SPATIAL DISTRIBUTION OF HUMAN CELL SURVIVAL AND OXYGEN EFFECT IN A

THERAPEUTIC HELIUM ION BEAM

PAUL TODD, PHD,* BAMBINO I. MARTINS, P H D , ~ JOHN T. LYMAN, P H D , ~ J.-H. KIM, MD,* AND CARTER B. SCHROY, MY

The survival of human kidney T-1 cells, based on colony formation, was deter- mined in the presence and absence of oxygen as a continuous function of depth in a beam of high-energy helium ions from the Berkeley 184-inch syn- chrocyclotron. Cells were exposed while attached to plastic coverslips in holders that spaced them 0.25 cm apart along the beam path. In some samples oxygen was removed from the medium by metabolic depletion by freshly-irradiated cells. The original beam energy (910 MeV) was degraded by absorbers so that the residual range was 10 cm; a reciprocating ridge filter then spread the Bragg peak out to a width of 5.5 cm. Cell survival decreased as a function of depth, and the RBE of this beam at maximum depth (highest LET) was 1.4. At this point the OER was 2.2 2 0.2, compared to 3.0 0.3 at the point where the minimum-LET beam entered the sample. At the minimum depth to which the Bragg peak was spread, the RBE was 1.1, and the OER was 2.3 2 0.3. Beyond the stopping point of the beam there was no cell death detectable within the sensitivity of the experiments; for example, surviving fraction was 0.99 f. 0.05 at a point 0.5 cm beyond where the dose was 765 rads.

Cancer 341-5, 1974.

N ORDER TO EXPLOIT THE ADVANTAGES PRE- I sented by high energy heavy ions for the radiation treatment of cancer and for other biomedical applications,* it will be necessary to deposit the dose to the treatment zone in multiple Bragg peaks by stopping the particle beam at various depths, as has been done, for

Presented at the Fifteenth Annual Meeting of the American Society of Therapeutic Radiologists, New Orleans, Louisiana, October 24-28, 1973.

From the Lawrence Berkeley Laboratory, The Penn- sylvania State University, and Memorial-Sloan-Ketter- ing Institute for Cancer Research, New York.

Supported in part by the US. Atomic Energy Com- mission a t the Lawrence Berkeley Laboratory.

* Associate Professor of Biophysics, The Pennsyl- vania State University.

t Assistant Professor of Radiology, University of Maryland School of Medicine.

f Biophysicist, Lawrence Berkeley Laboratory. $Resident in Radiation Therapy, Memorial Hospital

for Cancer and Allied Diseases. IlResearch Assistant, T h e Pennsylvania State Univer-

sity. Address for reprints: P. Todd, 618 Life Sciences

Building, University Park, PA 16802. The authors are grateful for the generous coopera-

tion and encouragement of Dr. G. J. D’Angio and Dr. C. A. Tobias, and gladly acknowledge the skilled technical assistance of Mr. J. Howard and Mr. R. Roisman. They also thank Dr. M. R. Raju for help- ful discussions.

Received for publication January 7, 1974.

1

example, in the treatment of cervical tumors with protons.5 This type of “transformed Bragg peak” treatment has recently been ap- plied to experimental treatments of human lung nodoles.7 Using the 910 MeV helium ion beam of the Berkeley 184-inch synchrocyclo- tron, Berry and Andrews reported evidence that at maximum depth of penetration that beam is less dependent upon oxygen than gamma radiation in the inactivation of P-388 murine ascites tumors.2 Using in vitro ex- posure and colony-formation assay of human kidney T-1 cells, Raju et al. reported that helium ions in a transformed Bragg peak had OER of 1.8 f 0.3 for this end-point, compared with 2.5 2 0.3 for gamma rays.6 Dose-survival curves were reported at three depths along the path of the beam, which was transformed by a reciprocating ridge filter to place the maxi- mum depth of penetration at 10 cm, with constant dose from 4.5 to 10 cm, the region over which the Bragg peaks were spread. Sur- vival curves were of similar shape. RBE at maximum depth was 1.4; at 5.0 cm it was 1.3, where the shallowest Bragg peak stopped.

