EGFR Gene Overexpression Retained in an Invasive Xenograft Model by Solid Orthotopic Transplantation of Human Glioblastoma Multiforme Into Nude Mice

Figures & data

Figure 1  MRI and histopathologic features of human GBMs. MRI reveals irregularly and nonhomogeneously enhancing mass (black arrow) in the right hemisphere zone, and edema zone surrounding solid tumor sometime could be detected in contrast-enhanced T1-weighted imaging. Histopathologically, patient tumor morphology is mitotically active and includes pleomorphic cells, nuclear atypia, abundant microvasculars (black arrow), endothelial proliferation, and necrotic foci (HE staining).

Figure 2  Immunohistochemical analysis of EGFR protein expression in human GBMs. Compared with human normal brain tissues from surgical decompression without EGFR expression, the 4 human GBMs maintain the genetic property of EGFR overexpression. PBS instead of primary antibodies is used as negative controls.

Table 1  Subcutaneous and Intracranial Implantation: Xenograft Origin, Number of Mice and Testing Strategy

Patient tumor number	Subcutaneous xenograft line^a	Intracranial xenograft line^b	MRI^c	Histopathological/ molecular tests^d
1	1	1	10	20
2	2	2	10	20
3	3	3	10	20
4	4	4	10	20

^aSubcutaneous xenograft line 1, 2, 3, and 4 derived from corresponding patient tumor 1, 2, 3, and 4, respectively, and each patient tumor was xenotransplanted into 2 mice flanks with one graft per mouse.

^bIntracranial xenograft line 1, 2, 3, and 4 derived from correspondingly subcutaneous xenograft line 1, 2, 3, and 4, respectively, and grafts in each xenograft line were transplanted into 30 mice brain.

^c10 mice per intracranial xenograft line were scanned for brain MRI.

^d 20 mice per intracranial xenograft line were performed for histopathological and relevant molecular analysis.

Figure 3  Histopathologic features of flank xenografts. Compared with the human surgical materials, GBM xenografts in 4 different xenograft lines displayed pleomorphic cells, presence of mitotic activity, necrotic foci, and mild microvasculars (black arrow), but endothelial proliferation with multilayering of endothelial cells was not observed.

Figure 4  MRI and histopathologic features of intracranial xenografts. The 4 different intracranial xenografts generated from 4 corresponding flank GBM xenograft lines. MRI reveals irregularly and nonhomogeneously enhancing mass (black arrow) in contrast-enhanced T1-weighted imaging, which shared the similar MRI features with its corresponding human tumors. In HE-stained sections, the intracranial xenografts show mitotically active, high cellular density, poorly differentiated pleomorphic cells, necrotic foci, and some multinucleated cells, as well as mild or profuse microvessels (black arrow). Microvessels appear to consist of a continuous single layer of endothelial cells in CD34 immunostaining (black arrow), but endothelial proliferation is not observed compared with human tumors.

Table 2  Difference in Numbers for Necrotic Foci, Vascularization, Invasion and Percentages for EGFR Protein Expression Between 4 Different Intracranial Xenograft Lines

	Xenograft Line 1	Xenograft Line 2	Xenograft Line 3	Xenograft Line 4	p value
Necrotic foci	1.5 ± 1.6	1.2 ± 1.6	1.0 ± 1.3	1.4 ± 1.5	p > .05
Vascularization^a	6.2 ± 1.0	5.7 ± 0.8	6.1 ± 0.9	5.9 ± 0.9
Invasion^b	10.0 ± 1.4	9.7 ± 1.7	9.0 ± 1.8	9.8 ± 1.6
EGFR	69.8 ± 2.9	68.4 ± 2.6	68.5 ± 3.5	67.4 ± 1.2

^aVascularization was determined by counting the microvessel density in the intracranial xenografts.

^bInvasion was determined by counting the number of small satellite tumors surrounding the intracranial xenografts.

Figure 5  Highly invasive characteristics of intracranial xenografts. The 4 different intracranial xenografts generated from 4 corresponding flank GBM xenograft lines. Macroscopically, tumors (black arrow) grow up to the surface of ipsilateral cortex, blurring the border with surrounding normal host brain. In HE-stained sections, a large number of tumor cells can be seen migrating through the corpus callosum and extending into the opposite hemisphere, and the migrating tumor cells (black arrow) are clearly entering the normal host brain tissue, suggesting an invasive phenotype of intracranial GBM xenografts. EGFR immunostaining shows that single or clusters of tumor cells (buffy, black arrow) are infiltrating the surrounding normal host brain parenchyma.

Figure 6  Immunohistochemical analysis of EGFR protein expression in intracranial xenografts. Compared with normal mice brains tissues without EGFR expression, the 4 different intracranial xenografts generated from 4 corresponding flank GBM xenograft lines contain overexpressed EGFR protein. Compared with Figure 2, the intracranial xenografts retain the genetic property of human EGFR overexpression. PBS instead of primary antibodies is used as negative controls.

Figure 7  EGFR transcription and expression in xenografts and its original tumors. Compared with human tumors, the overexpressed EGFR gene is retained in the GBM xenografts of 4 different lines by RT-PCR and western blot analysis. No template for RT-PCR is as negative control. Brain tissues from human specimens of surgical decompression without EGFR expression are as normal control.