drug sensitivity and metastatic ability in b16 melanoma cells

CLIN. EXPL. METASTASIS, 1991 VOL. 9 NO. 4 393-402

Drug sensitivity and metastatic ability in B16 melanoma cells

A. J A N G and R. P. H I L L ?

Division of Experimental Therapeutics, Ontario Cancer Institute, Department of Medical Biophysics, University of Toronto, 500 Sherbourne St., Toronto, Ontario, Canada M4X 1K9

(Received 27 March 1991; accepted 16 May 1991)

We have previously reported that highly metastatic cell lines derived from KHT fibrosarcoma (KHT 35L1) and B16 melanoma (B16F10) are more resistant to N-phosphonacetyl-L-aspartate (PALA) and methotrexate (MTX) than the parental cell lines. This correlation between drug resistance and metastatic ability suggested the possibility that both phenotypes might have arisen in parallel as a result of a similar mechanism. In this study, we examined this possibility by reproducing the selection procedure for B16F10 cells (by serial passage of B16F1 cells as lung nodules) and testing the cells at each passage for changes in resistance to PALA and MTX. The results confirm that serial passage of B16F1 cells as lung nodules (LP) selects for cells with increasing metastatic ability (100-fold after seven passages), but these ceils did not develop increased resistance to PALA and became more sensitive to MTX. For comparison B16FI cells were also serially passaged (six passages) as leg tumors (LT). These cells became slightly more metastatic (3-fold) than B16F1 cells maintained in tissue culture, and demonstrated a small increase in sensitivity to MTX, as in the LP lines. There was also an apparent increase in resistance to PALA. In no instance was there a parallel increase in drug resistance and metastatic ability indicating that these two phenotypes do not necessarily arise in parallel in this cell line.

Introduction Two characteristics of malignant cells, which have been associated with tumor

progression, are the abilities to metastasize and to develop resistance to anti-cancer drugs. Our previous studies indicated a possible correlation between the development of these two phenotypes [2]. In B16 melanoma, higher rates of generation of drug-resistant and metastatic variants were found in the more metastatic subline B16F10, than the parental cell line B16F1 [2,7]. In an unrelated cell line, K H T fibrosarcoma, it was also noted that a highly metastat ic clone, 35L1, generated metastatic and drug-resistant variants at rates higher than the parental line [3, 6]. We suggested that this correlation might be due to similar mechanisms being involved in the development of both phenotypes. A potential mechanism could be gene amplification, since the rates of generation of drug-resistant and metastatic variants were in the range of 10 -5 per cell per generation [2, 3, 6, 7], rates which are characteristic of gene amplification events [9]. Fur thermore in the case of drug resistance, the drugs used for testing were M T X and P A L A , where resistance is usually due to amplification of the dihydrofolate reductase (DHF R ) [1] and carbamyl phosphate syn thase-aspar ta te t ranscarbamylase-dihydroorotase (CAD) [20] genes, respectively.

tTo whom correspondence should be addressed. �9 1991 Rapid Communications of Oxford Ltd.

394 A. Jang and R. P. Hill

The current investigation was designed to examine further the possible correlation between drug sensitivity and metastatic ability. If a correlation exists between the two phenotypes (perhaps due to an underlying common mechanism), it might be expected that parallel increases in metastatic ability and drug resistance would be observed. Accordingly, we selected increasingly metastatic cell lines by serial passage of B16F1 cells as lung nodules following a protocol similar to that used in the original selection of B16F10 cells [5]. At each successive passage we tested the sensitivity of the cells to PALA and MTX. As a control for in vivo passaging, we also passaged B16F1 cells sequentially as leg tumors and tested them for metastatic ability and drug sensitivity at each passage.

Materials and methods

Tumor cell lines The B16 melanoma cell line (obtained from Dr I. J. Fidler) has been

characterized elsewhere [5]. B16F1 melanoma cells were maintained in nucleo- side-free, low folate (0-1 mg/1) o~-minimal essential medium (oL-MEM-) supple- mented with 10% dialyzed fetal bovine serum (D-FBS) and streptomycin and penicillin (100 mg/1). Cell lines obtained by serial passage of B16FI cells as lung nodules (LP) or leg tumors (LT) were maintained in the same medium. All tests with PALA or MTX were done using o~-MEM- plus 10% D-FBS.

