Chemotherapeutic drugs as indirect oxygen radiosensitizers

03scr-30’6/83/0~751-o7M3.~/0 Copyright Q ‘983 F’erymm Preu

Inr. J. Radiation Omwlw ffiol. Phys.. Vol. 9. pp. 75 I-757 Rintd in the U.S.A. All rights resewed

Ltd.

??Brief Communication CHEMOTHERAPEUTIC DRUGS AS INDIRECT OXYGEN RADIOSENSITIZERS FRED W. HETZEL,

PH.D.

AND

NATHAN

KAUFMAN,

M.A.

Division of Radiobiology, Henry Ford Hospital, Detroit, MI We bave characterized the oxygen distribution io V-79 spheroids which were grown by tbe spinner Iask method. Using ultnmicroelectrodes with tip diameters of l-5 microns and a perfusion system whereby the spberoids’ milieu could be nrritined and cq&olled, we found plateau p0, values of less than 10 mm Hg in spberohk of greater than 500 microns in diiter. Data from experiments using respiration inhibitory drugs indicate that tbe cbaracterktics of tbe outer layers of cells is tbe major determinant of tbe oxygen profile ia tbe interior. P8nllel control ndiobhlogical experiments confirmed tkcontrol p02 measurements, and formed tbe basis for tbe experime&s using the poteothl indirect radiation sensitizers: Cblorambucil and Mustargen. Spheroids, Mustargen, Indirect sensitizer.

INTRODUCTlON The multiceiluiar spheroid system, as developed by Sutherland and co-workers,24 has come into increasingly widespread use over recent years.48’2*26 The value of the system is in its potential as an intermediate path of investigation between in vivo and in vitro single ceil work, offering a possible means of correlating results from the two methodologies. It is thought that the central cells of spheroids may suffer nutritional stress similar to those ceils distant from capillaries in nodular carcinomas,‘7,25 probably causing the conditions of necrosis and chronic hypoxia found in both cases. Consequently, this “in vitro tumor model” has proven valuable in studying basic radiobiological phenomena such as repair and reoxygenation, ‘“.“322in addition to being uniquely useful in examining the effects of agents such as chemotherapeutic drugs ‘.9.‘3.‘5.‘*and hyperthermia,‘*6 both alone and in conjunction with radiation. Interpretation of experimental results has been complicated by several factors. First, spheroids have been grown from many diverse cell lines, both rodent and human, and by several different methods.23’2~27 Second, the different techniques which may be used to perform the same radiobiological procedure (for example, irradiating in stirred or unstirred media), can have an impact on the radiation response. Also, while the presence of a radiore-

METHODS AND MATERIALS The cells employed in the monolayer experiments and in the growth of spheroids were V-79 Chinese hamster fibroblasts.” Themethods for growth, irradiation and subsequent assay of spheroids as well as for the handling of the monolayer cultures have been described elsewhere.‘4*‘6The drugs employed in this study were Chlorambucil and Mechlorethamine HCL.+ Each drug was tested for single cell toxicity in exponential, confluent,

Presented at the American Society of Therapeutic Radiologists Annual Meeting, held at Miami Beach, Florida, October 12-16,198l. This work supported in part by Grant CA25781 from the National Cancer Institute and DHEW. Reprint requests to: Fred W. Hetzel, Ph.D., Division of Radiation Biology, Henry Ford Hospital, 2799 W. Grand Blvd., Detroit, MI 48202.

Acknowledgemenrs-The authors wish to thank Mr. Mike Mensinger for excellent technical support of this project and Ms. Susan Larson for her secretarial help in the preparation of this manuscript. Accepted for publication 10 December 1982. *The drugs were supplied by the Burroughs Wellcome Co. (Leukeran(Chlorambuci1)) and by Merck and Co. (Mustargen(Mechlorethamine HCL)).

sistant fraction of cells in the spheroid has been clearly demonstrated,s*‘3.23 it cannot be conclusively stated from the indirect radiobiological evidence that these cells are indeed hypoxic. We have developed a system in which the direct microphysiological determinations can be made. We have used this system in conjunction with radiobiological experiments to determine the presence of hypoxia in spheroids of various sizes.‘* Similar studies have also recently been reported by Mueller-Klieser and Sutherland.20*2’ We have also performed parallel experiments to determine whether these hypoxic areas would reoxygenate when the spheroids were treated with currently employed chemotherapy drugs that were found to inhibit cellular respiration at doses which were previously determined to be non-toxic.

