic acid–Schiff dual staining showed positive expression for CD34 in tumor cells (black arrow) and blood vessel endothelium (white arrow). (B) In the adjacent section stained with glial fibrillary acid protein (GFAP), the tumor cell showed GFAP-positive expression, the proliferated vessels work as internal negative control (black arrow). Magnifications: A = x400; B = x200.

 
Limited necrosis was found in the tumor samples examined in the case with VM PAS-positive pattern.


    Discussion
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 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 
According to the criteria of Maniotis et al. (1999)Go, VM channels are patterned networks of interconnected loops of PAS-positive extracellular matrix that may be solid or hollow. In the latter, red blood cells could been detected. Endothelial cells are not identified within these matrix-embedded channels. On the basis of these criteria, VM channels were found in some of the astrocytomas examined here. Laminin reaction further identified the similarity between the VM PAS-positive pattern in astrocytoma and melanoma, and the GFAP expression characterized tumor cells as cells of astrocyte lineage. Lee et al. (2002)Go reported that extensive PAS-positive networks and loops were identified at the light microscopic level in oral malignant melanoma; many of these loops were confirmed ultrastructurally to represent intratumor microhemorrhages. Their study suggested that extravasation of erythrocytes into the melanoma tumor interstices can produce abnormal vessel-like spaces and, in this instance, also produce a pattern that emulates the interconnecting vascular channel loops and networks as formed by vasculogenic mimicry.

Maniotis et al. (1999)Go reported highly aggressive primary and metastatic tumor cells in in vitro reconstituted channels that were interconnected into the patterns seen histologically in tissue samples of metastatic melanoma. By contrast, poorly aggressive uveal melanoma cells were incapable of generating channels under identical culture conditions as the aggressive cell lines. Furthermore, highly aggressive and metastatic melanoma cells were frequently observed aligned outside the vascular channel wall. The differential ability of highly invasive and metastatic melanoma cell lines to generate patterned vascular channels (in comparison with poorly invasive melanoma cell lines) provided a biological basis for the histological appearance of patterned, matrix-lined vascular channels in melanomas that are not lined by endothelial cells.

Indeed, some studies based on scanning and transmission electron microscopy demonstrated that endothelial cells in tumor vessels are highly disorganized and loosely connected, with formation of intercellular gaps, transendothelial holes, vesiculovacuolar organelles, and endothelial fenestrae (Dvorak et al. 1988Go; Hashizume et al. 2000Go). We did blood–brain barrier ultrastructure observation of 18 gliomas (including astrocytoma WHO grade II [five cases], grade III [three cases], grade IV [three cases] and oligodendroglioma WHO grade II [five cases] and grade III [two cases]) and two brain-metastases originated from lung cancer under transmission electron microscopy. We have not found the phenomenon described previously in a viable area of these astrocytomas. In contrast, we found the perfect tight conjunction between endothelial cells: complete basement membrane. Endothelial fenestrae, intercellular gaps, and vesiculovacuolar organelles could not be found in 18 gliomas; however, quite a few endothelial fenestrae and vesiculovacuolar organelles could be easily found in two brain metastasis (Yue et al. 2000Go). The blood–brain barrier ultrastructure observation of these astrocytomas does not suggest the extravasation of erythrocytes through the abnormal endothelial cell and the secondary PAS- positive patterns formation after intratumor microhemorrhages formation. From light microscopic observations, erythrocytes (microhemorrhages) were not found in solid PAS-positive pattern network of VM, indicating that extravasation of erythrocytes inducing the secondary PAS-positive patterns formation as suggested by Lee et al. (2002)Go was not the case, at least in astrocytoma. In contrast, our study suggested it could be a special biological feature of the tumor cell to form the VM PAS-positive pattern.

Maniotis et al. (1999)Go demonstrated that the patterned channels generated by aggressive uveal melanoma cells in vitro were capable of conducting dye over a short distance. Maniotis et al. (1999)Go suggested that VM PAS-positive pattern in uveal melanoma was indeed a form of tumor microcirculation. In this study, erythrocyte shadows could be easily found in the PAS-positive CD34-negative channels, supporting that the PAS-positive pattern in astrocytoma were indeed a form of tumor microcirculation.

Maniotis reported the colocalization of CD34 to PAS positive pattern networks. The primary uveal melanoma tissue section showed CD34 colocalizes to the PAS-positive loops by staining the lumen contents rather than endothelial cells. The blood column also stained with multiple putative endothelial cells markers such as factor VIII–related antigen and Ulex, as well as CD31and KDR (Maniotis et al. 1999Go; Folberg et al. 2000Go). In astrocytoma, we have found erythrocytes in some channels of the PAS-positive pattern, but the CD34-positive signals around these erythrocytes or blood column was really weak. In areas of VM PAS-positive pattern networks, some CD34-positive signals locate close to the channel walls that were PAS-positive; the other CD34-positive signals form a loop or slit-like channel with thin PAS-positive channel wall. They look like the endothelial cells of small capillaries that link directly to the PAS-positive pattern channel of VM. The CD34-positive signals in the networks of PAS-positive pattern suggest the incorporation of VM channel and vessel with endothelium. In this study, location and morphology of CD34-positive signals do not suggest the CD34-positive signal in the network of PAS-positive pattern is the lumen content of VM channel.

Lee et al. (2002)Go reported that they found CD105 (an active endothelial cell-specific marker) positive expression in some melanoma cells, suggesting that active melanoma cells may potentially show endothelial cell-like characteristics, which results in VM. Some researchers (Maniotis et al. 1999Go; Bittner et al. 2000Go; Hendrix et al. 2001Go; Seftor et al. 2002Go) have described that aggressive melanoma cells were genetically deregulated cells and may express inappropriate markers not expected in cells of melanocytic lineage. It is therefore possible that the deregulated melanoma cells may lose expression of S100 protein or Melan-A as they acquire expression of CD34. For example, the expression of MiTF, a melanoma marker of differentiation, decreased in more aggressive cutaneous melanomas (Salti et al. 2000Go). In glioblastoma, the focus of tumor cells appeared with lack of GFAP expression. It is assumed that these foci reflect new clones of neoplastic astrocytes with additional genetic alteration (Fujisawa et al. 2000Go). In this study, some astrocytoma cells showed CD34 expression and some lost GFAP expression, suggesting that genetically deregulated tumor cells in astrocytoma could lose the astrocyte-specific protein GFAP and express inappropriate markers not expected in cells of astrocyte lineage, such as CD34. However, this phenomenon needs further investigation.


    Acknowledgments
 
This study was funded by the grants from Academic Development Program of Sun Yat-sen University (2002) and 211 program.


    Footnotes
 
Received for publication September 9, 2004; accepted February 1, 2005


    Literature Cited
 Top
 Summary
 Introduction
 Materials and Methods
 Results
 Discussion
 Literature Cited
 

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