We show that directed, vascular killing of tumor neovascular endothelium by use of short-range (~60–80 μm) cytotoxic alpha particles is an effective treatment modality that enhances subsequent chemotherapeutic efficacy. We describe a mechanism whereby 225
Ac-E4G10 induces vessel remodeling and leads to improved penetration of subsequently administered small molecules. A number of studies have shown the additive effects of anti-angiogenic compounds and chemotherapy in preclinical and clinical trials; however, the mechanisms to explain the results were unclear and controversial (10
). The data here suggest that the order of administration of the two types of agents, anti-vasculature and anti-tumor, is critical to successful outcomes and may have an important impact on the design of combination therapies for clinical trials.
Anti-angiogenic agents, including anti-VEGF or anti-VEGFR mAbs, have been studied extensively preclinically and bevacizumab has been successful when combined with chemotherapy. One mechanism that explains the additive effects observed with bevacizumab and chemotherapy is vascular normalization with remodeling caused by anti-angiogenic therapy, leading to improved drug accumulation. In mice, treatment with DC101, an anti-VEGF receptor-2 mAb, increased pericyte coverage, reduced microvessel density, reduced interstitial pressure and improved oxygenation, however, these studies stopped short of proving enhancements in drug accumulation (10
). Improved accumulation of TRITC-labeled albumin has also been demonstrated after bevacizumab, suggesting improved perfusion, and increased accumulation of subsequent topotecan has been reported by Dickson et al.
in neuroblastoma xenografts in mice (10
). However, the use of bevacizumab in mouse models is problematic as the antibody does not bind or neutralize murine VEGF-A (23
). While bevacizumab can effectively block VEGF derived from the implanted human tumor cells, it has been previously demonstrated that host-derived VEGF significantly contributes in a model-dependent manner, to the angiogenic process(24
). Thus, while improved drug accumulation or uptake in tumors has been speculated as a mechanism for anti-angiogenic therapies, little direct pharmacokinetic evidence for this phenomenon exists.
In contrast to anti-angiogenic agents, 225
Ac-E4G10 is a cytotoxic, vascular targeting agent that selectively binds to the neo-vasculature of tumors, depleting the endothelial cells within tumors, as well as their progenitors in the blood and bone marrow, but not the tumor cells themselves(9
). Endothelial progenitor cells (EPCs) have been shown to play a role in vasculogenesis within tumors, and vascular depletion, as well as the anti-tumor effects observed in our study, could be a result of specific killing of both endothelial cells within tumors and killing of EPCs. The relative contribution of the EPC population is an interesting question for future studies.
The specificity of E4G10 to the neovasculature allowed us to isolate the effects of anti-vessel therapy from other anti-tumor effects in our study, but may not, consequently, generalize to all anti-angiogenic therapies. However, like anti-VEGF therapies, 225Ac-E4G10 treatment also results in phenotypic vascular normalization without targeting a highly redundant angiogenic pathway. Thus 225Ac-E4G10 treatment has the potential to avoid some of the pitfalls observed with classical anti-VEGF pathway therapeutic drugs. However, further experiments are necessary to determine whether 225Ac-E4G10 treatment might be a second line alternative for anti-VEGF-resistant tumors.
Unlabeled E4G10 at >4000 times the dose of 225
Ac-E4G10 used here has some anti-tumor effect, but it did not at 10 times the dose we used (shown in control groups)(19
). In addition, alpha emitters on non-specific mAb were ineffective. These data show that 225
Ac-E4G10 was not operating by binding to, or blocking VE-Cadherin, but by delivering the alpha-emitter selectively to kill the targeted endothelial cells.