752

Radiation Oncology0 Biology0 Physics

and spheroid cultures as a function of both concentration and exposure time. In addition, each drug was tested for its ability to inhibit cellular respiratory activity at a relatively non-toxic level (approximately 80% survival for necessary exposure time). The methods for the determination of cellular respiratory activity and direct readings of p0, in spheroids have been reported in detail recently.‘“‘* Briefly, all pOz measurements were made with gold-in-glass microelectrodes with tip diameters of I to 5 cccoated with membranes of Formvar and Rhoplex. In single cell studies, continuous recordings were made in a closed system with a known volume of medium and a defined cell number (106/ml). Changes in cell respiration following addition of drug were calculated from the slopes of the p0, vs. time graph produced. Figure I is a schematic illustrating the system that was used to measure p0, distribution in spheroids under controlled conditions. The spheroid was removed from a spinner flask and placed directly under a calibrated microelectrode in a Plexiglas chamber (total volume capacity 10 ml) which was perfused with media maintained at 37°C at a rate of 20 ml/min. The spheroid rested on a polyethylene mesh throughout the experiment, thus allowing virtually its entire surface to be perfused. The media entered the chamber from the side on a plane just below that of the supporting mesh and was collected from

May 1983. Volume

9, Number

5

the top of the chamber on the opposite side. In this manner the spheroid was perfused directly from the side and also from the bottom as the media was drawn up and out. It is likely that turbulence existed in some areas around the spheroid making it less like the normal growth conditions; however, the magnitude of any changes in p0, observed as a result of drug addition would likely be similar since the perfusion rates would be constant in both cases. The electrode was introduced stepwise from the top of the spheroid with the aid of a hydraulic microdrive while being viewed with a stereomicroscope at a magnification of 70x-140x through a glass window in the front of the chamber. A temperature probe and the reference electrode were held in the chamber via a rubber stopper on the side. For the initial oxygen profiles, the electrodes were inserted a good deal deeper than the plateau depth in order to ensure the reading of the lowest possible p0, value. This was not always done for the drug experiments, because the achievement of long term, stable readings prior to introduction of drugs was more important than reading minimum p0, values. Radition studies were performed with an orthovoltage X ray machine (250 Kvp, 15 ma, l/4 mm Cu, 1 mm Al) at a dose rate of 337 rad/min or with 9-MeV electrons at a dose rate of 950 rad/min with appropriate buildup (leucite).

8.

1iI: :x :A

h.

Fig. I. Overall view of system used for measuring microphysiological parameters in spheroids. a) Chamber: water jacketed with stainless steel or polyethylene mesh (150 +) on which spheroid is placed. b) Microelectrode. c) Micromanipulator base and electrode holder. d) Hydraulic microdrive. e) Reservoir. f) Water bath heating chamber and column from reservoir. g) Chemical microsensor. h) Recorder. i) Faraday cage. Not shown are temperature probe, cables and connections, binocular microscope, and fitting for bubbling gas into reservoir.

753

Indirect sensitization 0 F. W. HETZEL AND N. KAUFMAN

90

DEPTH vs. MEAN pO2 -

18 Spheroids (500 ~800

--

bydiameter)

8 Spheroids (300 ~~-500 JI diameter)

70

5 5 5

50

c 3c

1c (

I

,I

I

20

I

60

r

I

100

I

I‘1

140

180

I1

220

m

11

260

300

“‘1

340

380

Depth (~1 Fig. 2. Each data point represents the mean value of p02 obtaiped for a specific depth in either 8 or 18 spheroids as shown.

pO2 vs. MEDIAN DEPTH AT PLATEAU

6~ .

t

\ \ \ \

cb 40.

\

\

m

\

\

\

??

20-

.*

-

18 Spheroids

( 300 p-500

JI diameter)

( 500 p- 800 )I diameter)

??

\

\

\

.

\

\* \

m

105

8 Spheroids

\ \

z E

o??

135

165

\

\

195

225

255

285

315

345

375

Microns Fig. 3. Each data point represents the actual value of p& obtained at the midpoint of the p0, plateau within each single spheroid.

