INT . J . RADIAT . BIOL.,

1975,

VOL .

27,

NO .

4, 377-387

The lung-colony assay : Extension to the Lewis Lung Tumour and the B16 Melanoma-Radiosensitivity of B16 Melanoma cells R. P . HILLf and JUDITH A . STANLEY Biophysics Department, Institute of Cancer Research, Clifton Avenue, Sutton, Surrey SM2 5PX, England

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(Received 14 .Yanuary 1975 ; accepted 6 March 1975) Experiments are described which demonstrate that a lung-colony assay can be used to study the viability of unknown cell populations from the B16 Melanoma or the Lewis Lung Tumour . It is shown that the number of lung colonies formed can be increased by the addition of plastic microspheres to the injected cell suspension or by pre-irradiating the lungs of the recipient mice . The colony technique has been used to isolate melanotic and amelanotic cell-lines from the B16 Melanoma which were found to have different growth-rates . In vitro radiation survival curves for B16 Melanoma cells have also been established, and these have parameters in the usual range for mammalian cells .

1. Introduction In 1969 a technique for determining the viability of KHT sarcoma cells by an in vivo lung-colony assay was described (Hill and Bush 1969) . This assay had the advantage that it took only 18-21 days, compared with 60-90 days for an end-point dilution assay (Kallman, Silini and van Putten 1967) . Since then such assay systems for experimental tumours have become more in demand in experimental chemotherapy and radiotherapy . For example, Bruce and Lin (1969) have described an approach to chemotherapy research which is dependent on having such assays available for a range of different tumours . This paper describes the extension of the lung colony assay for use with the Lewis Lung (L.L.) Tumour and the B16 Melanoma, both extensively studied in chemotherapy research, and compares these tumours to the KHT sarcoma . The use of the assay to determine the in vitro radiosensitivity of B16 Melanoma cells and to isolate in vivo melanotic and amelanotic variants from the tumour is described . 2.

Materials and methods

2 .1 . Tumours

The B16 Melanoma arose spontaneously in a C57BL mouse at the Jackson Laboratory (see Handbook on Genetically Standardized Jax Mice) and has been serially transplanted through many generations . Tumours were transplanted into female C57BL mice (18-21 g) by injecting about 10 6 tumour cells subcutaneously, and after two weeks they were used for experiments having reached a volume of 100-300 mm 3. The L .L. Tumour also arose spontaneously in a C57BL mouse in 1951 (Sugiura and Stock 1955) and has also been serially transplanted . It has been f Address for correspondence : Physics Division, Ontario Cancer Institute, 500 Sherbourne Street, Toronto, Ontario, Canada M4X 1K9 R .B .

3 c



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R. P. Hill

and J. A . Stanley

maintained by injecting approximately 10 6 cells subcutaneously into female C57BL mice . Such an implant reaches a size of 100-300 mm 3 about 12 days after injection .

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2.2.

Preparation o f cell suspensions

Tumours were excised and the capsules stripped off and discarded . The tumour material (1-3 g) was minced with iris scissors and washed twice with ice-cold PBSA (Dulbecco's PBS Solution A) . It was then resuspended in 25 ml PBSA containing 0 .2 per cent trypsin (Difco 1 : 250 Bactotrypsin) and 0. 01 per cent (w/v) DNAse (Sigma : crude from beef pancreas) . The suspension was incubated for 10 min in a 37 °C water-bath with gentle shaking, and after the pieces of tumour had settled, the fluid was discarded . A further 25 ml of PBSA with trypsin and DNAse was added, and the incubation continued with periodic shaking at 37°C (for 45 min for B16 Melanoma or for 20 min for L .L. Tumour) . After incubation the suspension was given four or five hard shakes to free cells loosened by the enzyme treatment and 2 .0 ml foetal calf serum and 1 ml of a 2 .5 mg/ml DNAse solution was added . The fluid was carefully poured off through a 200 mesh/in stainless-steel screen and centrifuged . The pellet was resuspended in 10 ml Eagle's basal minimal essential medium (BME) supplemented with 10 per cent foetal calf serum (ice-cold) by gentle pipetting . After further centrifugation the cells were resuspended in 5 ml of cold BME medium plus serum containing 0 . 25 mg DNAse to help prevent aggregation of the cells . Dilutions of the cell suspensions were then prepared, and cells which did not take up the dye erythrosin were counted in a haemacytometer. This method yielded 3-4 x107 B16 cells/g of tissue with non-staining cells being about 90 per cent of the total recovered, and 1-2 x 10' L .L. cells/g of tissue with 80-85 per cent unstained . This compares with a recovery of about 10 8 KHT cells/g of tissue with 95 per cent unstained, using the method described in the previous paper (Hill and Bush 1969) . 2.3 .

