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Inr. _I. Radiation Oncology Bid. Phys.,, Vol. 5, pp. 130F1312 @ Pergamon Press Ltd., 1979. Printed m the U.S.A.

??Adriamycin

THE CYTOTOXIC ACTION OF ADRIAMYCIN AND CYCLOPHOSPHAMIDE ON TUMOR CELLS IN VZZXO AND IN VZVO W. M. CRAIG MARTIN, M.R.C.P.,

F. R. C. R.

AND

NICHOLAS J. MCNALLY, Ph.D. Gray

Laboratory of the Cancer Research Campaign, Mount Vernon Hospital, Northwood, HA6 2RN England

Middlesex,

The ability of Adriamycin (ADR) to kill tumor cells were studied using two transplantable murine tumors adapted for growth in tissue culture. Cells were treated either in viva as subcutaneous (SC) tumors or lung colonies up to 2 mm in diameter or in \?tro. ADR in contact with cells in vitro for one hour readily killed cells from both CBSAF and WHFIB tumors. Cells plated from tumors were more resistant in vitro than cells kept in culture. The D,,‘s were 0.17 and 0.1 pg/ml, respectively. The resistant tail in WHFIB at lo-’ survival was not inherited by surviving cells. In log phase the D,, was reduced. However, in I~YJ an IP or IV dose of 18 pgiml ADR to mice bearing CBSAF tumors produced a surviving fraction as high as 0.64 for SC tumors and 0.3 for lung colonies. Hypoxic tumor cells in l’itro are more resistant than aerobic ones; there may also be a tumor size effect because lung colony cells treated in \*itro has a sensitivity intermediate between SC tumors and cultured cells; but even both factors together cannot explain the lack of effect in viva relative to in vitro. Preliminary studies with cyclophosphamide suggested that it, in contrast to ADR, was highly effective in killing tumor cells both in SC tumors and in lung colonies. Adriamycin,

Cyclophosphamide,

fn vitro, In ~‘ivo, Cytotoxicity

“feeder layer” cells form colonies in Petri dishes with a plating efficiency of 70-80% for WHFIB and lO-40% for CBSAF. The methods used to grow cells and tumors and to prepare cell suspensions have been described bef0re.j Tumors could also be grown in pulmonary metastases by injecting 104-IO5 cells with lo” radiation-killed cells into mouse tail veins. Subcutaneous tumors were treated at 6-8 mm mean diameter and lung tumors at 2 mm maximum diameter. Drugs were administered intraperitoneally (IP) at the appropriate time before excising the tumor to measure cell survival; alternatively, tumor diameters were measured 3 times a week to obtain growth curves following treatment. For in tzitro studies, cells in exponential growth were exposed to ADR for one hour at varying concentrations, or for varying times at the fixed concentration of 0.5 pgiml. At the end of treatment the cells were rinsed with Hanks’ solution and fresh medium added. They were then incubated at 37°C for 9-10 days when colonies were stained and counted.

INTRODUCTION

There are situations in which potential cytotoxic drugs are highly effective in vitro, but not in \,ivo. Whereas Adriamycin (ADR) is known to be a highly effective drug in certain human tumors, especially sarcomas and breast carcinomas,2,3,6 lymphomas, Balconi,’ using the closely related drug Daunorubitin, found a poor in vi\v-in vitro correlation using a C,,H spontaneous mammary carcinoma. This study is concerned with determining the effectiveness of ADR and cyclophosphamide (CY) on two murine sarcomas, and with investigating the causes of the discrepancy between the effect of ADR on the tumor cells treated in \>itro or in vi\v. METHODS

AND

MATERIALS

The two tumor lines used, denoted WHFIB and CBSAF, have been adapted for growth either in t,ivo or in \~itro from sarcomas of spontaneous origin in our WH/Ht and CBA mouse colonies. Cells either from tumors or bottle culture, when plated on to AcXno~c~l~~dgPrr~rnts-This work Cancer Council.

was financed by the Research Campaign and the Medical Research The authors thank Montedison Ltd. for providing

the Adriamycin and Ward-Blenkinsop Ltd. the cyclophosphamide. Jennifer de Ronde Minchinton provided technical assistance. 1309

for providing and Andrew

Radiation Oncology 0 Biology 0 Physics

1310

August 1979, Volume 5, Number 8

100

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concentration

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FIG. 1. Surviving fraction as a function of Adriamycin concentration in the WHFIB cultured cells. Exposure for