Drugs N-phosphonacetyl-L-aspartate (PALA) was obtained from the US National

Cancer Institute (courtesy of the Drug Synthesis and Chemistry Branch). Methotrexate (MTX) was obtained commercially (Lederle, Pearl River, NY). The sensitivity of B16F1, LP, and LT cell lines to MTX or PALA was measured by plating the cells (up to 104 cells per 100 mm plate) in the continuous presence of the drug. The resistant colonies were counted 10 days later, after fixing and staining with methylene blue. The drug-containing media were made up freshly prior to each experiment. An aliquot from a stock solution of MTX was added to c~-MEM- to get the appropriate concentration for each experiment. The required amount of PALA was weighed out exactly and added to ol-MEM- for the experiments with the LP lines. Because of the variability observed from experiment to experiment (see Figure 3), in the experiments with the LT lines, a concentrated solution of PALA was prepared and the required amount of PALA was then added to c~-MEM-. PALA prepared in this manner resulted in less variability in the survival curves from experiment to experiment.

Experimental metastasis assay The metastatic efficiencies of the cell lines were measured using an experi-

mental metastasis assay. Cells (up to 5 • 104) in 0.2 ml of medium were injected into the tail vein of 8- to 12-week-old female C57BL/6J mice (Jackson Laboratories, Bar Harbour, ME). The lungs were excised 14-16 days later, fixed in Bouin's fluid, and then stored in 95% ethanol until scored. The number of nodules on the surface of the lung was counted with the aid of a dissecting microscope, and the metastatic efficiency expressed as the number of lung nodules per clonogenic cell injected (EME).

Drug sensitivity and metastatic ability 395

Serial passage of B16F1 cells The procedure for the isolation of increasingly metastatic cells from B16F1

was adapted from that previously described [5]. B16F1 cells (5 x 104) were injected into the tail vein of C57BL/6J mice. Two weeks later the lungs were removed aseptically and digested in an overnight digestion procedure [21]. The resulting cell suspension was put into a tissue culture flask for in vitro growth. The (tumor) cells (LPn, n is the passage number as lung nodules) were grown for 2 weeks in culture and then were tested for their metastatic ability and their resistance to MTX and PALA. At the same time, the cells were reinjected into fresh mice for the next passage (LPn+a). B16F1 cells maintained in tissue culture were used as control cells for each passage.

B16F1 cells were also serially passaged as leg tumors. Cells (2 x 105) were injected intramuscularly into the left hind leg of C57BL/6J mice. Two weeks later, the tumors were excised aseptically and digested with trypsin and DNAse [16]. The tumor cells (LT~) were tested immediately for their resistance to PALA or MTX, and for their metastatic ability, and they were reinjected into animals for the subsequent passage (LT~+I).

Results

Metastatic ability The results in Figure 1 show that by serially passaging B16F1 cells as lung

nodules, we were able to isolate cell lines which were increasingly metastatic, thus reproducing the results reported by Fidler [5]. We were able to select cells which were 100 times more metastatic than the starting population after only seven passages. Also shown in Figure 1 are the results for B16F1 cells passaged as leg tumors. There was an initial 3-fold increase in the metastatic ability with no further increase despite several passages intramuscularly. During these studies, the metastatic efficiency of the B16F1 control population fluctuated from 1 to 6 x 10 -4 lung nodules per clonogenic cell injected.