The imminent possibility of using trans- formed-Bragg-peak helium ions in trial treat-

CANCER J u l y 1974 Vol. 34 2

Tl

‘ VARIABLE R I D G E SAMPLE ABSORBER F I L T E R I O N I Z A T I O N CONTAINER

CHAMBER

FIG. 1. Experimental arrangement for irradiation of cultured human cells attached to plastic coverslips in rectangular containers with 910-MeV helium ions transformed by a ridge filter. (No portion of the sketch is to scale.)

ments of patients prompted confirmation of the above observations. This work reports re- sults of measurements of human cell survival and OER as a continuous function of depth in approximately tissue-equivalent material. Attempts were made to improve oxygen re- moval and spatial resolution.

MATERIALS AND METHODS

Cells Human kidney T-1 cells were used as previ-

ously described.9 Medium was Eagle’s MEM? supplemented with 10% fetal calf serum.” Cells used for oxygen depletion were grown to a titer of 5 X 10’ cells in 32-oz prescription bottles, harvested by trypsinization, irradiated with 4500 rads of W C o gamma rays, and added

* Chemicals and solutions, including serum, obtained from Grand Island Biological Co., Grand Island, N.Y., or Microbiological Associates, Bethesda, Md.

~~

F B J.

HELIUM LBL

t

l - 1 2 4 6 8 10

DEPTH, VI

FIG. 2. Helium depth-close profile used in this study. Dose was measured with a small diode scanning in a rectangular water phantom. The profile was produced by use of a ridge filter as previously described.5 “F” marks the position of the front of sample containers, and “B” marks the back. Samples were spaced at 0.25-

to containers of test cells at a density of 1.2 x 100 cells/ml. Oxygen depletion was allowed to proceed for 5.5 hours prior to exposure to the helium ion beam. This technique of oxy- gen depletion has given OER equal to or greater than 3.0 in exposures of human kidney cells to “Co gamma rays.

Dosimetry The 910-MeV helium-ion beam of the Berke-

ley synchrocyclotron was focused, collimated, and passed through a variable water absorber and through a ridge filter moving back and forth in the beam out of synchrony with the beam pulse. Three-foil ionization chambers were used for absolute dosimetry; the de- livered dose was read digitally from an elec- trometer, which automatically interrupted the beam after the pre-set dose was delivered. These methods have been described in detail previously.6 A sketch of the beam layout is given in Fig. 1. The resulting depth-dose pro- file in water, given in Fig. 2, is in agreement with that reported previously, using the same physical system.” Positions of front (“F”) and Back (“B”) of cell containers in the depth- dose profile are marked for further reference under RESULTS. A transverse scan of the beam, shown in Fig. 3, indicated that the dose was uniform within 2.5% over the 22-mm sample width.

100

W m 0 n

2 W

5 50

3 2 1 0 1 2 3 CM FROM CENTER

FIG. 3. Dose as a function of position across the horizontal midline of the beam at a depth of 5.0 cm in water (see Fig. 2). Thick bar indicates the width and position of cell samples. Dose varies less than 3% across the sample region. Measured by diode, as in

cm intervals between the two arrows during exposures. Fig. 2.

No. 1 CELL SURVIVAL & OXYGEN EFFECT IN HE ION BEAM - T o d d et al. 3 Preparation of Cells for Irradiation

Cells were trypsinizied from a log-phase (1-day-old) monolayer, counted, and seeded in predetermined numbers on 20 x 22 mm plas- tic (LUX, Microbiological Associates, Inc.) coverslips in 0.5 ml of complete medium in 35 mm plastic (Falcon Plastics, B-D Corp., Los Angeles, Calif.) petri dishes. These cells were incubated for 7.0 hours. The coverslips were then transferred to slotted rectangular poly- carbonate containers, which spaced the cover- slips 0.25 cm apart along the length of the container with the coverdips in vertical ori- entation, as was reported previously.3 (Fig. 1). The containers were fabricated in the shop of the Naval Research Laboratory Cyclotron Laboratory, Washington, D.C. After cover- slips were inserted (40 per 10-cm container, and 10 per 3-cm container), the containers were completely filled with medium (with or without irradiated cells for oxygen depletion), sealed, and incubated for another 5.5 hours before exposure to helium ions. At this time there were approximately 1.3 cells/colony. Containers were placed in the transformed beam so that coverslips were distributed, per- pendicular to the beam, from the point marked “F” (front) to the Point marked “B” (back) in Fig. 2. Some coverslips were deliber- ately placed beyond the range of the particles. After irradiation, coverslips were transferred to 35 mm petri dishes containing 3 ml each of complete medium. These cultures were fed every 4 days. CoIony formation was completed at 9-10 days, at which time the coverslips were stained with methylene blue, and the colonies were counted under a dissecting microscope. Colony counts on groups of adjacent cover- slips were averaged to calculate local survivals.