754

Radiation

Oncology 0 Biology 0 Physics

RESULTS An important step in the definition of our system was determination of detailed oxygen profiles in spheroids of various sizes. Figure 2 is a summary of a depth vs. pQ study on 26 spheroids divided into two size classifications. The depths were chosen so as to encompass the descending plateau and a portion of the ascending segment of the p0, profiles. Note that each point is a mean value taken from different sized spheroids within each classification. Consequently, there are no values of 0 mm Hg; however, the trend of depth versus p0, is clearly evident. Figure 3 is perhaps the most salient treatment of the data obtained from the same two groups of 26 spheroids. Each point represents the mean depth (i.e. the midpoint) of the

100

MUSTARGEN 0 Exponential ??

1 hr. Ceils

Confluent Cells

May

1983, Volume 9, Number

plateau of minimum pOz in each spheroid, and the pOz corresponding to that plateau. Note that six of the values are actually 0 mm Hg. Before being able to use any respiration inhibitory drug on the spheroid, the drug’s toxicity, respiration rate effects, and radiation survival curve effects had to be determined. We have to date completed all of these procedures on a number of drugs, the most promising of which are Mustargen and Chlorambucil and for which we are reporting the detailed results. Drug toxicity determinations were made for each drug under a variety of conditions of growth and time sequencing. Figure 4 shows the results of exposing exponential or confluent cells to various concentrations of Mustargen for one hour. From this type of experiment a “working” dose was chosen for subsequent experiments. Employing a dose of .75 &ml, toxicities for Mustargen were determined as a function of time in each of the three cell growth states. As seen in Figure 5, exponential cells appear to be most resistant and spheroids most sensitive. Qualitatively similar results were seen for Chlorambucil.” The appearance of a “resistant tail” was noted in all cases and was

100 1c

5

WSTAWEN

0.75 pa/ml

?? ConfllJent cells 0 Exponent&l Cells ~-spheroids WOp)

2 ._ 5 * 1.c ap

0.’

0.0

--.5-- ’ l:o

’ 1:5

Concentration @g/ml) Fig. 4. Toxicity for a one hour exposure of Mustargen to exponential or confluent cells as a function of drug concentration. Error bars are shown when larger than the plotted symbol.

Time (hr) Fig. 5. Toxicity as a function of time for exponential, confluent and spheroid ceils exposed to 0.75 ccg/ml Mustargen. Error bars are shown when larger than the plotted symbol. This figure is redrawn from ref. 13, and included here for clarity and completeness.

755

Indirect sensitization 0 F. W. HETZEL AND N. KAUFMAN

determined to be a result of inactivation of the drug in the medium.” After complete curves were obtained for each drug, one concentration-time combination was chosen for each drug for all subsequent studies: Mustargen (.25 pg/ml) for one hour and Chlorambucil (30 pg/ml). Employing concentrations of drug described above, single cell respiratory inhibition was determined. In each case a control respiration rate curve was determined. Pooling all of the control curve data, a mean value was obtained of 6.2 x IO-” moles 0, set-‘/cell. The addition of drug to the media resulted in average percentage inhibitions of 43% for Mustargen and 77% for Chlorambucil. It must be noted that all of these values were

Table 1. Calculated values of Do for the upper (oxygenated) and lower (hypoxic tail) regions of complete spheroid survival curves. Each set of data represents the combined results of two sets of experiments which were subsequently normalized (for comparative purposes only) so that the upper portions of the curves were superimposed.

DRUG

I

I

MUSTARGEN

220

CHLORAMBUCIL

220

Do

Do

665

255

2.6

610

300

2.0

(NOTE Numerical values corrected for geometry and dosimetry)

obtained within one half-hour of drug addition at concentrations already shown to be relatively non-toxic. For this

easily modulated. It has been pointed out that whether the media is stirred or not can affect the radiation response of

reason subsequent experiments were performed after 30 minute exposures. Table 1 shows the results.when Mustargen (.25 Kg/ml) or Chlorambucil (30 r(8/ml) was added to spheroid cultures for 30 minutes prior to and during irradiation (the exposure time was reduced based upon the rapid inhibition observed in the microelectrode studies). It is clear from this table that no effect is seen in the upper part of the survival curve, but the resistant, hypoxic tail is shifted significantly by each of the drugs. The terminal portions of the control and drug treated curves yield drug enhancement ratios (more properly an oxygen enhancement ratio) of approximately 2.0 for Chlorambucil and 2.6 for Mustargen. In order to test the effect of these drugs on the oxygen distribution within spheroids, (ie. to confirm that the change in the resistant slope was because of oxygen) the procedure for obtaining pO1 profiles was repeated. When the p0, reached a plateau or a suitably low steady level, the electrode tip was. left at the same depth for a long enough period of time to ensure that there was no upward drift in the readings. The perfusate was then switched to the reservoir containing drug-media and the p0, was continually monitored at the same fixed depth. Table 2 summarizes the findings in six experiments using Mustargen.