Sensitivity to M T X and PALA In addition to metastatic efficiency, the sensitivity of the cells to PALA and

MTX was measured at each passage. Survival curves for cells plated in the presence of different concentrations of MTX are shown in Figure 2. After the first passage in the lung, the cells became more sensitive to MTX and this sensitivity remained unchanged despite several more passages in the lung. When we plated LP cells in the presence of PALA, we found that there was considerable variability in response from experiment to experiment (Figure 3). This was probably due to the method of preparing the PALA solution for each experiment. Since the sensitivity of both the LP line and the control B16F1 population plated in the same experiment varied in a similar manner, there was no significant difference when the LP line and the B16F1 control, plated on the same day, were compared. This is illustrated in Figure 4A which shows the ratio of drug doses in each experiment which resulted in a survival level of 10% for the lung passaged and control B16F1 cells. The results for MTX have been analysed in the same manner and are also shown in Figure 4A. For comparison, the ratios of metastatic ability for the various lung passages (from Figure 1) are also given.


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Figure 1. Effect of serial passaging as lung nodules or leg tumors on the metastatic efficiency of B16F1 melanoma cells. The changes in metastatic efficiencies are shown by plotting the ratios of the EME of each lung (0) or leg (5) passaged line to the EME of B16F1 cells maintained in tissue culture.

The B16F1 cells which were passaged intramuscularly (the LT lines) were also tested for sensitivity to MTX and PALA. The results, shown as the ratio of drug doses to give 10% survival for the leg passaged and control B16F1 cells plated in the same experiment, are given in Figure 4B. For comparison the ratio of metastatic efficiencies of the LT lines (from Figure 1) is also shown. A slight increase in sensitivity to MTX and an apparent increase in resistance to P A L A occurred during the passaging.

Stability of the metastatic and drug-resistant phenotypes of the lung-selected cells was determined using the subline LP6 which was initially 30 times more metastatic than the control B16F1 population. This cell line had been frozen at the time of the initial test of metastatic ability. It was recovered from frozen stock and passaged continuously in culture for several months. The metastatic ability and sensitivity to MTX and P A L A were tested at various times during the passaging. The results indicate that all three phenotypes remained stable during this time (Table 1).

Drug sensitivity and metastatic ability

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Figure 2. Survival curves for cells from each serial passage of B16FI cells as lung nodules (LP) plated in various concentrations of MTX. The control B16F1 cells were maintained in tissue culture and the survival curve is plotted as the mean + SE. The hatched lines of the survival curves of the LP lines represent maximal values.

The spontaneous metastatic efficiencies of the B16F1 and the LP6 line were examined in a single experiment in which 2 • 10 5 cells were injected intramuscularly into groups of mice. When the tumors reached a size of 17 mm the animals were killed and examined macroscopically for metastases. Individual lung suspensions were prepared and the cells plated for assessment of microscopic disease. The tumors in the mice injected with LP6 cells reached the size of 17 mm/about 2 days before those in mice injected with B16F1 cells even though the doubling time of the two cell populations is not different in vitro. Two of four animals injected with LP6 cells showed evidence of spontaneous metastases while only one of four mice injected with B16F1 cells did so.

Discussion Metastatic variants have been shown to be generated and lost during the

growth of tumors [6 ,7] suggesting that unstable genetic events are involved. In general, studies have shown that genetic instability, as measured by mutation


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Figure 3. Survival curves of B16F1 control cells and the individual lung passages plated in different concentrations of PALA. The range of surviving fractions for the B16F1 controls is represented as the upper (B16FIU) and the lower (B16F1L) limits, and it is clear that the individual lung passages (LP) demonstrate the same range of sensivity to PALA as the parental cell line.

rates or amplification rates, is greater in tumorigenic (malignant) cells than normal or transformed cells [8, 14, 17]. The finding by Cillo et al. [2, 3] that highly metastatic clones derived from KHT fibrosarcoma and B16 melanoma cells were also more resistant to the drugs PALA and MTX is consistent with the hypothesis that increasing genetic instability of tumor cells leads to tumor progression [11]. Furthermore we have demonstrated that exposure to hypoxia [22] and other environmental stresses such as acidosis and glucose starvation [15] can transiently increase the metastatic ability of tumor cells while Rice et al. [13] have reported increased resistance to MTX and doxorubicin in cells exposed to hypoxia.