Assay procedure

After the counting of the cell suspensions the required dilution was made and, in most cases, a large number of heavily-irradiated (' 10 000 rad 60Co y-rays) tumour (HR) cells and plastic microspheres (15µm diameter 3M Company) were added to the viable cells . A small volume (0 .2 ml) of this suspension was then injected into one of the tail veins of each of about 7 mice, resulting in viable cells, 1-2 x106 HR cells and 10 5-10 6 microspheres (MS) being injected per mouse . About three weeks (18 days for the L .L. cells and 22 days for B16 cells) later, macroscopic tumour nodules had grown in the lungs . The lungs were removed and fixed in Bouin's fluid for 24 hours before being transferred to 95 per cent alcohol for counting and storage . The lobes were separated, and the total number of colonies per lung was counted using a dissecting microscope . 2 .4. Isolation o f melanotic and amelanotic variant sub-lines

B16 Melanoma cells were injected intravenously into mice which were sacrificed 21 days later . Colonies were then selected according to melanin content and were implanted, using a trocar, subcutaneously into recipient mice .



Lung-colony assay

379

These subcutaneous colonies were allowed to grow until they reached a suitable size (- 1 cm diameter), and one tumour was then used to prepare a cell suspension . Known numbers of single cells of the desired line were then injected intravenously into fresh mice . Two cell-lines were thus established and maintained through successive clonings . Colonies which were black or grey in appearance have been designated `melanotic ', whereas those which seemed, under a dissecting microscope, to have no pigmentation have been called ` amelanotic '. The relative numbers of melanotic and amelanotic colonies in the lungs were counted after each cloning for each cell-line . Int J Radiat Biol Downloaded from informahealthcare.com by Mcgill University on 12/14/14 For personal use only.

2 .5 . Irradiation technique

The irradiation of tumours in vivo or cells in vitro was carried out with 60 Co y-rays using a dose-rate of approximately 250 rad/min . For the irradiations in vivo the mice were irradiated to the whole-body and rotated by 180° half-way through the irradiation to reduce dose variations . For the lung irradiation, the thoracic cavity was exposed to 230 kV X-rays with the head and rest of the body shielded . Dosimetry in both cases was done with a Baldwin Farmer Sub-Standard Dosemeter . 3. Results 3 .1 . Development o f the assay system

Initial studies with the L .L. Tumour indicated that colonies had reached a suitable size for counting by 18 days after injection, and that when about 10 6 cells were injected per animal, only 15-20 colonies were obtained, giving a colony efficiency (number of colonies per non-stained cell injected) of approximately 10 -5 (see tables 1 and 2) . This is very much lower than the value obtained Number of cells injected

