Antiangiogenic Therapy for Glioma

Table 1

A critical summary of the major in vitro and in vivo models to study tumor angiogenesis.

Cellular models	Characteristics	References
Human tumor cell lines	There are a total of 60 cell lines representing nine distinct tumor types. However, this model does not reflect the complexity of the real tumor environment.	[6, 7]
Multicellular tumor spheroids	Genes associated with cell survival, proliferation, differentiation, and resistance to therapy are differentially expressed in cells grown as multicellular spheroids versus 2D cultures.	[8–13]
	The capacity for spheroid outgrowth in 3D matrices is an interesting parameter to study the migratory behavior of tumor spheroid cells; however, this parameter can only be used for rapidly migrating cells (e.g., glioblastoma spheroids).
	Endothelial cell spheroids are increasingly used for evaluating the pro- and anti-angiogenic potential of drugs.
	Cospheroids of HUVEC and human fibroblasts are used for angiogenesis studies.
	Tumoral spheroids cocultured with endothelial cells potentiate tumor angiogenesis by upregulating proangiogenic factors that are absent in multicellular tumor spheroids alone or in monolayers.
	Another advantage is the possibility to use tumor spheroids from biopsies. This is useful for the study and development of patient-specific therapies and for the presence of tumor-initiating cells and tumor progenitors stem cells in tumor spheroids.
Xenograft models	Characteristics	References
Chicken chorioallantoic membrane tumor assay	The CAM tumor model could allow for a prescreening of drugs and subsequently reduce the number of animals used for in vivo experiments. This model is much faster than animal models. Histological analyses of the CAM tumors revealed a well-organized tumor tissue that strongly resembled clinical specimens of human tumors. The CAM model allows for the formation of tumors comparable to patient samples, with a degree of fidelity to human disease that is impossible to achieve with other nonanimal models; it combines the advantages of an in vivo environment with the simplicity of an in vitro experiment
	The duration of the follow-up period is limited due to the hatching of the chick 21 days after incubation.	[14]
RG2 and F98 rat cell lines	Tumors were produced by Wechsler in Koestner’slaboratory by the i.v. administration of a single dose of ethyl-nitrosourea (50 mg/kg b.w.) to a pregnant CD Fischer rat on the 20th day of gestation. The isolated clones retain individual characteristics, including the differentiation status, despite repeated propagations in vitro, elevated mitotic index and an increased nuclear-cytoplasmic ratio consistent with glioma cells in culture. When injected, these tumors have been refractory to chemotherapy and radiotherapy and adaptive to immunotherapy and exhibit an infiltrative pattern of growth within the brain. These characteristics closely resemble those of human glioblastoma.		[15]
Subcutaneously implanted human tumor xenografts	Tumors obtained from the direct implantation of the human cell lines or patient tumor biopsies are models that allow the monitoring of tumor growth. However, growth can be too slow; in xenografted models, the microenvironment and host immune responses are altered, and this may influence the tumor response.	[16, 17]
Orthotopic xenograft models	This model mimics the morphology, growth characteristics of clinical disease and metastatic processes more efficiently. There are several studies that report differences in the therapeutic responses between subcutaneous and orthotopic models.	[18, 19]
J3T-1 and J3T-2 orthotopic mice and rat models	The traditional orthotopic models for brain tumors did not aggressively invade healthy brain tissues; for this reason, we do not have an ideal GBM animal model that incorporates all of the human GBM features. Spontaneous canine glioblastoma approximates the human disease characteristics. However, it is not trivial to study a large number of spontaneous canine glioblastomas. The orthotopic xenograft implant of the two GBM cell lines, J3T-1 and J3T-2, into immunosuppressed mice and rats histologically recapitulated two invasive and angiogenic phenotypes: angiogenesis-dependent and angiogenesis-independent invasion observed in human glioblastoma.	[20, 21]
Spontaneous/genetic models	Characteristics	References
Pten-, Rb1-, Tp53-deleted mice	The HGA murine models with Pten, Rb1,  or Tp53 deletion are relevant to human disease, reflecting a spectrum of tumor histology and molecular features. Thus, molecular and other complex processes including specific contributions of the tissue microenvironment, such as tumor angiogenesis, can appropriately mimic human disease in these spontaneous tumor models.	[22, 23]
VM-M3 spontaneous tumors of the VM mouse strain	The inbred VM mouse strain is unique in exhibiting a relatively high incidence (1.5%) of spontaneous brain tumors. The VM-M3 brain tumor arose spontaneously in the forebrain of a VM mouse and expresses properties of microglia/macrophages similar to that seen in several types of invasive cancers of neural origin. Similar to high-grade human gliomas, the VM-M3 tumor cells, highly invasive, can be grown in the syngeneic VM mice with reproducible growth rates and have genetic similarities to human GBM. In addition, the tumor cells are labeled with the firefly luciferase gene allowing for noninvasive detection and quantitation of tumor growth.	[24]
Canine spontaneous glioma	GBM is the most common primary brain tumor in dogs and brachycephalic breeds such as Boston terriers and Canine Boxers are genetically predisposed to develop these tumors. Spontaneous gliomas may provide a valuable large animal model for the investigation of novel delivery and therapeutic strategies for intracranial tumors. The presence of pseudopalisading necrosis and endothelial proliferation that closely resemble those found in human GBMs suggests the presence of a hypoxic environment in canine GBM. The large size of the canine brain compared to the rodent brain would be more useful for preclinical assessment of doses, comprising more relevant volumes needed to implement novel therapies. However, spontaneous GBM in dogs is not a tumor model that is as easily accessible as the rodent GBMs. These models have variable penetrance, resulting in lack of synchrony in tumor development. The variability in time to progression represents a limitation in its use for drug testing.	[20, 25, 26]
F98 rat glioma	This tumor is produced by the administration of a single i.v. dose of ethylnitrosourea to a pregnant rat. It has been classified as an anaplastic and undifferentiated glioma. It is refractory to chemotherapy and radiotherapy. This model is effective for the evaluation of survival rate. However there are serious limitations in directly applying data from rat tumor models to any clinical treatment for human brain tumor.	[15, 27]