In this series of experiments, we have studied the distribution patterns of Tc-labeled Corynebacterium parvum in normal and tumor-bearing mice. C57BL/6 mice were given i.V., i.p., or s.c. injections of 250 /ug of "'"Tclabeled C. parvum and killed at 10 min, 1, 4, and 24 hr. After i.v. administration, labeled organisms were distrib uted primarily to the liver, the lungs, and the blood (46% of injected dose), followed by the gastrointestinal tract, the spleen, and the kidneys (11%). Total recoverable radioactivity, which was defined as the percentage of injected dose that was recovered, ranged from 59% at 10 min to 15% at 24 hr. In contrast to this, TcS colloid, an inert particulate material, was localized almost entirely in the liver, and the amount recoverable remained constant over 24 hr. One hr after i.p. administration of Tc-labeled C. parvum, the gastrointestinal tract accounted for 27% of the injected radioactivity, followed by liver, blood, and spleen (12%). This was rapidly excreted between 4 and 24 hr, at which time only 12% of the injected dose was recovered. The skin accounted for 54.6% of the injected radioactivity 1 hr after s.c. injection, 6% 4 hr after s.c injection, and 0.8% at 24 hr after s.c. injection. Eight percent of the recoverable radioactivity was detected in the lymph nodes at 1 hr and by 24 hr this had increased to 50%. Total recoverable radioactivity was 68% at 1 hr, 31.6% at 4 hr, and 8.5% at 24 hr. The distribution of i.v.administered C. parvum in C57BL/6 mice that carried the B16 melanoma was similar to that in normal animals, although the amount localized in liver was somewhat less. The tumor itself accounted for 8.7% of the recovera ble radioactivity at 1 hr and increased to 39% at 24 hr. After intralesional injection, the tumor accounted for 25% of the injected radioactivity and 58% of the recoverable radioactivity at 1 hr, and this increased to 28% of the injected radioactivity and 85% of the recoverable radio activity at 24 hr. Although the immunopotentiating activity of C. parvum in tumor-bearing hosts may be attributable primarily to a generalized effect on the reticuloendothelial system, localization and retention of the organisms within the tumor itself also may have some relationship to its immunotherapeutic effectiveness. ' Supported by Grants CA 16503-03 and GM 22814-01 from the NIH. Presented in part at the Sixty-first Annual Meeting of the Federation of American Societies for Experimental Biology, Chicago, III., April 4 to 8, 1977 (23). 2 To whom requests for reprints should be addressed, at Department of Pathology and Laboratory Medicine, Mount Sinai Medical Center, Mil waukee, Wis. 53201. Received June 27, 1977; accepted September 27, 1977. INTRODUCTION Corynebacterium parvum has been used as an immunostimulatory agent in both experimental animals (16, 17, 20, 21, 25) and humans (14). Its effects on the immune system have been well documented (13, 16, 24), but until recently there were no data on clearance and tissue localization of the organisms. Dimitrov ef al. (8) and Scott and Milas (22) have reported recently on the distribution of 125l-labeled C. parvum in mice, and their data provide important informa tion on the in vivo fate of the organisms. Although distribu tion varied somewhat with the route of administration, C. parvum was localized primarily in the liver, the spleen, the lungs, and the gastrointestinal tract. Relatively small amounts of radioactivity were localized in the kidneys, the thymus, the lymph nodes, and the bone marrow. After i.v. administration to tumor-bearing hosts, radiolabeled orga nisms were found in the tumor itself, but the amount that was taken up appeared to be unrelated to its immunothera peutic effectiveness (22). The fact that the distribution of sodium [125l]iodide was completely different from that of 125l-labeled C. parvum indicated that the radioactivity was organism associated and not free. Parallel studies with fluorescein isothiocyanate-labeled C. parvum revealed dis tribution patterns that were qualitatively similar to those of 125l-labeled organisms (22). Sadler ef al. (19) have taken a different approach and labeled C. parvum by propagating the organisms in a medium containing [3H]TdR.3 The organisms were distrib uted primarily to the liver, the lungs, and the spleen follow ing i.v. injection and to the gastrointestinal tract following i.p. administration. Recoverable