RESULTS

Hypoxic Survival

The stopping power of helium ions of 110 MeV (such as at the point of entrance of the sample containers) is 30 MeV-cm2/g. For such radiation the OER should be 2.8 or greater.’,g The observed OER over the first 4 cm of beam path in the sample containers was therefore taken as a measure of the adequacy of the metabolic depletion technique for achieving hypoxia. The samples irradiated at higher LET at ,greater depth were in the same con- tainers as those in which hypoxia was tested on the basis of low-LET survival. The data

PLATEAU HELIUM

0 500 loo0 1500 2000 2500 DOSE , RADS

FIG. 4. Survival curves for cells irradiated in the presence (empty circles) and absence (filled circlcs) of oxygen in the minimum-ionizing region, where helium ions had residual range between 6.0 and 8.5.

are presented in the form of dose-survival curves in Fig. 4. The low-LET OER was found to be 3.0 2 0.3.

Survival at Various Depths Helium-ion doses of 240, 330, 555, and 810

rads were deposited at the points marked “F” in Figs. 2 and 5, and the full depth-dose pro- file in Fig. 2 was delivered to containers of cells on coverslips. The resulting surviving frac- tions were then plotted as a function of depth, expressed as cm of residual helium-ion range in water. In Fig. 5, 0 on the abscissa is the depth where 50% of the most energetic helium

1

k OXYGENATED rt-

B .I.

V I I I 1 I I I f 1 I ”

10 9 8 7 6 5 4 3 2 1 0 RESIDUAL RANGF . CM

Fic . 5. Depth-survival profiles for oxygenated cclls exposed to (from top to bottom) doses of 240, 390, 555, and 810 rads delivered at thc position marked “F”. Data were used for survival curves of Figs. 4 and 7. Plating efficiency was 55% relative to unmanipiilated rontrol cclls.

4 CANCER July 1974 Vol. 34

ions stop, The manipulations associated with this portion of the experiment resulted in plating efficiency that was 55% that of con- trol, unmanipulated cells, which in turn had 88% plating efficiency.

A set of containers bearing hypoxic cells was similarly treated. Doses of 600, 800, 1100, and 2300 rads were deposited at position “F.” The resulting depth-survival profiles are shown in Fig. 6. T h e plating efficiency was 75% that of unmanipulated control cells.

Survival Curves at Various Depths The data shown in Figs. 5 and 6 were re-

plotted in the form of survival vs. dose, using only survival points from 0.00 to 0.75 cm re- sidual range, so that survival curves under oxygenated and hypoxic conditions were ob- tained for helium ions at the very “back” of depth-dose profile, as shown in Fig. 7. This should be the region with the highest fraction of Bragg-peak ions and corresponds to “Posi- tion 3” of Raju et al.G The survival curve for oxygenated cells indicates that helium ions at this point are 1.44 times as effective as mini- mum-ionizing helium ions (compare to Fig. 4). At this depth the OER is 2.2 2 0.2.

Fewer survival points are presently avail- able for comparison of other parts of the pro- file on a survival curve basis, owing to the uni- formity of the doses. Preliminary survival

1,o

z 0

I01

RESlDUAL RANGE , CM FIG. 6. Depth-survival profiles for hypoxic cells ex-

posed to (from top to bottom) 600. 800, 1100 , and 2300 rads delivered at the position marked “F”. Data were used for survival curves of Figs. 4 and 8. Plating efficiency was 75% relative to unmanipulated control cells.

1,o

t V

2 0 , l

I 0 1

b - PEAK HELIUM OER = 2,2 t 0,2 \ \

\‘ \ -

-

0 500 1000 DOSE , RADS

FIG. 7. Survival curves for cells irradiated in the presence (empty circles) and absence (filled circles) of oxygen in the region between 0 and 0.75 cm residual range. OER = 2.2 * 0.2.

curves for the region 3.5 to 5.5 cm from the position “B” indicate that this portion of the profile is about 1.1 times as effective as the plateau, and the OER lies between 2.0 and 2.6. This region corresponds roughly to “Posi- tion 2” of Raju et a1.6

The values of OER and RBE (assuming constant survival curve shape) are summarized in Table 1.