FIG. 2. Surviving fraction as a function of Adriamycin concentration in the CBSAF cultured cells. Exposure for one hour.

one hour. RESULTS Adriamycin in vitro Both tumor cell lines, when exposed to varying concentrations of ADR for 1 hour, gave exponential survival curves (Figs. 1 and 2). WHFIB cells showed a biphasic response with a “break-point” at a drug dose of about 0.7 pg/ml, when the surviving fraction was about 10e3. The initial sensitive region had a D,, of 0.11 pgiml and the resistant tail a D,, of 0.6 pg/ml. CBSAF cells were slightly less sensitive with a D,, of 0.14 pgiml and no evidence of a biphasic response down to a surviving fraction of 5.10p4. Both cell lines showed a biphasic response when exposed to 0.5 pgiml for variable times up to 4.5 hours (data not shown). Adriamycin in vivo Cells from subcutaneous tumors were assayed for cell survival in vitro at various times after IP ADR injection. For drug doses between 1.3 and 18 mg/Kg there was very little cell killing in either tumor (Fig. 3). The LD,,, in both strains is about 18 mg/Kg. If drug-killed cells are rapidly removed, this could lead to an artificially high estimate of cell survival in vitro. While estimates of cell yield are imprecise in

either tumor, there was no evidence of a decrease in cell yield with time following ADR treatment. There was also little effect of ADR on 2 mm lung mestases. A dose of 18 mg/Kg of ADR did not reduce the surviving fraction below 3x 10-l regardless of when tumors were excised. In an attempt to increase the effect of ADR on dividing cells of these small tumors, 2 or 3 repeated injections of ADR to a total dose of 18 mg/Kg were given at 6 or 8 hour intervals and cell survival was assayed at various times after the last injection. There was no increase in cell kill over that resulting from a single injection of the drug. Measurements of ADR concentration in subcutaneous tumors gave approximate values of 12, 4 and 0.25 pg/ml 4, 9 and 28 hours after IP injection of 9 mg/Kg. These levels in vitro even for one hour would cause extensive cell killing. In spite of these relatively large tumor concentrations of the drug, there was never less than 30% of cells surviving, even in small lung colonies. It follows that the majority of cells in the tumors were resistant to ADR. In WHFIB subcutaneous tumors, the hypoxic fraction is at least 50% and in CBSAF tumors it is about 10% (McNally, unpublished data). We therefore tested whether chemo-resistance could be as-

Action 0

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,

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FIG. 3. Surviving fraction in 6-8 mm subcutaneous tumors in each tumor line, following varying doses of Adriamycin,

excised at varying times after injection, assayed in \Vtro.

.-6

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z ~

> .7 5 WI

sociated with hypoxia. WHFIB cells in spinner culture were exposed in the log phase at 37°C to 0.5 pgimi ADR for varying times, either when rendered hypoxic by passing nitrogen over the cells or when aerobic. The results are shown in Fig. 4. In both cases, the response was biphasic, but in both regions of the survival curve hypoxic cells were more resistant than aerobic cells (Fig. 4). However, this increased resistance was insufficient to account for the enormously increased resistance to ADR of cells in tumors. A more likely explanation is that many of the cells, even in small tumors, may be out of cycle and hence resistant. It has not been possible to maintain WHFIB or CBSAF cells in plateau phase. Therefore, this possibility was tested in V79 Chinese hamster cells. These were exposed to different concentrations of ADR for one hour when either in exponential or (fed) plateau phase (Fig. 5). Plateau phase cells were significantly more resistant. This may be because cells in plateau phase may take up significantly less ADR.” It is possible that non-dividing cells in tumors also do not take up the drug and this could be the explanation for the absence of significant cell kill in these two tumors, in spite of the extreme sensitivity of the tumor cells in vitro. If this is the explanation, it implies that even in 2 mm lung metastases a high proportion of the cells are out of cycle. Autoradiograph studies should give information on this. Cyclophosphamide

Cyclophosphamide (CY), by contrast, is highly effective in virao on both tumor lines. The doseresponse curve for WHFIB subcutaneous tumors excised 2 hours after injecting the drug and assayed in vitro is linear with a D,, of 25 mg/Kg. Cells from CBSAF tumors were slightly more sensitive with a

lo-*-

C

10-j

-

4

10-L

0

O-5 )-lg/ml Adriamycin

1 Time

2 /

3

4

hours

FIG. 4. Surviving fraction of cultured WHFIB cells in log phase in Spinner Culture under oxic or hypoxic conditions as a function of time of exposure to 0.5 pgmsiml Adriamycin.

