As new anti-angiogenesis drugs enter the clinic for cancer treatment and an even larger number of candidates progress through preclinical and clinical development, it is important to obtain a better understanding of their effects on tumor blood vessel patency and their potential interactions with traditional cancer chemotherapies. In the current study, we investigated axitinib, an oral anti-angiogenic drug and potent and selective inhibitor of VEGF receptors-1, -2, -3, which has shown substantial antitumor activity in multiple preclinical models and in several clinical trials (17
). Specifically, we investigated how axitinib affects tumor blood perfusion in human prostate cancer PC-3 xenografts, and the potential impact of such changes have on the efficacy of axitinib in combination with the cancer chemotherapeutic prodrug cyclophosphamide. PC-3 tumor blood vessel patency was significantly reduced by axitinib, which substantially blocked tumor cell access to pimonidazole, Hoechst 33342 and 4-OH-CPA, the active, cytotoxic metabolite of cyclophosphamide, all delivered via tumor blood flow. Despite the unfavorable impact on tumor uptake of 4-OH-CPA, the antitumor activity of axitinib in combination with cyclophosphamide was found to be greater than that of either agent alone. The present study also suggests that the intrinsic vascularity of a given tumor may influence its responsiveness to angiogenesis inhibition, as PC-3 tumors, which are poorly vascularized, showed substantially greater sensitivity to axitinib treatment () than 9L gliosarcoma, which are highly vascularized (21
). Finally, studies with PC-3 tumors, and with 9L tumors expressing the cyclophosphamide-activating P450 2B11 (19
) revealed that the relative timing of axitinib and cyclophosphamide administration can significantly impact the antitumor activity of the combination. Thus, optimization of combination therapies will require careful consideration of the effect of intrinsic tumor vascularity and the diverse interactions that may occur between angiogenesis inhibitors and cytotoxic chemotherapeutic agents.
Multiple methods have been developed for measuring tissue oxygenation, including polarographic needle electrodes, fluorescent fiber probes (29
), electron paramagnetic resonance oximetry (30
), and hypoxia-specific dyes (31
). Pimonidazole, a 2-nitroimidazole derivative (HypoxprobeR
), is enzymatically reduced at low oxygen tensions and forms protein adducts, which can be visualized using specific antibody (32
). Pimonidazole is widely used for detection of hypoxia in vivo, including tumor hypoxia induced by anti-angiogenic drugs (4
). However, the present findings demonstrate that the reliability of pimonidazole as an indicator of tumor hypoxia can be compromised by drug-dependent reduction in tumor blood perfusion, which impacts the tissue penetration of pimonidazole itself. For example, untreated PC-3 tumors showed extensive hypoxia staining following i.p.
delivery of pimonidazole, which is consistent with direct measurements of oxygen tension in PC-3 tumors using polarographic electrodes (33
). Interestingly, this staining became undetectable after chronic axitinib treatment. Although certain anti-angiogenic drugs can increase tumor oxygenation by inhibiting tumor cell mitochondrial respiration (34
), this effect has not been reported for axitinib. Moreover, our monitoring of PC-3 tumor hypoxia with the endogenous hypoxia marker Glut-1, whose expression co-localized with pimonidazole staining in untreated PC-3 tumors and some other tumor types (35
), revealed an increase in tumor hypoxia following axitinib treatment. Perfusion with the fluorescent dye Hoechst 33342 revealed strong staining of discrete cell clusters surrounding patent blood vessels in untreated PC-3 tumors that was abolished by axitinib treatment. In addition, the PC-3 tumor content of liver-activated, blood-delivered 4-OH-CPA was reduced more than 70% by long-term axitinib treatment. Thus, the decrease in pimonidazole staining seen in axitinib-treated PC-3 tumors is not due to a decrease in tumor hypoxia per se
, but rather, results from the inability of the dye to efficiently penetrate into the tumor via blood circulation. Therefore, when strong inhibition of tissue blood perfusion may occur, exogenous hypoxia-sensitive dyes such as pimonidazole cannot be reliably used and alternative methods, such as staining with Glut-1 or other endogenous hypoxia markers, or direct pO2
measurements, should be employed.
