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Purpose: Metabolic changes induced by radiation therapy (RT) provide a biological measure of tumor response to treatment beyond anatomic changes. In this preclinical study we investigated these changes through longitudinal monitoring of tumor response to varying doses of RT with 18F-Fluorodeoxyglucose Positron Emission Tomography (FDG-PET) and Computed Tomography (CT), as well as bioluminescence imaging (BLI). We sought to establish the metabolic alterations in irradiated tumors at early timepoints following treatment. Materials & Methods: Human HT-29 colon carcinoma cells were inoculated subcutaneously on both sides of the back of nude mice. Tumors were grown for two or three weeks. One tumor per animal was treated with 10 or 20 Gy of RT on day 0. The first ten mice were imaged with both FDG-PET and CT on day -1, 1, 3, 9, and 14. The other 10 mice were imaged with BLI once a week during tumor growth and on the same days as the PET-CT study. These findings were correlated with histology. Results: At the beginning of treatment tumors exhibited low FDG uptake. The volume of the treated tumors (TT) determined by CT images, remained stable or decreased (for dose of 20 Gy), whereas the untreated tumors (UT) continued growing. Surprisingly, 9 days after RT, the FDG-uptake increased in the TT and remained low in the UT. We observed a clear decrease in BLI signal in TTs appearing also 9 days after RT. Conclusion: We have applied preclinical imaging and radiotherapy methods to study tumor response to treatment. The surprising rise in FDG uptake at day 9 after RT could be the result of several mechanisms including in inflammation, that still remains to be determined with immunohistochemistry. This study raises the possibility of FDG PET producing false negatives for early response. The preliminary data reported here will be used to establish future technical and biological approaches towards monitoring cancer therapies