spheroids,7*20 presumably by alterations of the oxygen distribution via the presence or lack of convection of oxygen. This point is amplified by the observation (not shown) that there is a significant drop in the p0, of the media in the area surrounding, but not in, the spheroid even at a flow rate of 5 mljmin in a 10 ml chamber. As the delivery of oxygen to the whole spheroid may affect the internal oxygenation, so may the delivery of oxygen to the interior cells of the spheroid be affected by two important factors. First is the morphology of the spheroid. The degree of cell packing, or cell density, and the corresponding size of the extracellular spaces probably has a great impact on how much oxygen can diffuse into the spheroid.2’ Obviously, the method of spheroid cultivation must be taken into account, since the agar gel and agar-underlay methods result in looser packing than the spinner flask method. The different morphologies of our respective spheroids is perhaps the major cause of the discrepancies between our results and those of Carlsson et 4L3 (the recent work of Mueller-Kleiser and Suther1and’9*” confirms our findings in V79 spheroids). As Durand’ noted, “the diffusion gradients of metabolites and oxygen present in such spheroids (grown in gel or agar-underlay cultures) may well be very different than those in tightly-packed, spontaneously occurring spheroids in suspension culture (and cells in tissue?).” The second factor is the respiration rate of the cells in the outer layers of the spheroid. Naturally, the cell line used is important in this regard, since spheroids cultivated from different cell lines have been found to contain different widths of the outer, viable rims of cell~.‘~ We have been able to manipulate the respiration rate of the outer cell layers using respiration inhibitory drugs. Each drug altered the p0, being measured in the spheroid interior; the effect correlates well when compared with results from parallel, radiobiological experiments performed in our laboratory as well as others.8*9.‘3 The methods and rationales for choosing certain drugs and the rather lengthy series of steps in the drug screening procedures have been previously discussed.‘3*‘sIt is impor-

DISCUSSION We have made oxygen determinations in over 100 spheroids during the course of several experimental procedures. Within experimental error our observations currespond to our initial finding of radiobiological hypoxia in spheroids of greater than 500 p diameter. It has been noted that several methodological factors may affect the radiation response of spheroids.7.20.2’ Our system has been developed so that the flow rate, composition, temperature, chemical makeup, and respiratory gas partial pressure of the media, and other parameters which may affect the microphysiological makeup (and consequently the radiation response) of the spheroid may be controlled and

tant

to note

the

following

for

the

completed

series

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Radiation Oncology 0 Biology 0 Physics

May 1983. Volume 9, Number 5

Results obtained when single intta spheroid oxygen tensions were monitored before and during the administration of .21 or .42 rg/ml of Mustargen. Both pre and post drug p0, values were steady state values obtained at a particular point within the spheroid without moving the electrode. Table 2.

.42vg/ml

.2 1ug/ml

MUSTARGEN

Pro Drug PO2

0

5

Port Drug ~02

18

15

50

80

7aop

7ooJJ

time

21

20

1

88

42

58

18

30

40

40

38

52

to maximum

effect (min)

Spheroid Dkmeter

700~800~500~~80~

1

.

described in this paper: In this study it has been shown that the concentration of drugs employed to produce the effect are sufficiently low to preclude direct cell killing as the major reason for the response. In addition, it is clear that on the transient basis observed, the oxygen allowed to diffuse into the spheroids when peripheral respiration is inhibited is not totally consumed by previously lowly respiring cells in the interior. The modification in radiation response seen in Table I therefore can be attributed directly to increased oxygenation in the interior of the spheroids because of respiratory inhibition. For the drugs shown, the only effect observed is respiratory inhibition

without modification of the single cell survival curve at a dose of drug chosen to be of minimal direct cytotoxicity (approximately 80% survival). The results reported provide evidence that current chemotherapeutic drugs do result in modification of cellular radiation response, both directlyI and indirectly. To determine the possible clinical uses for these responses, it will also be necessary to elucidate whether similar effects can be obtained in vim It is important to note, however, that the technology and procedures developed and used in this study are easily adaptable to more complex physiological systems.

