Interruption of the paracrine communication between tumor cells and tumor vasculature either through the blockade of vascular endothelial growth factor receptor-2 (VEGFR2) or through the neutralization of tumor-associated VEGF enhances antitumor activity of ionizing radiation1–11
. Although this concept is not new, the optimal scheduling of these two modalities in relation to each other remains uncertain. Preclinical animal studies1–3,5,6,10
as well as clinical trials4,7,8–11
have called attention to the possibility that depending on the sequence of the applied treatments, anti-VEGFR2 and anti-VEGF therapies combined with ionizing radiation can be synergistic, additive, have no effect, and in some cases, can even compromise the outcome of radiotherapy9,10
Total radiation doses, dose per fraction, overall treatment time as well as the amount of anti-angiogenic therapy in combined treatments can impact the outcome12
. However, timing and the sequence of therapeutic regimens are key factors, which ultimately dictate the outcome. For example, Williams et al.13
used ZD6474, a potent VEGFR2 inhibitor, to augment radiation therapy of non-small-cell lung cancer in concurrent and sequential regimens. The tumor growth delay after the sequential schedule was significantly enhanced compared to the concurrent treatment. Impaired tumor reoxygenation between fractions in the concurrent protocol was suggested as the reason for these differences. Authors concluded that the clinical efficacy of ZD6474 as the adjuvant to radiation therapy will strongly depend on the course of therapy. In contrast, in a related study Brazell et al.14
reported virtually identical tumor responses regardless of the treatment scheme.
Radiation alone can result in the intensification of the angiogenic processes15
and contribute to the direct up-regulation of the VEGF expression in cancer cells1
. This is a manifestation of the overall cellular response to stress associated with the induction of various genes, transcription factors and the activation of growth factors, and allied receptors. Tumor-associated host cells are attracted to cancer cells by VEGF and engage in a continuous exchange of molecular information with cancer cells, affecting tumor response to therapy, tumor invasion and metastasis. One of the outcomes of this complex communication is angiogenesis with VEGF as a dominant proangiogenic protein16–18
VEGF stimulates angiogenesis through cooperative mechanism involving VEGF-induced proteins in endothelial cells including osteopontin (OPN)19
. OPN has diverse functions such as cell adhesion, chemoattraction, immunomodulation, and upregulation of the endothelial cell migration induced by VEGF. OPN expression is also correlated with tumor hypoxia20
. Efforts of the past 25 years to develop VEGF-and VEGFR-targeted imaging probes are yet to produce clinically useful agents. Data suggest that surrogate markers are needed. Tumor-derived OPN shows positive correlation with VEGF and can trigger VEGF-dependent tumor progression and angiogenesis19,20
. The reported here studies were designed to evaluate if similar positive correlation between OPN and VEGF exist in irradiated tumors and to establish the validity of OPN as a surrogate marker.
Of the three recognized receptors for the VEGF family of ligands, the activation of VEGFR2 (KDR, Flk-1) is sufficient to elicit all proangiogenic, proliferation and survival effects associated with VEGF21–23
. The induction of VEGF by ionizing radiation is implicated in processes protecting tumor blood vessels and contributing to tumor radioresistance1,12,24
. VEGF mRNA levels in irradiated Lewis lung carcinoma persisted at elevated levels two weeks after irradiation1
. Anti-VEGF antibodies improved tumor response and this synergistic effect was attributed to the increased radiation-induced death of endothelial cells. Similar conclusions were derived from studies in glioblastoma cells, in which radiation-enhanced VEGF secretion was attributed to their increased radioresistance12,24
. These and other preclinical studies have shown that therapeutic benefits of radiation therapy may be greatly enhanced when used in combination with the inhibitors of the VEGFR2 pathway. Based on these findings several clinical protocols have been undertaken7,8,11,25–26
. Early results are mixed. The emerging sense is that because the mechanism of the VEGF-VEGFR2 interactions with ionizing radiation is only partially defined, the design of some clinical studies may have been suboptimal9,10
. A recent report on the effects of VEGF in several endothelial cell lines concluded that VEGF does not confer any significant level of radioprotection to these cells27
suggesting that not all anti-VEGF therapies target tumor vasculature as it was previously hypothesized.
Here we demonstrate that irradiation of xenografts in athymic mice is associated with the induction of VEGF and VEGFR2. We also show that the expression of OPN, which cooperates with VEGF in proangiogenic processes, is significantly induced in response to ionizing radiation and in its expression parallels levels of the host VEGF in tumor suggesting OPN as an excellent alternative marker. This study reports for the first time the significant upregulation of the VEGFR2 expression by ionizing radiation in cancer cells and its direct relationship to the therapeutic response. Observations reported here also contribute the model of angiogenic regeneration12,15,28
, wherein the radiation-induced expression of VEGF, its allied receptor and the downstream proteins are a factor in the failure of radiotherapy by intensifying the vascular regrowth.