Antiangiogenic approaches, such as by metronomic chemotherapy, may hold promise in the treatment of highly angiogenic gliomas. The ability to follow the effect of metronomic chemotherapy using markers that could be assessed repeatedly during therapy could be used to optimize metronomic schedules and guide the combination with other therapeutic modalities, such as radiotherapy.
We have systematically investigated the effect of weekly CPA treatments on the subcutaneous 9L tumor pO2 and growth over days. We sought to further take advantage of an increase in tumor pO2 to establish an optimized schedule for radiation therapy in the context of weekly antiangiogenic CPA treatments. The 9L tumors were hypoxic and a further decrease in the tumor pO2 with increase in tumor volume was evident in the control group. No significant effect of 4 Gy × 5 on 9L tumor growth indicates the radioresistant nature of these tumors. Treatment with four weekly doses of CPA led to a significant increase in the tumor pO2 which remained at an elevated level throughout the treatment. A similar increase in the tumor pO2 was also observed with two weekly treatments of CPA. These treatments also resulted in a relatively slow increase in the tumor volume as compared to the control for up to two weeks followed by significant tumor shrinkage in the subsequent weeks.
The increase in the tumor pO2
during metronomic CPA is likely due to vessel normalization resulting in an increase in blood flow to the tumor. A unique feature of metronomic cyclo-phosphamide schedule is that it has both a direct cytotoxic and antiangiogenic effect unlike most angiogenic inhibitors (15
). Both, the weekly CPA schedule (21
) and continuous CPA in the drinking water were shown to induce antiangiogenic activity (11
) and are thought to generate severe hypoxia or anoxia in tumors (27
). Unlike the continuous CPA treatment such as through the drinking water which maintained hypoxia (28
); we have earlier observed an increase in tumor oxygenation and drug uptake with two weekly CPA thus suggesting a functional improvement of tumor vasculature (10
A significant decrease in the VEGFR2 content in the 2CPA group occurred on day 14. These results are in agreement with earlier reports of an increase in tumor pO2
which coincided with an increase in the antiangiogenic markers (29
). The changes in the tumor pO2
were used to schedule radiotherapy when the tumors were well oxygenated or hypoxic to determine the therapeutic outcome. The irradiation of the tumors at the time of an increase in tumor pO2
led to a significant delay in the tumor growth as compared to the CPA and irradiation alone groups. Under most antiangiogenic treatments, the repetitive or continuous administration of angiogenic inhibitors prevents endothelial cells from recovering, thus leading to capillary ‘drop out’ and tumor collapse. In the case of weekly CPA schedule, such as ours, the increased CPA uptake and oxygenation has increased the radiotherapeutic sensitivity of tumor cells as well as endothelial cells leading to dramatic tumor shrinkage. The mechanism of similar effects on the tumor growth in 2CPA/Irrd/Oxic and 2CPA/Irrd/Hypoxic groups could be due to several factors such as inhibition of angiogenesis, inhibition of cell proliferation and stimulation of apoptosis. It is likely that the combination of such effects masked the effect of tumor hypoxia on radiotherapeutic response; this warrants further investigation.
A significant increase in the tumor pO2
was also evident in mice treated with 2CPA/Irrd/Oxic and 2CPA/Irrd/Hypoxic groups as compared to 2CPA alone. Several possible mechanism have been suggested for oxygenation post-irradiation such as reduced oxygen consumption by radiation-damaged cells, cell loss leading to tumor shrinkage and improved micro-circulation (31
). Nevertheless, these results confirm that the antiangiogenic effect of metronomic CPA will enhance tumor pO2
, thereby will provide a therapeutic window that could be exploited to merge it with other therapeutic approaches. However, it should be emphasized that the antiangiogenic effect is likely to vary with the drug used, dose, interval between doses, and also with tumor types. Using polarographic electrodes or hypoxic markers, a decrease, no change, and also an increase in tumor pO2
has been reported using various angiogenic inhibitors in different tumor models (32
In conclusion, EPR oximetry provides temporal changes in the tumor pO2
during metronomic CPA treatment of 9L tumors. Results indicate a significant increase in tumor pO2
i.e., a therapeutic window, during weekly CPA treatments. A significant delay in the tumor growth was achieved when metronomic chemotherapy was combined with fractionated radiotherapy. With non-invasive repeated tumor oximetry by EPR, it is now possible to systematically investigate the effect of antiangiogenic approaches on tumor pO2
in pre-clinical models and develop protocols that could potentially enhance therapeutic efficacy in a clinical setting. EPR oximetry is currently being tested for repeated tumor pO2
measurements in patients with peripheral tumors undergoing chemo and/or radiotherapy (18
). We anticipate that EPR oximetry will be a valuable tool to study the effect of various treatments on tumor oxygenation and use this vital information to optimize such treatments and guide its efficacious combination with other therapeutic approaches.