Reduction of Lung Metastases by Na[trans-RuCl4(DMSO)Im] is not Coupled With the Induction of Chemical Xenogenization

The effects of the treatment of tumor cells of MCa mammary carcinoma and TLX5 lymphoma with the ruthenium complex Na[trans-RuCl4 (DMSO)lm] for several transplant generations were studied on tumor growth and metastases formation. On TLX5 lymphoma cells, treatment was performed in vitro prior to in vivo inoculation of tumor cells in intact or immunesuppressed mice. Either considering tumor take and growth or its capacity to invade the brain of the inoculated hosts, Na[trans-RuCl4(DMSO)lm] did not induce any significant modification. Conversely, in mice with MCa mammary carcinoma, the in vivo treatment of tumor cells in immunesuppressed hosts caused a progressive increase of DNA activity and, starting from the 4th transplant generation, a significantly increased susceptibility of lung metastasis formation to a further treatment in intact mice. These data seem to suggest that Na[trans-RuCl4(DMSO)Im] does not induce chemical xenogenization of tumor cells nor its repeated treatment induces resistance in tumor cells. Conversely, it appears that Na[trans-RuCl4(DMSO)lm] may select a tumor cell population which maintains its capacity to metastasise to the lung but with enhanced sensitivity to the antimetastatic properties of this compound.


INTRODUCTION
The effects of a new generation ruthenium(Ill) complex, Na[trans-RuCl4(DMSO)lm], are different from those of cisplatin in that, unlike cisplatin that is equally active on primary tumor growth and lung colonies, Na[trans-RuCl4(DMSO)lm] is markedly effective only on spontaneous metastases (1)(2)(3). The selectivity of Na[trans-RuCl4(DMSO)lm] on lung metastases is marked also on advanced metastases and accounts for a significant prolongation of host's survival time, particularly in the experiments in which drug treatment is associated with surgical removal of primary tumor. This effect is not associated with any residual effect on primary tumor cells after treatment discontinuation whereas it tends to reduce the metastatic ability of the same tumor (3).
Either by means of vivo-vivo bioassays or by microscopical examination it appears that the growth of lung tumors is markedly reduced whereas the growth of the i.m. primary tumor is much less affected and histologically not detectable. These effects account for the prolongation of the survival time and for the cure rate observed and highlight the pharmacological properties of this compound for the control of solid tumor metastases, an effect that was shown to be similarly exerted also on advanced tumor metastases (4,5). Metastases represent the greatest obstacle to cures after surgery and/or radiotherapy in that they often show a low chemosensitivity to the available anticancer drugs (6). The lack of success derives from the fact that tumor metastases are always treated with drugs that have been specifically developed by studying their activity in reducing primary tumor growth rather than by examining their efficacy on the more selective metastatic population (7). The aim of the present investigation was that of examining the cumulative effects of repeated treatments for several transplant generations on primary tumor growth and lung metastasis formation using the MCa mammary carcinoma of CBA mouse. Tumor treatment will be performed in vivo either in mice immunesuppressed by DTIC or in intact hosts. Parallelly, in vitro treatments of TLX5 lymphoma cells for up to 13 transplant generations will be performed to ascertain the possible occurrence of chemical xenogenizing effects, similar to those caused by triazeno derivatives and nitrosoguanidine derivatives (8)(9)(10). This study will therefore Permanent address: Department of Biomedical Sciences, University of Trieste, via L. Giorgieri 7-9, 34127, Trieste Italy. E-mail: CALLERIO@univ.trieste.it. focus the attention on the possible intervention of antigenic modifications on tumor cells after treatment and on the susceptibility of treated tumor cells to further treatments in terms of modification of tumor growth and metastasis formation.

