Tumor irradiation with intense ultrasound

Ultrasound in Med. & Blot., Vo|. 4, pp. 337-341 Pergamon Press Ltd., 1978. Printed in Great Britain

TUMOR IRRADIATION WITH INTENSE ULTRASOUND F. J. FRY and L. K. JOHNSON Ultrasound Research Laboratories of the Indianapolis Center for Advanced Research, Inc., Indianapolis, Indiana 46202, U.S.A.

(First received 26 May 1978; and in final form 2 October 1978) Abstract--Tumors implanted in the hamster flank have been irradiated in vivo with intense focused ultrasound at a spatial peak intensity of 907 W/cm 2. A matrix of points was irradiated under c.w. conditions through the central plane of the tumor and perpendicular to the longitudinal axis of the sound field. A center spacing of 4 nun between matrix points and a time-on period of 2.5 sec at each point produced no cures. A spacing distance of 2 mm with 7 sec time-on period at each point increased mean survival time in non-cured animals and produced a cure rate of 29.4%. Combining the second regime of ultrasound treatment with administration of a chemotherapeutic agent (BCNU) 24 hr after irradiation did not increase mean survival time in the non-cured animals compared to the BCNU non-irradiated shams; however, the cure rate increased to 40~. Secondary tumors which were not seen in any ultrasound shams or controls were observed in all other regimes including BCNU non-irrndiated shams. The incidence of secondary tumors was inversely related to the cure rate.

Key words: Ultrasound, Tumor. INTRODUCTION

A rationale for ablation of a tumor mass as a potential cancer therapy involves reducing the total number of tumor cells in the mass to a level where the immune system might be capable of taking any remaining viable cells to extinction (Burnet, 1970; Thomas, 1959). Additionally, the immune system might be further activated due to the nature of the ultrasonically destroyed cells residual in the tumor mass, and thereby take the remaining viable cells to extinction. Apart from immune responses, the use of chemotherapeutic agents on the reduced tumor mass could lead to a further reduction or possible extinction of remaining viable tumor cells. It is also conceptually possible to destroy all the tumor cells so that no viable cells remain after an appropriate ultrasound irradiation. Most previous studies on the direct destruction of tissue with intense focused ultrasound have involved normal tissues (Fry et al., 1954; Ballantine et al., 1956; Lele, 1962; Bell, 1957; Linke et al., 1973; Lizzi et al., 1976; Fry and Dunn, 1956; Dunn and Fry, 1971; Chan and Frizzell, 1977; Frizzell et al., 1977). A few notable exceptions to this generalization include the Brown-Pierce tumor in the rabbit (Burov and Andreevskaya, 1956), malignant melanoma in the human (Burov and Andreevskaya, 1956), Horie's sarcoma in the rat (Wagai and Kaketa, 1971), and glioma in the mouse (Oka, 1977). Continuous wave irradiations of approx. 350 W/cm 2 were used on the Brownu1~m VoL 4. No. ,*..--n

Pierce tumor in rabbits (Burov and Andreevskaya, 1956; Burov, 1956) with the result that not only was the irradiated tumor eliminated, but the metastatic nodules which were not treated were either resolved, granulated or petrified. The cure rates obtained in this study are quoted as 60-80%. ResuRs on nine human patients with malignant melanoma (Burov and Andreevskaya, 1956; Burov, 1956) showed that although the irradiated sites were resorbed, the metastases were not affected. In one series of experiments involving irradiation of Horie's sarcoma (Wagai and Kaketa, 1971), the average survival time in the irradiated group was 81.8 days compared to 56.3 days in the controls. It was noted that the incidence of lung metastases was the same for the irradiated group as for the control group. In another study involving irradiation of implantable murine glioma (Oka, 1977) in the abdominal wall of the mouse, there was a considerable reduction in the growth of tumors irradiated at 1000 Wlcm 2 for 2 sec compared to controls. At 1000W/cm" for 1 sec, the growth rate was minimally affected. At 100 W/cm 2 for 10 sec, the growth was somewhat above that of controls. This study also showed that mice implanted with blocks of ghoma irradiated by ultrasound in vitro showed an active immunity proven by higher rejection rates at the second transplantation of untreated glioma. This study documents the use of intense

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F. J. Fltv and L. K. JOHNSON