These results suggested the possibility that the genetic instability of tumor cells may result in the parallel development of drug resistance and metastatic ability. We addressed this possibility by selecting cells with increasing metastatic ability and examining their sensitivity to P A L A and MTX at each stage of the selection procedure. Our results, demonstrating increasing metastatic ability in


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Figure 4. Summary of the changes in metastatic ability and drug sensitivity of B16F1 cells passaged sequentially as (A) lung nodules or (B) leg tumors. The experimental metastatic efficiency (EME) (H) plots were calculated as described in Figure 1. The sensitivity of the LP and LT lines to MTX (O) and PALA (&) is illustrated by plotting the ratios of the drug dose required to give a survival level of 10% for the LP or LT lines to that required for the control B16F1 cells tested on the same day.

TABLE 1. Stability of the metastatic and drug sensitivity phenotype of the LP6 line. The drug sensitivity data are presented as the ratio of the drug doses required to give a 10%

survival level for the LP6 line to the B16F1 control tested at the same time.

Weeks in culture

EME Ratio of drug dose (10% survival)

MTX 6ALA

B16F1 3.8 (+ 2.2) x 10 -4 1.0 1.0 LP6 (initial selection) 1.3 x 10 .2 0.64 0-97 1 1.2 x 10 .2 0.80 1.2 4 1-2 x 10 .2 0.82 1.3 11 1.1 x 10 .2 0.82 1.3

EME = experimental metastatic efficiency.

the lung-passaged lines without parallel changes in drug sensitivity, imply that these two phenotypes arise independently in this cell line. During these studies, experiments examining the possible role of gene amplification in the development of the metastatic phenotype in B H K cells were reported. Consistent with


our results the authors were unable to find a correlation between the generation of drug resistance and metastatic ability [4]. Thus our previous observations [2, 3] of increased drug resistance of highly metastatic populations may have been coincidental, just as in the present experiments we observed an increase in sensitivity to MTX. Differences in response to drugs between primary tumors and different metastatic subpopulations have been reported [18] and the increase in sensitivity to MTX by the LP lines after the first passage as lung nodules may be due to the selection and enrichment of a clone that is more sensitive to the drug but also highly metastatic. B16F1 cells passaged intramuscularly also demonstrated an increase in sensitivity to MTX although the level of sensitivity was not the same as the LP lines.

The small apparent increase in resistance to PALA by the LT lines may not be a direct result of passaging in vivo. An examination of the data indicated a slight decline in resistance to PALA in the B16F1 cells during the course of the experiments which may be due to the length of time that the cells were in culture. In contrast, the LT lines retained a level of sensitivity that is similar to that of the starting B16F1 population. Furthermore the lines passaged through the lung did not demonstrate increased resistance to PALA.

The small initial rise in metastatic efficiency of B16F1 cells passaged as leg tumors may be due to adaptation of cells, which have been extensively cultured in vitro, to growth in the animal. The protocol of direct transfer from animal to animal without an intervening period of growth in vitro was specifically designed to address this possibility. A recent report by Volpe and Milas [19] showed that increases in metastatic ability cannot be achieved by repeatedly passaging cells from primary tumors as intramuscular tumors. Our failure to observe increasing metastatic potential in the LT lines supports the hypothesis that the increasing metastatic ability of the cells passaged as lung nodules is due to the selection for cells with this particular phenotype, and is not simply due to adaptation to growth in the animal [10, 12]. The stability of the phenotype of the selected population (LP6) during extended growth in culture is consistent with this interpretation.

In summary, we were able to select for increasingly metastatic cells by serial passage of B16F1 cells as lung nodules but not as intramuscular tumors. The resistance of the lung-passaged lines to MTX and PALA did not increase in parallel to the metastatic ability as we had predicted based on previous results which suggested that there may be a correlation between these two phenotypes. Our results suggest that the development of drug resistance and metastatic ability are independent events occurring in B16F1 cells, but whether there is a common mechanism involved is still unknown.

Acknowledgement This research was supported by grants from the Medical Research Council of

Canada and the Ontario Cancer Treatment and Research Foundation.

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drug sensitivity and metastatic ability in b16 melanoma cells

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