In this study, we describe the novel VEGFR2 peptoid antagonist, GU81. GU81 binds to VEGFR2 with a binding affinity of 12 nM and displays potent in vitro biological efficacy (Figure 1B&1C). We tested the therapeutic efficacy of GU81, both alone and in combination with doxorubicin in the extensively characterized spontaneous and aggressive MMTV-PyMT model of breast cancer . While GU81 had little therapeutic efficacy when used alone, animals treated with the combination of GU81 and doxorubicin had decreased tumor burden (Figure 2A), significantly reduced tumor invasion (Figure 3), increased tumor fat content (Figure 4B), and a lower tumor growth index (Figure 5E) compared to animals from all other treatment groups.
We began therapy when mice reached 6 weeks of age, and at this time most of the mice harbored primary neoplasia that developed to the hyperplastic stage [14, 24]. In this case, the combination of GU81 + doxorubicin was able to delay tumor progression and prevent transition from a premalignant to an invasive phenotype. This striking delay in tumor progression can be attributed to our use of a "double-edged sword" to target tumor cells (doxorubicin) and endothelial cells (GU81). In using this approach we were able to achieve significantly better results compared to either agent alone. Although we do not see a decrease in vessel number following treatment with GU81 either alone or in combination with doxorubicin, GU81 did induce a decrease in total vascular area and vessel size (Figure 6). Tumor blood vessels are dilated, leaky, and inefficient at delivering both oxygen and chemotherapeutic agents to the tumor [reviewed in ]. Vessel dilation is decreased following treatment with GU81, which leads us to the hypothesis that GU81 may be effectively normalizing the vasculature, decreasing hypoxia, and increasing doxorubicin delivery into the tumor.
Interestingly, GU81 does not demonstrate in vivo efficacy as a single agent in the MMTV-PyMT model of breast cancer . This is consistent with our previous studies in the MMTV-PyMT model  and human data, which has shown that anti-VEGF therapy provides little clinical benefit as a single agent in breast cancer . In contrast, GU40C4, the parent compound, demonstrated single agent efficacy in the A673 Ewings' sarcoma xenograft model . GU81, however, does demonstrate in vivo efficacy as a single agent in the 4T1 syngenic breast cancer model, where it effectively reduces tumor size and MVD . There are several possible explanations for the variation in response to GU40C4 and GU81. Most notable is the difference in tumor model systems. The MMTV-PyMT model is a spontaneous model that develops in the mammary fat pad, which is highly vascularized. While the A673 model is a subcutaneous xenograft model that is highly dependent upon VEGF activity , such that even low doses of anti-VEGF agents have a striking effect on tumor growth . The 4T1 model is a syngeneic, highly metastatic inflammatory breast cancer model. Interestingly, recent work has identified VEGF as an autocrine survival factor for these cells under hypoxic conditions, which may explain the efficacy of GU81 as a single agent in this model . It is possible that a higher dose given for a longer period of time would be more effective at reducing microvessel density in MMTV-PyMT tumors. Furthermore, it is important to highlight that treatment with GU81 alone increased tumor VEGF expression in this model, which may explain its inability to control tumor burden as a single agent (Figure 6B). We decided to further investigate this increase in tumor VEGF expression and confirmed our in vivo results in an in vitro system (Figure 7C, D). Tumor cells in primary MMTV-PyMT tumors express low levels of VEGFR2 (data not shown), however it is difficult to argue that either VEGF or GU81 may be having a direct effect on tumor cell proliferation or survival as we see no change in either proliferation or apoptosis markers following treatment with GU81 as a single agent. Met-1 cells also express VEGFR2 and increased VEGF expression following treatment with GU81 suggests that there may be an intact negative VEGF:VEGFR2 feedback loop in these cells. Treatment with GU81 could inhibit such a negative feedback loop, resulting in increased VEGF expression.
Macrophage infiltration is associated with poor prognosis in a number of different tumor types, including breast cancer [30, 31]. After establishing that VEGF levels were increased following treatment with GU81 alone (Figure 6B), we decided to investigate what effect this increase has on macrophage infiltration, given that VEGF stimulates macrophage chemotaxis into the tumor microenvironment. GU81 increases macrophage infiltration as a single-agent , however, this effect is abrogated when GU81 combined with doxorubicin (Figure 6D). This increase in macrophage infiltration is puzzling as we and others have shown that anti-VEGF therapy can reduce macrophage infiltration in a number of pre-clinical models [9, 10, 23, 32, 33]. Additionally, we show that macrophages harvested from a tumor-bearing animal express both VEGFR1 and VEGFR2, whereas those harvested from non-tumor bearing mice are only VEGFR1+ [9, 23]. When VEGFR2 is expressed, it becomes the dominant receptor driving VEGF-induced chemotaxis and specific blockade of the VEGF:VEGFR2 interaction is sufficient to inhibit chemotaxis [9, 23]. The most plausible explanation for the observed increase in macrophage infiltration may be attributed to the detected increase in VEGF expression and we have not ruled out the possibility that GU81 may reduce macrophage infiltration, if given at a dose that could compensate for the increased VEGF expression. We also cannot rule out the idea that another unidentified cytokine may be contributing to this increased macrophage infiltration following GU81 therapy in this model.
Future studies to determine the maximum tolerated dose (MTD) of GU81 in the MMTV-PyMT transgenic breast cancer model are currently underway. Once this has been established, mice will be treated at the MTD for longer periods of time to optimize the efficacy of GU81 both as a single agent and a combination therapy. Furthermore, we are interested in the mechanism by which GU81 increases VEGF expression and macrophage infiltration in vivo and are currently investigating this phenomenon.