Surviving blood vessels in PC-3 tumors are reduced in size following axitinib or axitinib-cyclophosphamide combination treatment, suggesting there is a more pronounced impact of axitinib on larger blood vessels. Similar decreases in tumor blood vessel size have been observed with other anti-angiogenesis drugs (5
). The anti-angiogenic effects of axitinib seen in PC-3 tumors were not manifested as a decrease in blood vessel counts, despite the pruning of dilated blood vessels and the significant drop in tumor blood perfusion revealed by three separate functional assays, namely, an increase in tumor hypoxia, a decrease in Hoechst 33342 staining and a decrease in 4-OH-CPA uptake. These observations support the conclusion that changes in tumor microvessel density should be considered carefully when assessing the anti-angiogenic response of drug treatment (37
). Moreover, although highly vascularized tumors are often studied as therapeutic targets for anti-angiogenic drugs (38
), the importance of tumor vascularity as a predictor of the therapeutic efficacy of anti-angiogenesis treatments is still unclear (40
). In the present study, complete growth stasis was observed when axitinib was used to treat PC-3 tumors, which are hypovascularized, whereas only a transient tumor growth delay was seen in axitinib-treated 9L tumors, which are hypervascularized, despite the large decrease in 9L tumor microvessel density and the increase in 9L tumor hypoxia that accompanies drug treatment (21
). Several factors may contribute to the differential responses of 9L and PC-3 tumors to anti-angiogenic drug treatment, including tissue of origin, oncogenic background and vascularity. In particular, the low vascularity of PC-3 tumors may increase tumor susceptibility to anti-angiogenic drugs, such as axitinib, which may suppress tumor blood flow to a level where tumor growth cannot be supported. In contrast, the high vascular density of 9L tumors may provide a reservoir of excess blood flow capacity, which makes complete arrest of tumor growth by angiogenesis inhibition alone more difficult to achieve. Differential responses to anti-angiogenic treatments have been described in a limited number of other hypervascularized vs. hypovascularized tumor models (41
). Further investigation of the impact of intrinsic tumor vascularity on the response to angiogenesis inhibition will be of great importance to the identification of suitable patient populations and the selection of treatment schedules for this new class of anti-cancer drugs.
Certain anti-angiogenesis treatments may normalize tumor vasculature and thereby facilitate the delivery of cytotoxic drugs and oxygen to the tumor for a limited period of time (8
) and thereby sensitize tumors to chemotherapeutic agents and radiation therapy (4
). In combination therapies where anti-angiogenic drugs alone have limited efficacy, cytotoxic chemotherapeutics are likely to dominate the antitumor response. Consequently, any decrease in tumor blood flow induced by an anti-angiogenic agent may compromise the efficacy of the combination therapy. For example, in the case of 9L tumors treated with metronomic cyclophosphamide, which as a monotherapy induces substantial tumor regression, combination with axitinib decreases tumor uptake of 4-OH-CPA and blocks cyclophosphamide-induced expression of endogenous angiogenesis inhibitor thrombospodin-1 in host cells, thereby interfering with metronomic cyclophosphamide-induced 9L tumor regression (21
). Similar decreases in drug or nonspecific IgG uptake have been observed in other axitinib-treated tumors, despite the morphological normalization of the tumor vasculature, which is indicated by decreased tortuosity, a closer association between endothelial cells and pericytes, and reduced vessel leakiness (28
In the present study of PC-3 tumors, however, despite an even greater decrease in tumor uptake of 4-OH-CPA following axitinib treatment, and the blocking of metronomic cyclophosphamide-induced host cell thrombospondin-1 expression in combination therapy-treated PC-3 tumors (data not shown), a significantly improved antitumor response was achieved with the axitinib-cyclophosphamide combination. Thus, the strong anti-angiogenic action of axitinib on PC-3 tumors, which was manifested as a substantial decrease in blood flow to a tumor that is already hypovascularized, dominates the overall antitumor activity of the combination therapy. Consistent with the strong intrinsic anti-PC-3 tumor activity of axitinib, PC-3 tumors displayed several responses to the axitinib- and the combination drug treatment not seen in the cyclophosphamide alone treatment group. For example, metronomic cyclophosphamide decreased the number of TUNEL-positive cells and increased the occurrence of enlarged cell nuclei in PC-3 tumors, while both axitinib monotherapy and the combination treatment substantially increased apoptosis and induced a population of cells with condensed nuclei. Thus, despite the negative impact of axitinib treatment on tumor drug uptake, the cytotoxicity conferred by the residual intratumoral 4-OH-CPA supplements the antitumor activity of axitinib, which collectively enhance overall therapeutic response. Similarly, when axitinib was added to fractionated radiation therapy, despite an increase in tumor hypoxia, which would be expected to decrease tumor cell radiosensitivity, the overall antitumor effect was increased (46