D,, of 17 mg/Kg (Fig. 6). Figure 7 shows growth curves for subcutaneous WHFIB tumors treated with 100, 200 and 300 mg/Kg. With 300 mg/Kg all mice died by 6 days, but with the lower doses all mice survived. There was significant growth delay in all cases, and the regrowth times to 2 mm above initial diameter was 13.6, 9.6 and 3.1 days for 200, 100 mg/Kg and the controls. Tumors exposed to 200 mg/Kg, excised 2 hours later and assayed in vitro gave a survival fraction of about 3 x 10e4. If tumors were excised 24 hours after giving the drug the surviving fraction was about lo-‘. This increase in survival may reflect repair of potentially lethal damage. DISCUSSION In both tumor lines ADR is very effective in vitro and ineffective in vivo. This is not related to failure of drug delivery at the tumor, but may be due to drug not being taken up into non-cycling cells, the proportion of which may be high even in small lung colonies. Hypoxia may be a factor, but not the principal one. Cyclophosphamide is extremely effective in vivo on both tumors. At 2 hours after injection (with in vitro assay), massive cell kill is observed. By 24 hours, survival has increased by a factor of about 30, probably as a result of recovery from potentially lethal damage.

1312

Radiation Oncology 0 Biology 0 Physics

August 1979, Volume 5, Number 8

100

100

lo-’

lo-'

;

10-2

,o-2

a” z

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.-F

8 A

- 10-3 .-F .-> > 5 Ul 10-4

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A

1E4

\

1 hour

IT5

0

\

I

I

.*

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concentration

1o-5( pg/ml

FIG. 5. Surviving fraction of V79 379A cells in plateau phase or in log phase as a function of Adriamycin concentration. Exposure for one hour.

Cyclophosphamido

dose

mg / Kg

FIG. 6. Surviving fraction as a function of dose of cyclophosphamide, in both tumor lines, assayed in \Vtro at 2 hours after injection.

REFERENCES 1. Balconi, G., Bossi, A., Donelli, G., Filippechi, S., Franchi, G., Morasca, L., Garattini, S.: Chemotherapy of a Spontaneous Mammary Carcinoma in Mice; Relation Between in vitro-in vivo Activity and Blood and Tumour Concentrations of Several Antitumor Drugs. Can Chemother. Reports, Part 1, 57: No. 2, 115-124, 1973. 2. Carter, S.K.: Adriamycin-A Review. J. Nutional Cancer Inst.

55: 1265-1274,

1975.

3. Gottlieb, J., Bodey, G., Sinkovics, J.: An effective new 4-drug combination (CY-VA-DIC) for metastatic sarcomas. Proc. Amer. Sot. Clin. One. (Abstr. 713), In Proc. Amer. Cancer Res. 15: 162, 1974. 4. Harris, J.R., Timberlake, N., Henson, P., Belli, J.A. Adriamycin uptake and release in V79 Chinese hamster cells. Abstr. Proc. Radiat. Res. Sot., Dd-5, 26 Ann. Meeting, Toronto, 1978. N. J. et al. Effect of hyperthermia on 5. McNally, cytotoxicity of the radiosensitizer Ro 07-0582 in a solid mouse tumour. Br. J. Cancer 35: 372-375, 1977. 6. Rosen, G., Sunansirikul, S., Kwon, C. et al.: High-dose methotrexate and citrovorum factor rescue and adriamycin in childhood osteogenic sarcoma. Cancer 3: 1151-1163,

1974.

7. Twentyman, P.R.: Sensitivity to cytotoxic agents of EMT6 tumours in viva: Tumour volume versus in vitro plating. I. Cyclophospamide. Br. J. Cancer 35: 205-217, 1977.

CBSAF Cycbphosphamide

0

Controls

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100 rq/Kg

0

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II

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300

11

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0

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0

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0

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I

6

8

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12

14

Days FIG. 7. Growth curves for CBSAF subcutaneous tumors treated with varying doses of cyclophosphamide.