Additions

Number of colonies observed

Colony efficiency

8 .8 x 10 5 L.L . cells 4 .4 x 10 5 L.L . cells

1 . 6 x 10 6 HR 4 .4 x 10 5 MS

16 . 3 51 . 3

1 . 9 x 10-5 1 . 2 x 10-4

-

125 . 3 56 . 0 8 .5

5 . 2 x 10 -4 2 . 3x10-8 3 . 5x10-8

2 .4 x 10 5 B16 cells 2 .4 x104 B16cells 2 . 4 x10 3 B16cells

9x10 5 MS 9x10 5 MS

Table 1 . with the KHT sarcoma (2-4 x10-2) under similar conditions (10 3 unstained cells + 2 x 10 6 HR cells). Consequently, an attempt was made to raise this low colony efficiency . Firstly, 4 . 4 x 10 5 plastic microspheres were mixed with the viable cells before injection . This gave an increase in the colony efficiency by a factor of ten (see table 1) . Secondly, the lungs of the recipient animals were irradiated before the injection of the tumour cells . It has been shown by Brown (1973) that such an irradiation increases the colony efficiency of injected KHT sarcoma cells, and the results of a number of experiments using the L .L . Tumour cells are shown in figure 1 . In these experiments the lungs of the recipient mice were irradiated 24 hours before injection of tumour cells with or without



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3 80

R . P. Hill and J . A . Stanley

log0

500

1000

1500

2000

Dose (rad) of X-rays to lung of recipient

Figure

1 . The increase in colony efficiency for Lewis Lung Tumour cells as a function of radiation dose given locally to the lungs of the recipient animals . All the doses were given 24 hours before the injection of the tumour cells. In the case of the 500 rad dose, the irradiation was given to the whole body . Circles represent results of experiments in which microspheres were added to the injected suspension ; squares show results of experiments in which no microspheres were used .

admixed microspheres . It can be seen that the colony efficiency rises as a function of dose, such that for a dose of 2000 rad there is an increase of about a factor of ten, which is over and above that obtained with microspheres alone . Thus, by these two procedures, it was possible to raise the colony efficiency for the L . L. Tumour cells from 2 x 10 -5 to 2 x 10 -3. For the B16 Melanoma the initial experiments indicated that it was necessary to wait about 22 days after injection before counting colonies and that when viable cells alone were injected, the colony efficiency was about 5 x 10 -4 (see table 1) . The addition of 9 x 10 5 microspheres increased this to about 3 x 10-3 (see table 1) . If the recipient mice were given a dose of 500 rad to the whole body, 24 hours before the injection of cells plus microspheres, a further increase by a factor of three in the colony efficiency was seen, in line with the results shown in figure 1 . Experiments were conducted with both tumour systems to investigate whether it was possible to attain a constant level in the number of colonies that develop with time after the injection of a given number of unstained cells . The results of these experiments are shown in table 2, and they indicate that over the range of times studied there was no significant change in the number of colonies observed . There is a suggestion in table 2, that, for irradiated B16 Melanoma cells, the attainment of this constant level was delayed . A probable delay in growth for irradiated cells can also be seen in figure 2, in which distributions of colony-size are plotted for treated and untreated cells . The colony diameter was measured using a dissecting microscope with a graticule . Colonies from untreated cells



381

Lung-colony assay

Days after injection

Mean number of colonies (95 per cent C.L . calculated on colony count) Lewis Lung Tumour

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9 . 1 x 10 6 N .S .t cells injected

15 16 17 18 19 20 21 22

21 . 1 16 . 1 13 . 7 22 . 5 13 . 0

104 N .S .t cells+9 x 10 5 MS injected (recipient mice 500 rad 24 hours before injection of cells) 5.2 8.8 3.9 5.1

(12 . 2-30 . 0) ( 9 . 0-23 . 2) ( 0. 0-32 . 0) (10 . 3-34 . 7) -

(2 . 0- 8 . 4) (3 . 8-13 . 8) (0 . 1- 7 . 7) (0 . 0-10 . 3)

9 . 9 (5 . 9-13 . 9) 10 . 2 (3 . 4-17 . 0) B16 Melanoma

Untreated cells 2 x 10 3 N.S .t cells+ 1 . 2 x 10 6 HR cells+ 1 . 8x10 5 MS 18 19 20 21 22 23 24 25 26 27 28 32