). Together, these observations suggest that for axitinib, and perhaps other receptor tyrosine kinase inhibitors as well, inhibition of tumor blood perfusion can itself elicit a substantial antitumor response. In such a case, the overall antitumor activity of the combination therapy will reflect the impact of angiogenesis inhibition and the response to tumor cell exposure to cytotoxic agents. Sub-optimal delivery of the cytotoxic drug may be more than compensated by anti-angiogenesis-induced tumor cell starvation, so that overall antitumor activity is still enhanced, despite the decrease in tumor cell exposure to the cytotoxic drug. This antitumor mechanism is likely to be most important in combination therapies utilizing potent anti-angiogenic agents, such as axitinib, and in tumors where a low level of tumor vascular patency can readily be achieved. By contrast, in tumors where the cytotoxic drug dominates the antitumor response, addition of an anti-angiogenic agent may decrease the overall antitumor effect, as exemplified by our earlier study with 9L tumors given axitinib in combination with metronomic cyclophosphamide (21
). Moreover, the present studies suggest that therapeutic responses may need to be optimized in a tumor type-related manner by appropriate adjustment of the schedules of anti-angiogenic and chemotherapeutic drug treatment.
Cytotoxic drugs induce severe damage to both tumor cells and tumor-associated endothelial cells (22
). The repair processes initiated during the drug-free recovery period that follows a cycle of chemotherapy could become another target for anti-angiogenic treatment in a combination therapy setting (2
). Presently, the addition of axitinib as an adjuvant following MTD cyclophosphamide treatment improved the overall antitumor response, as could be anticipated. Surprisingly, however, substantial improvement in the antitumor response to MTD cyclophosphamide was also seen when a short course of axitinib therapy preceded MTD cyclophosphamide treatment, in particular for 9L/2B11 tumors. The superior 9L/2B11 antitumor activity of this neoadjuvant axitinib treatment cannot be explained by a change in the intrinsic chemosensitivity of 9L tumor cells to 4-OH-CPA (21
), or by functional normalization of the tumor vasculature, since axitinib actually decreases tumor uptake of 4-OH-CPA. Timing-dependent antitumor effects are also seen when the anti-angiogenic drug SU11657 is combined with radiation therapy (47
), suggesting there are intrinsic, beneficial interactions between the two therapeutic agents under this neoadjuvant schedule. These interactions not only counterbalance the negative impact of the decrease in tumor oxygenation and the reduced exposure to 4-OH-CPA, they further amplify the intrinsic antitumor activity of anti-angiogenesis and/or cytoreductive regimens. Given that VEGF receptor-positive endothelial cells are the primary target of axitinib action, axitinib pretreatment could increase the chemosensitivity of tumor-associated endothelial cells in a manner that increases both vascular damage and tumor cell damage following cytotoxic drug treatment. Another possible explanation involves bone marrow-derived endothelial cell progenitors, whose mobilization is induced by MTD cyclophosphamide treatment (48
) but is blocked by anti-angiogenic treatments including axitinib (14
). It has yet to be determined if axitinib, when used as an adjuvant, retains its impact on endothelial cell progenitor mobilization.
In conclusion, the VEGF receptor tyrosine kinase inhibitor axitinib induced a significant but reversible decrease in PC-3 tumor blood perfusion, which was sufficient to arrest tumor growth. This potent anti-angiogenic effect appears to serve as the major antitumor factor in combination therapies applied to PC-3 tumors, where co-administration of cyclophosphamide further enhanced the overall antitumor response despite a substantial decrease in tumor uptake of the active chemotherapeutic agent. The relative timing of anti-angiogenesis treatment and cytotoxic drug administration was also shown to affect therapeutic activity, and is an important consideration for the development of combination therapies. Finally, the low intrinsic vascularity of PC-3 tumors may be an important factor in the response to angiogenesis inhibition, insofar as a further decrease in blood supply may render tumor growth unsustainable. Further understanding of the relationship between tumor vascularity and the clinical efficacy of anti-angiogenic drugs may provide important guidance in patient stratification and regimen selection in cancer treatment.