<div>Abstract<p><b>Purpose:</b> The success of enzyme/prodrug cancer therapy is limited by the uncertainty in the delivery of the enzyme <i>in vivo</i>. This study shows the use of noninvasive magnetic resonance (MR) and optical imaging to image the delivery of a prodrug enzyme. With this capability, prodrug administration can be timed so that the enzyme concentration is high in the tumor and low in systemic circulation and normal tissue, thereby minimizing systemic toxicity without compromising therapeutic efficiency.</p><p><b>Experimental Design:</b> The delivery of a multimodal imaging reporter functionalized prodrug enzyme, cytosine deaminase, was detected by MR and optical imaging in MDA-MB-231 breast cancer xenografts. Stability of the enzyme in the tumor was verified by <sup>19</sup>F MR spectroscopy, which detected conversion of 5-fluorocytosine to 5-flurouracil. The optimal time window for prodrug injection determined by imaging was validated by immunohistochemical, biodistribution, and high-performance liquid chromatographic studies. The therapeutic effect and systemic toxicity of this treatment strategy were investigated by histologic studies and tumor/body weight growth curves.</p><p><b>Results:</b> The delivery of the functionalized enzyme in tumors was successfully imaged <i>in vivo</i>. The optimal time window for prodrug administration was determined to be 24 h, at which time the enzyme continued to show high enzymatic stability in tumors but was biodegraded in the liver. Significant tumor growth delay with tolerable systemic toxicity was observed when the prodrug was injected 24 h after the enzyme.</p><p><b>Conclusion:</b> These preclinical studies show the feasibility of using a MR-detectable prodrug enzyme to time prodrug administration in enzyme/prodrug cancer therapy.</p></div>
To investigate the in vivo and in vitro properties of 99mTc when labeled to antibodies via one direct and one indirect method, the B72.3 and C110 IgG antibodies were radiolabeled directly via stannous ion reduction and indirectly via the hydrazino nicotinamide chelator and compared in vitro and in vivo. Antibody avidity (but not immunoreactive fraction) appeared to be independent of labeling methods for both antibodies. Following stannous ion reduction, antibodies were fragmented by denaturing SDS PAGE although only slight evidence of fragmentation was found in vivo. The direct label was instable to transchelation to cysteine and glutathione in vitro and in vivo. Following intravenous administration, urinary excretion of activity was threefold greater for the direct label and was almost exclusively labeled cysteine and glutathione. Significant differences in the biodistribution of 99mTc were also observed: liver levels were lower, kidney levels were higher and clearance of label from blood and tissues was faster for the direct label. At Day 1, tumor accumulation was threefold lower for the direct label although most normal tissues were also lower. In conclusion, when labeled to two antibodies by one direct method, 99mTc is unstable towards transchelation relative to one indirect method. These relative instabilities greatly influenced the biodistributions in mice and may influence the quality of images obtained in patients.
Abstract Tumor hypoxia triggers signaling cascades that significantly impact on biological outcomes, resulting in resistance to radio- and chemotherapy. Therefore, understanding the hypoxic response of tumors is critical. In this study, we investigated links between hypoxia and different metabolites, such as lactate/lipid and total choline (tCho), in a human breast cancer model by combining in vivo magnetic resonance spectroscopic imaging (MRSI) with ex vivo optical imaging. Human MDA-MB-231-HRE-Tdtomato breast cancer cells, which were genetically engineered to express red fluorescent Tdtomato protein under the transcriptional control of hypoxia response elements (HREs) under hypoxic conditions, were orthotopically grown in female athymic nude mice. Both 3-dimensional (3D) water-unsuppressed chemical shift imaging (CSI) to determine tumor shape, and water-suppressed 3D CSI to detect metabolites, was performed. Each tumor was removed and sectioned to obtain serial slices throughout the tumor. Before sectioning, the tumor was embedded in a gelatine block containing straight fiducial marker lines for 3D reconstruction of serial optical images. Bright-field and Tdtomato fluorescence images of the same field of view were acquired from each tumor slice on a microscope to visualize hypoxia. We performed 3D reconstruction and registration of MRSI and optical images of 4 tumors. All metabolites were 3D-reconstructed from MRSI data with a 0.4 ppm spectral window size. Bright-field images from each serial section were aligned to each other by rigid body transformation based on the locations of fiducial markers. The tumor's edges were then detected and interpolated to reconstruct the 3D shape of the tumor. Accordingly, the 3D hypoxic region in the tumor was also reconstructed given the inherent co-registration of bright-field and fluorescence imaging. Then, the 3D tumor shape and the corresponding 3D hypoxic region were registered to 3D MRSI images of water-unsuppressed signal along with 3D metabolite images. Regions where hypoxia overlaps with a given metabolite were measured. tCho, glutamate/glutamine, lipid/lactate CH3, lipid CH2 and myo-inositol were detected by MRSI. The overlapping region between those metabolites and hypoxia was 35.1%, 12.7%, 9.8%, 16.7%, and 23.7% of the hypoxic region, respectively. A large proportion of high tCho-containing and myo-inositol-containing regions colocalized with the Tdtomato fluorescence in hypoxic regions, indicating that hypoxia can up-regulate tCho and myo-inositol levels in this breast tumor model. Hypoxia-driven up-regulation of choline kinase and tCho was previously shown by us in a prostate tumor model. Combining 3D MRSI and optical imaging proved useful to delineate the effects of tumor hypoxia on MRS-detectable metabolites, some of which may, in the future, serve as surrogate markers for hypoxia. This work was supported by NIH R01 CA134695. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr LB-381.