The importance of sequence of administration was evident when animals treated first with 225
Ac-E4G10 and then with LV/5-FU chemotherapy exhibited both decreased tumor burden and improved median survival as compared to animals receiving the inverse drug schedule. Histological examination of 225
Ac-E4G10-treated tumors showed smaller, less densely dispersed vessels. Additionally, vascular basement membrane (collagen IV) structures were present in the absence of endothelial cells with 225
Ac-E4G10 treatment, while control and chemotherapy treated tumors showed endothelial cells adjacent to the collagen scaffold, confirming that our treatment ablates endothelial cells(20
). These collagen IV structures have been implicated in the tumor revascularization that occurs once anti-angiogenic therapy is ceased by serving as a scaffold for the neo-endothelium(25
). Apoptosis was also higher in 225
Ac-E4G10 treated tumors, which could be a combination of endothelial cell death, tumor cell death as a consequence of endothelial cell death, or direct tumor cell killing, which might be expected given that the alpha-particle pathlength is several cell diameters(~60–80 m); the gross differences in tumor volume may be a consequence tumor cell death resulting from any of these phenomena.
Pericytes are known to contribute to the integrity of mature vessels, and when absent result in abnormal function and leakiness(26
). Morphologically, 225
Ac-E4G10-treated tumors showed significantly more pericytes surrounding smaller, less dilated vessels and higher pericyte coverage (pericytes per vessel) when compared to saline and LV/5-FU treatment. Interestingly, 225
Ac-E4G10-treated tumors showed a higher total number of pericytes adjacent to vessels, suggesting proliferation of intra-tumoral pericytes or recruitment of pericytes, rather than simply a product of immature vessel ablation. These histological results are consistent with what occurs during vascular normalization by anti-VEGF therapy.
Previous studies demonstrated that anti-angiogenic therapies have resulted in decreases in intratumor pressure, improved vessel function with decreased vascular leakiness, yet few have determined if there is improved penetration of the chemotherapy into the tumor upon vessel normalization(10
). Our findings show a novel and specific method for achieving vessel improvements in tumors. Furthermore, accumulation and homogeneity of tumor distribution of 111
In-DTPA by ex vivo
tissue harvest and autoradiographic analysis, and of Hoechst 33342 dye by immunofluoresence, were all improved in 225
Ac-E4G10 treated animals.
18FLT also entered tumors more effectively as measured by positron imaging and ex vivo tissue harvest in 225Ac-E4G10 treated animals than control. As 18FLT is metabolized by intracellular thymidine kinase, the total signal in tumors after treatment could be an amalgam of increased accumulation driven by 225Ac-E4G10–induced remodeling of tumor vasculature and increased metabolic activity in the tumors. If there is increased metabolic activity, this further supports the selectivity of the 225Ac-E4G10 for the vessel and not the tumor.
Since our drug results in vascular remodeling, improved perfusion, and improved small molecule drug accumulation within tumors, one might expect 225
Ac-E4G10 to cause a decrease in hypoxia as well. However, hypoxia levels of tumor sections by CAIX staining, were not significantly altered between treatments. Typically, we have found LS174T tumors to exhibit a much higher level of CAIX staining (at least 4 times more) than tumor sections derived from other cell lines that we have previously encountered (data not shown), and CAIX staining results may be a consequence of a high hypoxia baseline in these tumors such that more minor perturbations in hypoxia with 225
Ac-E4G10 would be masked if present. In addition, while anti-angiogenic treatment with inhibitors of the VEGF-pathway is hypothesized to cause vascular normalization and at least in one previously discussed setting improved drug uptake (14
), it is also frequently reported to actually increase hypoxia both in patients and in model systems (29
). This observation is often cited as a reason for the failure of these therapies and development of resistance and a more aggressive phenotype (33
). Our findings combined with results in the literature suggest that the effects of vascular changes on oxygen and drug transport can sometimes be decoupled. Given these differing data, it appears that our anti-vascular approach mimics certain effects on the vasculature previously observed with anti-angiogenic therapies—normalized vasculature and improved perfusion—while exhibiting no detectable effects on hypoxia.
In conclusion, 225Ac-E4G10 treatment potently killed neovascular endothelial cells, remodeled tumor vasculature without directly targeting an angiogenic signaling pathway, and improved the pharmacokinetics of subsequently administered small molecules in tumors, thus providing a mechanism for the improved therapeutic efficacy of combinations with chemotherapy. These observations also highlight the importance of therapeutic scheduling when employing agents targeting tumor vasculature.