Accessing key steps of human tumor progression in vivo by using an avian embryo model

Abstract

Experimental in vivo tumor models are essential for comprehending the dynamic process of human cancer progression, identifying therapeutic targets, and evaluating antitumor drugs. However, current rodent models are limited by high costs, long experimental duration, variability, restricted accessibility to the tumor, and major ethical concerns. To avoid these shortcomings, we investigated whether tumor growth on the chick chorio-allantoic membrane after human glioblastoma cell grafting would replicate characteristics of the human disease. Avascular tumors consistently formed within 2 days, then progressed through vascular endothelial growth factor receptor 2-dependent angiogenesis, associated with hemorrhage, necrosis, and peritumoral edema. Blocking of vascular endothelial growth factor receptor 2 and platelet-derived growth factor receptor signaling pathways by using small-molecule receptor tyrosine kinase inhibitors abrogated tumor development. Gene regulation during the angiogenic switch was analyzed by oligonucleotide microarrays. Defined sample selection for gene profiling permitted identification of regulated genes whose functions are associated mainly with tumor vascularization and growth. Furthermore, expression of known tumor progression genes identified in the screen (IL-6 and cysteine-rich angiogenic inducer 61) as well as potential regulators (lumican and F-box-only 6) follow similar patterns in patient glioma. The model reliably simulates key features of human glioma growth in a few days and thus could considerably increase the speed and efficacy of research on human tumor progression and preclinical drug screening.

Figures

Fig. 1.

Growth characteristics of experimental glioma on the CAM. (a) During the first 2 days after implantation, a well defined, solid tumor forms, which progressively becomes vascularized the following days. (b) Tortuous angiogenic tumor capillaries are visible at the surface of a D7 tumor. (c) Cross section of paraformaldehyde-fixed glioma in situ with adjacent CAM at D7. Areas of the tumor periphery and the center (rectangles) at higher magnification show angiogenesis (Ang), necrosis (Nec), and hemorrhage (Hem). (d) Histological analysis of D7 glioma reveals the presence of nucleated chick erythrocytes in tumor capillaries. (e) Glioma growth on the CAM is paralleled by the development of a large peritumoral edema (arrows, asterisk denotes tumor). CAMs close to the tumor are six times more swollen than controls. [Magnifications: a, ×10 (bar, 1 mm); b and c, ×63; d, ×100; and e, ×2.]

Fig. 2.

Expression of VEGFR-2 during experimental glioma development. (a) During the second day after implantation, the tumor becomes vascularized from the base by CAM vessels positive for the transcript of VEGFR-2. (b) ByD4, the tumor is almost completely invaded by VEGFR-2-positive capillaries. (c) At D7, overall VEGFR-2 reactivity decreases. (d–f). Higher magnification shows endothelial cells positive for VEGFR-2 (arrows). (Scale bars: a–c, 0.5 mm; and d–f, 0.1 mm.)

Fig. 3.

Immunohistological and confocal microscopy analysis of experimental glioma. (a) Frozen sections of D7 tumors were stained with antivimentin antibody (tumor cells, T), antidesmin antibody (pericytes, P), and fluoresceincoupled SNA-1 lectin (endothelial cells, E). Note the high tumor cell density, the irregular and dilated vessels (arrows), and pericyte coating of most capillaries. (b) Tumor capillaries form glomeruloid-like microvascular proliferations, covered by pericytes. (c) The presence of intercapillary pillars (arrows) in tumor capillaries shows intussusceptive angiogenesis. (d) Cooption of larger, dilated CAM blood vessels at the base of the tumor. (e) Flexion and orientation of a coopted vessel toward the tumor. (f) The presence of TN-C in the extracellular matrix of glioma identified by species-specific mAbs. (g) Lowpower magnification illustrates the invasive behavior of the tumor (arrows mark the borders of the CAM). (h) Individual glioma cells invade the CAM adjacent to the implantation site and migrate along blood vessels. (i) Experimental glioma do not exhibit apoptosis as revealed by physiological distribution of cytochrome c around cell nuclei (arrows). (Scale bars: a, d, and e, 100 μm; b and f, 50 μm; c, 20 μm; and i, 10 μm.)

Fig. 4.

Topical treatment of experimental glioma with receptor tyrosine kinase inhibitors Gleevec and PTK787/ZK 222584. (a) Biomicroscopy follow-up of control and treated tumors. Control tumors show progressive growth and vascularization, whereas Gleevec- and PTK787/ZK 222584-treated tumors appear white and show signs of tissue damage. (b–d) Confocal microscopy analysis of triple-stained tumors at D7. Tumor cells (T) are blue, pericytes (P) are red, and the endothelium (E) is green. (b) Control tumors show densely packed tumor cells within a well established pericyte-covered vascular network. (c) Gleevec-treated tumors show a strong decrease in tumor cell, pericyte, and vessel density. The increase in green fluorescence is caused by nonspecific lectin binding to necrotic cells. (d) In PTK787/ZK 222584-treated tumors, only a few pericyte-covered capillaries are found after treatment, and almost all tumor cells have disappeared. As in Gleevec-treated tumors, nonspecific green fluorescence increases. (e) Quantification of receptor tyrosine kinase inhibitor effects on glioma development. Bars indicate means of relative cell density and vessels counts. (Magnification: a, ×10; scale bar: b–d, 100 μm.)

Fig. 5.

Expression levels of four selected genes identified in the microarray screen were tested in patient GBM samples. Log-transformed expression levels (x-fold over ASII pool) are shown for each patient GBM. LUM, IL-6, CYR61, and FBXO6 follow the same expression tendency in GBM patients compared with AS experimental glioma on D4 compared with D2. Bar lines indicate median.