DISCUSSION

This study agrees in general with previous findings that high-energy helium ions in a

‘rARLE 1. Summary of RBE and OER from Figs. 4, 7, and 8, Using Depth-Dose Profile of Fig. 2

and Beam Positions as Defined by Maximum Residual Range, as

in Fig. 5

Residual range (cm) RBE OER

6.0-8.5 1.00 - 3 . 0 f 0 . 3 3.6-5.2 1.08 f 0.06 2 . 3 f 0 . 3 0.0-0.75 1.44 + 0.10 2 2 f 0.2

Ratios are dose ratios a t or near the 0.10 5urvival level uiing snrvival curves with extrapolation number = 2.5.

No. 1 CELL SURVIVAL & OXYGEN EFFECT IN HE ION BEAM - Todd et al. 5

transformed Bragg peak exhibit a slightly in- creased RBE and slightly decreased OER for mammalian cells.”G The OER of 2.2 f 0.2 at the end of the beam path appears to be realis- tic, in view of the success with which oxygen was removed as indicated by the high OER in the minimum-ionizing region. Evidently, as was also previously found, a reduced OER also exists at the front of the transformed Bragg peak.

Because the fraction of cells being killed by stopping particles increases from the front to the back of the profile, it is not surprising that RRE should increase. In order to produce uniform cell lethality over the treatment zone, it will be necessary to use rotation therapy or parallel opposed ports or a ridge filter de- signed to provide the depth-dose profile simi- lar to that sketched in Fig. 8, although further account will have to be taken of increased RBE at 5 cm as well. The ridge filter design suggested in Fig. 8 may require further refine- ments to account for fractionation in cases where parallel opposed irradiations are impos- sible.

The lack of exit-dose damage is also shown in this study. Cell lethality falls off sharply, as does the dose, beyond the range of the most energetic particles. Although there is not room to present the data in detail in Figs. 5 and 6,

1 , 3 3

1 , o o

W U-J 0 n

2 4

W

ic

w CT

I I I I I I I I I \ 0 2 4 6 8

DEPTH , CM FIG. 8. Depth-dose profile recommentled for single-

port irradiation with uniform cell survival throughout the treatment zone. T h e ratio 1.33 of dose at the front of the transformed Bragg peak to that a t the hack is the inverse of the ratio of RBE’s at t h e two positions, 1.44/1.08.

it was found in five separate exposures that survival beyond the beam never differed with statistical significance from 1 .OO. For example, less than 0.5 cm from the peak dose of 765 rads, the survival of oxygenated cells was 0.99 f 0.05. This treatment technique may be well suited for therapy very near radiosensitive or critical anatomical structures.

REFERENCES

1 . Barendsen, C. W., Koot, C. J., van Kersen, G. R., Bcwley, D. K., Field, S. B., and Parnell, C . J.: The effect of oxygen on impairment of the proliferative capacity of human cells in culture by ionizing radia- tions of differrnt LET. Int. I . Rarliat. Biol. 10:317- 327. 1966.

2. Berry, R . J., and Andrews, J. R.: The response of mammalian cells in vivo to radiations of differing ionizing densities (LET). Ann. N . X . Acad. Sci. 114:48- 59, 1964.

3. Brown, D. Q., Seydel, H. C., and Todd, P.: In- activation of cultured human cells and control of C3H mouse mammary tumors with accelerated nitrogen ions. Cancer 32:541-546, 1973.

4. Eagle, H.: Amino acid metabolism in mammalian cell cultures. Science I30:432-437, 1959.

5 . Karlsson, B. G.: Methoden zur Berechnung nntl

Erziehung einigrr fur die Tiefen therapie mit hochrn- energetschen Protonen gunstiger Dosisvcrteilungen. Stralilentherapie 124:481, 1964.

6. Raju, M. R., Gnanapurani. hf., Marlins, n. I., Howard J., and Lyman, J. T.: Measurement of OER and RBE of a 910-AleV helium ion beam using cul- tured cells (T-l). Radiology 162:425428, 1972.

7. Tobias, C. A., Lyman, J. T., and Lawrence J . H.: Some considerations of physical and biological factors in radiotherapy with high-LET radiations including heavy particles, pi mesons, and fast neutrons. Progr. Atomic M e d . 3:167-218, 1971.

8. Tobias, C. A,, and Todd, P.: Heavy charged par- ticles in cancer therapy. Natl. Cancer Ins t . ‘lfonogi-. 24:l-21, 1967.

9. Todd, P.: Heavy ion irradiation of cultured hu- man cells. Radint . Res,. [,Supp/.] 7:196-207, 1967.