2.0 5.2 2.3 1 .4 4.0

injected

(1 . 1-2 .9) (3 . 4-7 . 0) (1 . 4-3 . 2) (0 . 5-2 . 3) (1 .7-6 . 3)

Cells irradiated with 2000 rad in vivo 5 x 10 5 N.S .t cells+ injected 3 . 5x10 5 MS

1 . 6 (1 . 0-2 . 2) 2.7 4.2 3.3 6.6 4.1

3 . 8 (2 . 6-5 . 0)

(1 . 3-4 . 1) (3 . 5-4.9) (2 . 7-3 . 9) (5 .4-7 . 8) (3 . 5-4 . 7)

5 . 4 (3 . 6-7 . 2) 4 . 1 (3 . 3-4 . 9) 5 .0 (3 .2-6 . 8)

t N . S . = non-staining .

Table 2 .

gave a median diameter of 1 . 2 mm 22 days after injection, whereas colonies from cells irradiated in vivo with 2000 rad reached a similar median size on day 24, although a much greater spread of sizes was observed . Since the purpose of the assay is that it should indicate the viability of individual cells, it is important that a linear relationship exists between the number of unstained cells injected and the mean number of colonies obtained . The results of a study of this relationship for the L .L . Tumour are shown in figure 3 . It can be seen that there is a good linear relationship up to at least 50 colonies/lung as was the case for the KHT sarcoma . Two similar less extensive experiments for the B16 Melanoma indicated that the relationship was linear over the same range.



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0 Melanotic colonies

B16 Melanoma

® Amelanotic colonies Day 24 colonies from irradiated cells Median 1.2 mm (2000 rad in vivo) (305 colonies) 1

30

20

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10

Median

6 N Q 0

rn~ 30

ff

of

-OJi -rl m

Day 22 colonies from untreated cells (275 colonies)

20

10

0.2

0 .6

1.0

1 .4 1.8 2.2 Colony size (mm)

2 .6

3.0

Figure 2 . Size distributions for lung colonies growing from B16 Melanoma cells either untreated or irradiated in vivo with 2000 rad 60Co y-rays. Melanotic and amelanotic colonies are shown separately.

Figure 3 .

The mean number of colonies/mouse observed for different numbers of non-stained Lewis Lung Tumour cells injected . Mean ± S .E .



383

Lung-colony assay

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3 .2 . Use of the assay with the B16 Melanoma 3 .2 .1 . Melanotic and amelanotic colonies

The distributions of colony-size in figure 2 show the results for melanotic and amelanotic colonies separately . The numbers of amelanotic colonies were too small to draw precise conclusions about the relative sizes of the two types of colony, but the frequency of amelanotic colonies is different in the untreated (6 . 5 per cent) and radiation-treated groups (9 . 8 per cent) . In this sample the difference is not significant (0 . 1 >p > 0 . 05), however, an increase in amelanotic colonies has been noticed subjectively in other experiments with irradiated cells . Because of this difference, we decided to isolate a densely melanotic and an amelanotic sub-line from the tumour . This was achieved by repeatedly cloning the tumour cells in the lung as described in § 2 .4 . The relative frequency of melanotic and amelanotic colonies as a function of the number of clonings is shown in table 3 . The melanotic line showed no evidence of amelanotic colonies after the first cloning, whereas after four successive clonings, the amelanotic strain still gave some indication of melanin content . This may have been Number of clonings

Total number of colonies

Percentage amelanotic colonies

Percentage melanotic colonies

W, W2 W3 W4

7x 2 . 34 x 3 . 71 x 4 . 29 x

10 -4 10 -3 10 -3 10 -3

Amelanotic cell line 56 336 446 300

30 74 . 7 91 . 7 89 . 3

70 25 . 3 8.3 10 . 7

B, B, B3 B4

1 . 25 5 . 80 1 .47 7

10 -3 10 -4 10-3 10 -4

Melanotic cell line 201 81 205 49

0 0 0 0

100 100 100 100

6.5

93 . 5

Original experimental cell-line Table 3 .