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duration of heating. Int. J. Radiat.,OncoI. Biol. Phys. 4: 401-405, 1978. 7. Durand, R.E.: Variable radiobiological response of spheroids. Radiat. Res. 81: 85-99, 1980. 8. Durand, R.E., Biaglow, J.E.: Radiosensitization of hypoxic cells of an in vitro tumor model by respiratory inhibitors. Radiar. Res. 69: 359-366,

1977.

9. Durand, R.E., Biaglow, J.E., Greenstock, C.L.: Effects of sensitizers on cell respiration: II. The effects of hypoxic cell radiosensitizers on oxidative metabolism and the radiation response of an in vitro tumor model. Br. J. Cancer 37

(Suppl. III): 150-153, 1978. 10. Durand, R.E.. Sutherland, R.M.: Effects of intercellular contact on repair of radiation damage. Exp. Cell Res. 71: 75-80, 1972. 11. Durand, R.E., Sutherland, R.M.: Dependence of the radiation response of an in vitro tumor model on cell cycle effects.

Cancer Res. 33: 2 13-2 19, 1973.

lndirect sensitization 0 F. W. HETZELAND N. KAUFMAN 12. Haji-Karim, M., Carlsson, J.: Proliferation and viability in cellular spheroids of human origin. Cancer Res. 38: 14571464, 1978. 13. Hetzel. F.W., Brown, MS., Kaufman, N., Bicher, H.l.: Radiation sensitivity modification by chemotherapeutic drugs. Cancer Clin. Trials 4: 177-I 82, 198 1. 14. Hetzel, F.W., Kruuv, J., Frey, H.E.: Repair of potentially lethal damage in x-irradiated V-79 cells. Radial. Res. 68: 308-319,

20.

21.

1976.

15. Hetzel, F.W., Kruuv, J., Frey, H., Koch, C.J.: Radioresistance of anoxic cells and the effects of PNAP on the radiation response of an in vivo tumor model. Rod&. Res.

22.

56: 460-473, 1973. 16. Hetzel, F.W., Kruuv, J., McGann, L.E., Frey, H.E.: Expo-

23.

sure of mammalian ceils to physical damage: Effect on the state of adhesion on colony forming potential. Cryobiology 10: 206-211, 1973. 17. Inch, W.R., McCredie, J.A., Sutherland, R.M.: Growth of nodular carcinomas in rodents compared with multicell spheroids in tissue culture. Growth 34: 271-282, 1970. 18. Kaufman, N., Bicher, H.I., Hetzel, F.W., Brown, M.S.: A system for determining the pharmacology of indirect radiation sensitizer drugs on multicellular spheroids. Cancer Clin. Trials 4: 199-204, 198 I. 19. Mueller-Kheser, W.. Sutherland, R.M.: Frequency distribution histograms of oxygen tension in multicell spheroids.

24.

25.

26.

27.

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Presented at the Fifth Intl. Symp. of the Intl. Sot. for Oxygen Transport to Tissue. Detroit, Michigan, August 25-28, 1981. Mueller-Klieser, W.F., Sutherland, R.M.: Influence of convection in the growth medium on oxygen tensions in multicellular tumor spheroids. Cancer Res. 42: 237-242, 1982. Mueller-Klieser, W.F.. Sutherland, R.M.: Oxygen tensions in multicell spheroids of two cell lines. Br. J. Cancer 4& 256-264, 1982. Sutherland, R.M.: Select chemotherapy of noncyling cells in an in vivo tumor model. Cancer Res. 34: 3501-3503, 1974. Sutherland, R.M., Durand, R.E.: Hypoxic cells in an in vitro tumor model. Int. J. Radial. Biol. 23: 235-246, 1973. Sutherland, R.M., Inch, W.R., McCredie, J.A., Kruuv, J.: A multi-component radiation survival curve using an in vitro tumor model. Int. J. Radiat. Biol. 18: 491-495, 1970. Thomiinson, R.H.. Gray, L.H.: The histologic structure of some human lung cancers and the possible implications for radiotherapy. Brit. J. Cuncer 9: 539-549, 1955. Yuhas, J.M., Li, A.P.: Growth fraction as the major determinant of multi-cellular tumor spheroid growth rates. Cancer Res. 38: 1528-1532,1978. Yuhas, J., Li, A.P., Martinez, A.O., Ladman, A.J.: A simplified method for production and growth of multiceilular tumor spheroids. Cuncer Res. 37: 3639-3643, 1977.