0022-534 7/88/1404-0844$02.00/0 THE JOURNAL OF UROLOGY Copyright© 1988 by The Williams & Wilkins Co. Vol. 140, October Printed in U.S.A. INFLUENCE OF TRANSPLANTATION SITE ON METASTATIC ABILITY OF MOUSE BLADDER CARCINOMA SUBLINES JAMES E. KLAUNIG* AND BRUCE A. BARUT From the Department of Pathology, Medical College of Ohio, Toledo, Ohio ABSTRACT The influence of the primary implantation site on the metastatic behavior of a murine transitional cell carcinoma line (MBT-2) and three metastatic sublines (L3 F1, L3 F2, and L3F3) was studied. The parent MBT-2 cell line produced a low incidence of lung metastasis after intravenous injection and no metastases from the primary tumor when injected either subcutaneously in the right hind flank or in the footpad. Intramuscular implantation of the MBT-2 cells in the right hind flank resulted in a significant increase over the subcutaneous, footpad, and intravenous sites in the incidence and number of lung metastases. Three in vivo/in vitro selected metastatic sublines (L3 F1, L3F2, and L3F3) were highly metastatic when injected subcutaneously, intramuscularly, and intravenously. A low number of pulmonary metastases was observed after footpad implantation of the three sublines. This study demonstrated a definite implantation site-influence on the metastatic ability of the parent MBT-2 line and the three selected sublines. Intramuscular implantation was the most permissive implantation site for the development of spontaneous metastasis for the MBT-2 line and the L3Fi, L3F2, and L3F3 sublines (J. Ural., 140: 844-847, 1988) Metastatic models employing in vitro propagated cell lines that are transplantable and metastasize in laboratory animals have been developed 13 ' 24 ' 25 and used to explore the biology, therapy and mechanisms of metastasis. 24 • 29 The anatomic site of transplantation of metastatic cells appears to play an impor- tant role in determining both if the tumor cells will metastasize and to what extent metastasis will occur. Several studies have found implantation site-dependent differences in metastatic rate and number oftumors. 8 • 9 • 11 • 12 • 18 • 19 • 26 - 28 These studies, how- ever, were limited to the comparison of differences in metastasis in two implantation sites or were conducted with the intent of finding a metastatic favorable site for the particular cell line being examined. Analysis of the metastatic behavior of cells injected at different anatomic sites is important in both the characterization of new metastatic cell lines and in the inves- tigation of the metastatic process. We have recently established a metastatic model for transitional cell bladder carcinoma in the C3H mouse. 5 Development of this model involved the isolation of sublines of heterogenetic metastatic ability from the parent MBT-2 cell line when administered subcutaneously in the mouse leg. 5 The present study was undertaken to com- pare the tumorigenic and metastatic behavior of these bladder carcinoma sublines when administered in four different ana- tomic sites (intravenous, subcutaneous, in the footpad, or in- tramuscular). MATERIALS AND METHODS C3H/He male mice, six to eight week sold, were purchased from Charles River Breeding Laboratories (Portage, Ml). Mice were placed five per cage in polycarbonate cages with corncob bedding, and given water and food (Certified Purina Lab Chow) ad libitum. Four cell lines, the parent MBT-2 and three sublines L3Fi, L3F 2 and L3 F3 were used in the study. The MBT-2 cell line was established from a F ANFT-induced transitional cell carcinoma previously maintained by serial passage of transplantable tu- mor in syngeneic mice. 17 The three sublines were derived Accepted for publication April 13, 1988. *Requests for reprints: Dept. of Pathology, Medical College of Ohio, 3000 Arlington Ave., Toledo, OH 43699. Presented in part at the 77th Annual Meeting of the American Association of Cancer Research, May 7-10, 1986, Los Angeles, Califor- nia. through three sequential in vivo/in vitro selections of pulmo- nary metastases. 5 The four cell lines were grown in 79s medium (a modification of Ham's F-12) supplemented with 0.1 mM calcium, 5% fetal bovine serum (FBS), and gentamicin (25 μg./ml.) as previously reported. 5 Cells were maintained in a humidified 3% C0 2-97% air incubator at 36.5C. All cell lines used for metastasis exper- iments were in the fifth culture passage since establishment. Cells for injection were trypsinized from 50% confluent cul- tures, centrifuged at 1000 g for three min., and washed in phosphate buffered isotonic saline three times. Viable cells (1 x 10 5 ) from each line were injected either 1) intramuscularly (i.m.) in the right flank, 2) subcutaneously (s.c.) on the dorsal surface of the right hind thigh midway between knee and hip, 3) beneath the skin of the dorsal surface of the right hind footpad (fp) or 4) intravenously (i.v.) into the lateral tail vein approximately two cm. from the base of the tail. Ten mice were injected at each site with each subline. Primary tumor growth was measured weekly by averaging the maximum tumor length, width and depth to determine a mean geometric diameter of the tumor. 27 Mice were sacrificed five weeks after injection of the tumor cells. At sampling, mice were fatally anesthetized with sodium pentabarbital, weighed and necropsied. Primary tumors were dissected from each mouse, measured and weighed. Visceral organs were removed, grossly analyzed for tumors, fixed in 10% buffered formalin, dehydrated in ethanol, and embedded in paraffin. Sections were cut, mounted on slides and stained with hematoxylin and eosin (Hand E). The number of lung nodules was determined both grossly and histologically. The influence of transplantation site on DNA synthesis in the primary and metastatic tumors was assessed. Four hours prior to sampling, all mice were injected i.p. with a single pulse of tritiated thymidine (one μCi/g.b.w.;50 Ci/mM). All mice were sampled at the same time of day to minimize diurnal variation. Deparaffinized sections of the primary and secondary tumors were prepared for autoradiography. The slides were coated with Kodak NTB-2 nuclear emulsion (Eastman Kodak, Rochester, NY), exposed at 4C for two to four wk., developed, and stained with H and E. Five hundred tumor cells per slide were counted and the percentage of labeled cells greater than five grains per nucleus over background was determined. Only cells from viable tumor areas were counted. 844