Cloning efficiency

x x x x

Observations taken from several experiments

Variations in the proportion of melanin-producing cells in B16 Melanoma cell populations observed in consecutive clonings .

because of the relative difficulty of identifying amelanotic colonies before fixation . Another feature demonstrated in table 3 is the gradual but consistent improvement in the colony efficiency of the amelanotic cell-line, whereas the melanotic strain exhibits a more random fluctuation. Figure 4 shows the colony-size distribution for both cell-lines measured at 22 days after the injection of the cells . The amelanotic line had a mean diameter of 1 . 2 mm, whereas the melanotic line gave a mean diameter of 1 . 7 mm. These values are significantly different (p < 0 . 01). 3 .2 .2 . Radiation survival curve

The lung-colony assay has been used to study the radiosensitivity of both the L .L. Tumour (Shipley, Stanley, Courtenay and Field 1975) and the B16



384

R . P. Hill and J . A . Stanley

16

616

12

Melanotic Line Day 22 (89 colonies)

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Median diameter 12 mm .

Melanoma -

-

Amelanotic Line Day 22 (102 colonies)

12 L 8

4 0 02

0.6

1 .0

1 .4

1 .8

2.0

2 .4

2B

Colony diameter (mm)

Figure 4. Size distribution for lung colonies from melanotic and amelanotic cell-lines isolated from the B16 Melanoma .

B 16 Melanoma cells irradiation in vitro

Figure 5 . In vitro radiation survival curves for B16 Melanoma cells irradiated with 60Co y-rays . The different symbols represent results obtained in four independent experiments . Errors shown are S .E .



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Melanoma . In vitro survival curves for B16 Melanoma cells, both oxygenated and hypoxic, are shown in figure 5 . The data for oxygenated cells were obtained in three separate experiments, which agree well and give a Do of 158 rad (95 per cent C .L. 142-179 rad) and an extrapolation number of 8 (95 per cent C .L . 3-15) The data for the hypoxic cells are from a single experiment and should therefore be regarded as less reliable . They are, however, consistent with a dose-modifying action of oxygen, giving a Do of 496 rad and hence an o.e .r . of 3 . 2.

4. Discussion Experience with the lung-colony assay for the KHT sarcoma has shown it to be very useful for studies of the in vivo response of the tumour (Hill, Bush and Yeung 1971, Hill and Bush 1973, Milne, Hill and Bush 1973, Rauth and Kaufman 1975) . The system has been extended for use with both the L .L . Tumour and the B16 Melanoma . For both it was shown that a constant level was reached in the colony number as a function of time after injection and that, at this constant level, there was a linear relationship between the number of colonies obtained and the number of cells injected . The colony efficiency obtained for both tumours was lower than that obtainable with the KHT sarcoma and was significantly increased by the inclusion of 10 5-106 microspheres in the injected suspension, whether or not 10 6 HR cells were present . This is in contrast to the results with the KHT sarcoma, for which HR cells or microspheres gave the same increase in colony efficiency . However, the present practice with the KHT sarcoma is to mix the viable cells with both 2 x 10 6 HR cells and 8 x 105 microspheres before injection, and an analysis of results obtained during 1973 has indicated that the mean colony efficiency from 58 separate determinations was 3 .64 x 10-2 with a standard error of 0 . 18 x 10-2 . A similar analysis of 40 independent determinations over the same period of time during 1968-69, when only 2 x 10 6 HR cells were mixed with the viable cells, gave a mean of 3 . 18 (± 0 .30) x 10 -2 . These two means are not significantly different, confirming the previous observation that microspheres do not change the colony efficiency above that seen with HR cells alone, but the variances are significantly different (p

The lung-colony assay: extension to the Lewis lung tumour and the B16 melanoma--radiosensitivity of B16 melanoma cells.

Experiments are described which demonstrate that a lung-colony assay can be used to study the viability of unknown cell populations from the B16 Melan...
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