The overall goal of this project is to improve the methods of harvesting and radiolabeling blood cellular components, to study the kinetics and tissue distribution of the labeled cells, and to explore their diagnostic utility after reinjection. To date, the greatest clinical impact of imaging radiolabeled leukocytes has been the detection of foci of infection by labeled neutrophils, and in tumor localization by labeled lymphocytes. However, current methods suffer from serious technical difficulties including radioactivity leaking out of labeled cells, and cell damage from autoradiolysis of cell nuclei. We will continue the development of high specific activity radiolabeled (aminostyryl)pyridinium (ASP) membrane permeant dyes as agents with which to label mixed leukocytes for imaging sites of inflammation (131I, 129I, 111In, or 99mTc) and for labeling radiosensitive lymphocytes for in vivo tracking studies (131I or 111In). We have prepared and used radioiodinated membrane permeant dyes to label both lymphocytes and mixed leukocytes in high yield. Preliminary studies with the latter have demonstrated high localization in induced sites of inflammation in dogs. Since radionuclides are restricted to the cell membrane, labeled dyes will be less radiotoxic to lymphocytes than the radiopharmaceuticals used currently. By measuring changes in the migration pattern of rat T- lymphocytes labeled with increasing doses of radiolabeled dyes, we will determine the reduction in radiotoxicity compared to 111In-oxine. The ability of peripheral blood lymphocytes (PBLs) and tumor infiltrating lymphocytes (TILs) labeled with the new dyes to localize in tumors will be tested in EMT6 mammary carcinosarcoma tumors in Balb/c mice. The decreased radiotoxicity of the labeled dyes should allow higher doses of administered activity and thereby increase the information content of the images prior to adoptive immunotherapy. A now classic approach for loading fluorescent calcium-binding ligands into cells will be adapted for labeling leukocytes with 99mTc complexes. We will synthesize several ligands containing both a diaminedisulfide (N2S2) moiety and groups which can be hydrolyzed intracellularly. The overall lipophilicity of these new neutral 99mTcvO(N2S2) complexes will allow them to diffuse into leukocytes. Intracellular esterases will then convert the complex to a new charged 99mTcV(N2S2) complex thereby trapping the polar complex within the cell. Leukocytes labeled with the new agents will be tested in anesthetized animals bearing sodium urate induced gout and E coli. abscesses by diagnostic imaging techniques. Leukocytes labeled with these agents should provide improved images in regions of the body where leakage of 99mTc from 99mTc-HMPAO labeled leukocytes obscures the image. We will measure the effect of granulocyte colony-stimulating factor (G- CSF) on the uptake of radiolabeled leukocytes in induced sites of inflammation. If G-CSF increases neutrophil uptake in sites of inflammation, the procedure could ultimately be used to detect sites of infection in patients with leukopenia.
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