MATERIALS AND METHODS
Comlounds and treatments. Na[trans-RuCl(DMSO)lm] was prepared according to standard procedures (9,10). The compound was dissolved in 0.9% NaCI and was administered i.p. to mice in volumes of 0.1 ml/10 g body weight. The dose of Na[trans-RuCl(DMSO)lm] (100 mg/Kg/day), at the treated schedule used, is well tolerated by the treated animals and does not cause appreciable reduction of body weight gain vs untreated controls at the end of treatment.
MCa mammary carcinoma. The line of MCa mammary carcinoma of CBA mouse was obtained from the Rudjer Boskovich Institute, Zagreb, Croatia (11), and was maintained by biweekly passages of 10 s viable tumor cells into the calf of the left hind leg of CBA inbred female mice of 20fi2 g obtained from a locally established breeding colony. Tumor propagation for experimental purposes was similarly carded out using female mice 6-8 weeks old. Tumor cell suspensions were prepared from pdmary tumors of donors similarly inoculated two weeks before. In short, 2.5 g of freshly removed tumor was minced with scissors, finely dispersed in 20 ml Dulbecco's phosphate saline calcium and magnesium free (PBS) and filtered through a double layer of sterile gauze; after centrifuging at 250xg per 10 min, pelletts were resuspended in an equal volume of PBS and cell viability was checked by the trypan blue exclusion test: only cell suspensions with at least 55-60% viable cells were used.
Primary tumor and lung .metastasis evaluation. Pdmary tumors were measured by caliper and their weight estimated by the following formula: (n/6)ab [Equation 1], where a and b are two perpendicular axes (a<b) and assume tumor density equal to 1. The evaluation of the number and weight of lung metastases, spontaneously formed from the s.c. tumor implants, was performed after the killing of the animals by cervical dislocation. The number of lung metastases on the surface of the freshly removed lungs was counted by means of a low-power stereo microscope equipped with a calibrated grid. The weight of the metastatic tumor per mouse was calculated by determining the volume of each metastatic nodule by the formula of primary tumors reported above [ Equation 1]. Histolo.qical analysis. Pieces of primary tumor were collected immediately after killing of the animals and fixed in formalin. For light transmission observations, slices were stained with hematoxylin-eosin or with CajaI-Cailego mounted in Canada Balsam, and were observed in double blind with a Leitz-Orthoplan microscopy. TLX5 lymphoma. The TLX5 lymphoma line, originally obtained from the Chester Beatthy Research Institute, London, England, was maintained by weekly passages of 10 cells/mouse i.p. into CBA mice. For the experimental purposes, the tumor was collected from the peritoneal cavity of donor mice inoculated one week before, and washed twice with PBS. in vitro-in vivo bioassays. Tumor cell suspensions of TI_X5 lymphoma (10 s cells/ml) were kept in vitro at 37C for 60 rain under shaking in tissue culture tubes with 1.9 ml of PBS containing antibiotics (100 U/ml penicillin and 100 mg/ml streptomycin) to which were added 100 ml PBS containing the test compound for a final volume of 1.5 ml. At the end of the incubation, aliquots of 0.1 ml were injected i.p. into intact syngenic CBA mice of which survival time was recorded. Brain metastases. The determination of the occurrence of leukemic brain involvement was performed by means of a vivo-vivo bioassay. Briefly, whole brains of mice transplanted with TI_X5 lymphoma cells one week before, were aseptically removed after killing of the animal by cervical dislocation. Brains were subsequently transplanted s.c. in the flank of intact syngenic CBA mice by means of a sterile syringe with a 19x21 needle. The survival time of the transplanted mice gave an indirect measure of the amount of TLX5 lymphoma cells present in the transplanted brains (12,13). Cytofluorimetric analys!s. Propidium iodide staining was performed according to the procedure described by Krishan (14). Orange acridine staining was performed according to the methods of Dar-zynkiewicz (15). Statistical analysis. All data were subjected to statistical analysis by means of the computerized Student-Newmann-Keuls test or the t-test for grouped data. Statistical differences were accepted at the cut-off threshold of at least p<0.05. Groups B and C were further treated with the same dose and treatment schedule of Na[trans-RuCI,t(DMSO)lm]; group C was further processed as the initial treated group giving transplant generation 2 and so on. Pdmary tumor growth was measured on day 14 and lung metastasis formation was evaluated on day 25 after tumor transplantation.
The analysis of data obtained from all 8 transplant generations shows no significant modification of the response of primary tumor to Na[trans-RuCl(DMSO)lm], whereas lung metastasis formation was markedly affected starting from generation 4 onwards. A detail of the curve of pdmary tumor growth and of the reduction of spontaneous lung metastases is given in Figure 2, Panel B. The growth of MCa mammary carcinoma in untreated mice is regular at all transplant generations and is not statistically different of that of the intact untreated MCa mammary carcinoma, parental line in both intact and immunosuppressed mice. An example of the curves of tumor growth is given in Figure 2, Panel A. Days from tumor implantation  No modification of tumor cell shape, cell nucleus and staining was detected between untreated and treated mice. Furtheremore, no apparent difference of infiltrating leucocytes is noted between untreated or treated tumors or with increasing transplant generations.
Effects on TLX5 lymphoma. TLX5 lymphoma cells, treated in vitro with 10-M Na[trans-RuCl(DMSO)lm] for up to 13 transplant generations, do not show any reduction of tumor take and growth in both intact or immunosuppressed hosts ( Figure 5, Panel A). Similarly, no significant modification of the capacity of TLX5 lymphoma cells to metastasize to the brain was detected by means of a vivo-vivo bioassay of whole brains harvested from the test animals and aseptically transplanted into syngenic CBA intact mice ( Figure 5, Panel B). suggest such possibility, showing that the transplantation of tumor cells obtained by in vivo treated mice into intact syngenic recipients gave rise to a normal growth of primary tumor but to a pronounced reduction of lung metastasis formation. Therefore the present study was focused at evaluating the effects of the treatment for several transplant generations of the same tumor line on tumor growth and metastasis formation. TLX5 lyrnphoma, already shown to be a tumor line poorly responding to Na[trans-RuCl(DMSO)lm], was used because it offers good possibilities to highlight the occurrence of a chemical xenogenization similar to that described by the group of Fioretti (8,9). Despite 13 transplant generations in which TLX5 lymphoma cells were always exposed to 10M Na[trans-RuCl,(DMSO)lm] for hr no change of tumor growth and capacity to colonise the brain was detected. These data support the hypotesis that Na[trans-RuCl(DMSO)lm] does not induce new antigenicity to tumor cells and that lung metastasis formation of solid tumors are probably reduced by a mechanism different from xenogenization. In fact, data from a similar experiment performed with the solid MCa mammary carcinoma shows again that no apparent modification of tumor growth and of metastasis formation occur following in vivo treatment of tumor cells for up to 8 transplant generations. In the same model, however, it appears that the formation of spontaneous lung metastases becomes markedly more susceptible to the antimetastatic action of Na[trans-RuCl(DMSO)lm] after 4 transplant generations, as results by the examination of the response of the xenogenised tumor line to a further treatment with Na[trans-RuCl(DMSO)lm] in intact hosts. At the same time, it appears that the activity of DNA, but not that of RNA, in tumor cells markedly increased with the progression of the transplant generations.
Taken together these observations stress the already reported lack of cytotoxicity of Na[trans-RuCI(DMSO)Im] for tumor cells and suggest that the interaction of this compound with tumor cells does not induce resistence to the antimetastastic effect. Conversely it seems that the selection of a cell population with higher sensitivity to its antimetastastic action is unrelated to an appreciable modification of tumor take and growth in the syngenic hosts. ?2