radioactivity initially was considerably greater than that observed with 125l-labeled organisms, but the decline as a function of time was similar. Although 125Iand [3H]TdR may be suitable for tracking C. parvum in experimental animals, there are problems asso ciated with these radiolabels in humans. These include a long physical half-life with the attendant radiation risk to the patient for both radionuclides, less than optimum im aging properties for 125I, and an inability to detect 3H by scintigraphic methods. ""'Tc, on the other hand, has a short half-life (f,/2 = 6 hr) and generates a high photon flux; it is therefore an Â¡deallabel for detection by scintigraphy. We have used 99lllTc as a cell label in a variety of murine model systems (1-4). Our experience suggested that this radionuclide also could be used as a label to track C. 3 The abbreviations used are: [3H]TdR, thymidine; PBS, phosphatebuffered saline [0.15 M NaCI, 0.0076 M Na,HPO4, and 0.0024 M Na.HPO, (pH 7.4)]; Â¡.I.,intralesional. 32 CANCER RESEARCH VOL. 38 on April 16, 2017. © 1978 American Association for Cancer Research. cancerres.aacrjournals.org Downloaded from parvum (5), and in this series of experiments we have stud ied the distribution patterns of "'"Tc-labeled C. parvum in normal and tumor-bearing mice and compared these to the previously reported experiments in which 125Iand [3H]TdR were used as labels. MATERIALS AND METHODS Mice and Tumor. C57BL/6 mice weighing approximately 18 to 20 g were obtained from the Hall Mammalian Genetics Laboratory, University of Kansas, Lawrence, Kans. The B16 melanoma was purchased from The Jackson Laboratory. Bar Harbor, Maine. The tumor was propagated by biweekly s.c. transplantation into C57BL/6 mice. Preparation of "'"Tc-labeled C. parvum and Sulfur Col loid. C. parvum CN 6134 (Lot PX 398) was generously provided by Burroughs Wellcome and Co., Research Trian gle Park, N. C. One ml of the original suspension (7 mg/ml) was sedimented at 400 x g for 15 min, washed 3 times with 40 ml of PBS, sedimented, prereduced by incubation for 10 min at 37Â°with 100 jug of SnCL, and then washed 3 times with PBS. Labeling was accomplished by adding 10 mCi of Na""'TcOj that had been eluted from a molybdenum-technetium generator (Mallinckrodt Chemical Works, St. Louis, Mo.) with 0.9% NaCI solution. After an additional 10 min incubation at 37Â°,the C. parvum was sedimented at 400 x g, unbound radioisotope was removed by 3 washings with PBS, and the final concentration was adjusted to 1000 /ug/ ml. We prepared "'"TcS colloid from a commercially available kit (Tesuloid, E. R. Squibb and Sons, New York, N. Y.) by adding 0.1 ml of Na99l"Tc04 (10 mCi) to sodium thiosulfate, acidifying it with 0.25 N HCI, and then boiling for 10 min. Localization of '9 Tc-labeled C. parvum and Sulfur Col loid. The distribution of "'"Tc-labeled C. parvum was deter mined in normal C57BL/6 mice at 10 min and 1, 4, and 24 hr following i.v. injection and at 1, 4, and 24 hr following s.c. or i.p. injection of 250 /ng of radiolabeled organisms suspended in 0.25 ml of PBS. The distribution of "'"TcS Organ Distribution of ^"'Tc-labeled C. parvum was determined following i.v. injection of 1 mCi of radiola beled colloid suspended in 0.25 ml of 0.85% NaCI solution. Distribution of C. parvum in tumor-bearing mice was deter mined 2 weeks after s.c. implantation of B16 melanoma by which time the tumor volume was approximately 4 cu cm. Mice were given either i.v. or i.l. injections of 250 /ug of "'"Tc-labeled organisms and killed after 1, 4, or 24 hr. In all of these experiments, animals were bled via the retroorbital sinus immediately before they were killed by cervical dislo cation. The cpm for total blood volume were calculated by multiplying by 11. Thymus, muscle (0.5 g), brain, lymph nodes, heart, spleen, stomach, intestine, kidneys, liver, lungs, and femora were removed, and localization was determined by means of a Searle/Analytic Model 1185 gamma scintillation counter. The amount of radioactivity in the skin was determined for a circular piece 1 cm in diameter. For animals given injections s.c., an estimate of the total amount localized in the skin was determined by multiplying by 84. This calculation was based on the as sumption that a 20-g mouse had a body surface area of 66 sq cm (10). All data were corrected back to f,,, the time at which the first sample was counted.