Vascular endothelial growth factor expression promotes the growth of breast cancer brain metastases in nude mice

Abstract

Patients with breast cancer brain metastases cannot be cured and have a poor prognosis, with a median survival time of six months after diagnosis, despite developments in diagnostic and therapeutic modalities. In large part the progress in understanding the biology of breast cancer brain metastasis has been limited by the lack of suitable cell lines and experimental models. The objective of this study was to develop a reliable experimental model to study the pathogenesis of breast cancer brain metastases, using intra-internal carotid artery injection of breast cancer cells into nude mice. Brain metastasis-selected variant cells were recovered after three cycles of injection into the internal carotid artery of nude mice and harvest of brain metastases, resulting in variants termed MDA-231 BR1, -BR2 and -BR3. The metastasis-selected cells had increased potential for experimental brain metastasis and mice injected with these cells had significantly shorter mean survival than mice injected with the original cell line. Brain metastatic lesions of the selected variants contained significantly more CD31-positive blood vessels than metastases of the non-selected cell line. The variants selected from brain metastases released significantly more VEGF-A and IL-8 into culture supernatants than the original cell line, and more VEGF-A RNA when cultured in normoxic conditions. Mice injected with MDA-231 BR3 into the carotid artery were treated with the VEGF-receptor tyrosine kinase inhibitor PTK787/Z 222584. Oral administration of the inhibitor resulted in a significant decrease in brain tumor burden, reduced CD31-positive vessels in the brain lesions and incidence of PCNA positive tumor cells, and increased apoptosis in the tumor, as measured by TUNEL labeling. We conclude that elevated VEGF expression contributes to the ability of breast cancer cells to form brain metastases. Targeting endothelial cells with a VEGF-receptor specific tyrosine kinase inhibitor reduced angiogenesis and restricted the growth of the brain metastases.

Mystery of the brain metastatic disease in breast cancer patients: improved patient stratification, disease prediction and targeted prevention on the horizon?

“…Preclinical studies demonstrated the stem cell-specific signalling by the Wnt and Notch pathways to be associated with the development of BM [38,39]. Angiogenic factors-the vascular endothelial growth factor (VEGF), angiopoietin-2 and chemokine receptor type 4 (CXCR-4)-have been identified as the specific drivers of BM developmental process [40][41][42][43][44][45]. Dysregulation of these factors helps cancer cells to disrupt the blood-brain barrier followed by their extravasation and migration into the brain parenchyma.…”

Breast Cancer Metastasis to the Central Nervous System

“…A rat model of leptomeningeal colonization of Her-2-overexpressing SKBR3 cells was reported, although it requires considerable small animal surgical skills to obtain leptomeningeal metastases in a high percentage of animals. 37 That report, and the carotid artery injections of MDA-MB-231 BR1 to BR3 sublines, 35 demonstrate that certain models are sufficiently robust to provide quantitation of therapeutic effects of compounds in preclinical analyses. It may be possible to use certain models not only for basic molecular biology but for preclinical drug development experiments.…”

“…Recently, the laboratories of Zhang and colleagues, 33 Yoneda and colleagues, 34 and Kim and colleagues 35 performed successive rounds of culture of isolated brain metastases and reinjection into animals to produce sublines with enhanced brain metastatic potential and/or increased selectivity for brain compared to other metastatic sites. For MDA-MB-231 cells, six rounds of selection resulted in the MDA-MB-231BR subline that metastasized with 100% frequency to the brain but was undetectable in other organs.…”

“…34 The MDA-MB-231BR cells exhibited similar tumorigenicity in the mfp compared to a similarly derived bone-seeking subline, but variations were found in production of parathyroid hormone-related protein and in responsiveness to transforming growth factor (TGF)-␤ and insulin-like growth factor (IGF)-1 in vitro. Three rounds of selection via carotid artery injection performed in the laboratory of Kim and colleagues 35 resulted in the BR1, BR2, and BR3 MDA-MB-231 sublines, which exhibited an increasing incidence of brain metastases (82 to 100% of mice) and decreasing times after injection for mice to became moribund (59 to 41 days). A comparable MDA-MB-231 subline, selected for lung colonization, did not show increased incidence of brain metastasis or shorter survival, indicating that the results were due to specific selection for brain colonization ability.…”

“…VEGF also modulated the permeability of the endothelial cells. The laboratory of Kim and colleagues 35 reported that the BR2 and BR3 sublines of MDA-MB-231 exhibited increased microvessel densities in vivo compared to the parental line. The brain-selective lines also produced higher levels of the angiogenic factors VEGF-A and IL-8 in vitro compared to the parent line.…”