In vivo study to optimize treatment schedule for the lung stereotactic ablative radiotherapy based on the real-time changes in tumor hypoxia

Abstract

Introduction: An optimal interval between the fractionation sessions of stereotactic ablative radiotherapy (SABR) has not been established. Understanding of temporal changes in tumor hypoxia can provide clues for the optimal fractionation intervals and potentially lead to improved therapeutic outcomes by SABR. In this study, we hypothesized that serial in vivo measurements of tumor hypoxia after a single high-dose irradiation in a mouse model of subcutaneous and orthotopic lung cancer would be done by [18F]-misonidazole (F-MISO) positron emission tomography (PET) and hypoxia-responsive element (HRE)-driven bioluminescence imaging and we also hypothesized that tumor hypoxia would return to pretreatment levels at 6 hours ~ 6 days after irradiation in murine lung carcinoma model. Methods: Syngeneic Lewis lung carcinomas were grown either subcutaneously in the back or orthotopically in the lung of C57BL/6 mice and irradiated with a single dose of 15 Gy to mimic SABR used in the clinic. Serial F-MISO PET imaging was performed before irradiation (day ‒1), at 6 hours (day 0), and 2 (day 2) and 6 (day 6) days after irradiation for both subcutaneous and orthotopic lung tumors. For F-MISO, the tumor-to-background activity ratio (TBR) was analyzed. Serial HRE-driven bioluminescence imaging was performed at 6 hours, 1 day, 2 and 6 days after irradiation. Pimonidazole fluorescent activated cell sorting (FACS) analysis and Hoechst 33342 vascular perfusion combined with immunostaining, were also performed to further explain the findings of F-MISO PET and bioluminescence imaging. Results: In subcutaneous tumors, the maximum TBR was 2.87 ± 0.483 at day ‒1, 1.67 ± 0.116 at day 0, 2.92 ± 0.334 at day 2, and 2.13 ± 0.385 at day 6, indicating that tumor hypoxia was decreased immediately after irradiation and had returned to the pretreatment levels at day 2 after radiation. Hypoxic signals were too low to quantitate for orthotopic tumors using F-MISO PET or HRE-driven bioluminescence imaging. Pimonidazole FACS analysis also revealed similar patterns, in which pimonidazole-positive cell populations were decreased immediately after irradiation followed by a recovery to the pretreatment levels. Using Hoechst 33342 vascular perfusion dye, CD31 and cleaved caspase 3 co-immunostaining, we found a rapid and transient vascular collapse, which might have resulted in poor intratumor perfusion of F-MISO PET tracer or pimonidazole delivered at day 0, leading to decreased hypoxic signals at day 0 by PET or pimonidazole analyses. Conclusions: F-MISO PET and HRE-driven bioluminescence imaging can measure temporal changes of individual tumor hypoxia not in the orthotopic lung tumor model but in the subcutaneous lung tumor model. After a single high-dose of irradiation in murine subcutaneous lung carcinoma model, the level of tumor hypoxia returned to the pretreatment level by 2 days. Our results also indicate that a single high-dose irradiation can produce a rapid, but reversible, vascular collapse in tumors.