vertically in degassed mammalian Ringer's at 37°C. This method is described elsewhere (Fry et aL, 1978). This open procedure presumably would not be necessary if a more appropriate focusing system were used that avoided intense ultrasound at the skin level while providing sufficient intensity inside the tumor volume. For this animal preparation, a 4 M H z transducer with appropriate focal configuration would presumably avoid the skin opening procedure. For the BCNU (1,3 - bis(2 - chloroethyl)l nitrosourea); Bristol Laboratories, Syracuse, M A T I ~ J A I J AND Mitl'llOl~ NY) series, the drug was injected 24 hr after ultrasound irradiation (BCNU + ultrasound), Cells Hamster medulloblastoma cells (HM) or 24 hr after the same manipulation without (Rapp et aL, 1969) were maintained at 37oc as ultrasound (BCNU non-irradiated shams). monolayer cultures in sealed 75 c m 2 tissue The dose was 2 mg of BCNU per 150 grams culture flasks. Growth medium for the culture of body weight. This corresponds to 39°~ of a was BME, basic medium (Eagle), with Earl's single injection human dosage regime as salts and without L-glutamine or sodium recommended by the supplier (BiCNU Carbicarbonate (Grand Island Biological Co., mustine (BCNU) New Product Monograph, Grand Island, lqY). This medium was suple- 1977). mented with~fetal calf serum, sodium bicarIn order to provide the appropriate bonate, gentamicin and L-glutamine. ultrasound intensities, the tumor was irradiated at a number of sites placed in a recSolid tumor irradiation tangular matrix, since the tumor size is such The HM cells for innoculation of hamsters that no single position could appropriately (Ela: SYR hamster; Engle Laboratories, cover the necessary area. Selection of the Farmersburg, IN) were harvested with tryp- 4 mm spacing distance for the matrix sites sin-EDTA solution and resuspended in fresh means that most of the tissue is irradiated at media at a concentration of 107ceils/ml. a level between the spatial peak intensity and Hamsters were innoculated subcutaneously 6 d B down from this value. Multiple small in the left flank with 106 cells in a volume of intermatrix point regions receive less than the 0.1 ml. The area around the innoculation site 6 dB down level. With the 2 mm spacing dis* was shaved to facilitate injection. Within a tance the bulk of the tissue is irradiated at a period of 7-14 days, tumors reached what level between the spatial peak value and was considered the optimum size for irradia- 1.5 dB down from this value. No attempt was made to monitor tumor tion (an average of I cm in all dimensions). For irradiation, the hamster was anes- temperature or to record cavitation events. It thetized with Metofane (Pitman-Moore, Inc., can be presumed that the temperature and Broadview, IL) applied by a face mask, and cavitation events would be rather similar to the area around the tumor shaved and swab- those reported for ultrasonic irradiation of bed with alcohol. An incision was made over normal tissues at similar intensity levels the tumor, the skin pulled away and the (Lele, 1978a and 1978b). The automated system for irradiation is tumor exposed. This was done to prevent damage to the skin due to the intensity of the under computer control, and is described ultrasonic beam at the point of entry. The elsewhere (Fry et al., 1978). Irradiation site tumor was held to one side by a loop of positions, time-on period of the intense suture that had been carefully tied so as not focused ultrasound, and time period between to restrict the tumor's vascular supply in any irradiations are under computer control. The transducer was made from PZT No. way. (After irradiation, the loop of suture 5800 (Channel Industries, Santa Barbara, CA) was removed and the wound closed with a with a 7.5 cm aperture diameter, and the axial combination of 5--0 silk and 11 mm wound focal center at 13cm from the spherical clips.) For irradiation, the hamster was mounted on an animal holder and suspended transducer face. The operating frequency is

ultrasound in a mode to destroy a medulloblastoma implanted in the hamster flank. Animals were irradiated with ultrasound as the sole mediating agent, or with ultrasound in combination with a well-known chemotherapeutic agent. The aims of this study were to investigate the possibility of producing cures using ultrasound in tumor bearing animals, to evaluate increased survival time in non-cured animals, and to look for gross evidence of secondary tumor sites.

Tumorirradiationwith intenseultrasound 1. I 1 MHz, and the 6 dB beam width is 4 mm. Axially, the 6 dB beam length is 16 mm. For irradiation, the axial focal center was placed in a central plane of the tumor, which is approximately perpendicular to the beam axis. The matrix for irradiation was set up so that the tumor boundary was always covered by the maximum spatial beam intensity. Tumor growth or regression was recorded on all animals on a weekly basis by measurements made from the external skin surface. These data are not reported here. Additionally, a section of tissue from each tumor was removed at the time of death and placed in formalin. It is anticipated that a subsequent, more mechanistically oriented publication with additional temperature and cavitation data will include the above information.

DISCUSSION Introduction in the hamster flank with HM cells produces a tumor take of 95%. We judge the 5% lack of tumor take to be due most likely to a technical fault at the time of innoculation. No animal with a tumor take has ever had a spontaneous regression or lived longer than 79 days after innoculation. This tumor grows at a very rapid rate and, at the time of death, is usually larger than the animal itself. Calibration m e t h o d s

The 4 m m diameter, 6 d B focused beam used for tumor irradiation was calibrated by the radiation force method using a 1.98 mm diameter stainless steel ball mounted on a bifilar suspension (Dunn and Fry, 1972).

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itESULTS Results with the solid tumors in the hamster flank are shown in Table 1. There are six (6) groups of animals involved. Control animals are those which are innoculated and replaced in their cages. No subsequent procedures are performed except that of tumor growth determination from external skin measurements. Ultrasound shams receive all procedures involved in an ultrasound irradiation except exposure to the ultrasound. One ultrasound series involved a 4 mm spacing of the individual points in the irradiation matrix overlying the tumor region with a 2.5 sec time-on period at each point. When this spacing distance was reduced to 2 mm and the time-on period increased to 7 sec, the first cures (animals with no palpable tumor after 60 days and which continue to live out the normal life span) were in evidence. Secondary t um or sites occurred in 4 out of 9 animals in the 4 mm spacing series and in 3 out of 17 animals in the 2 mm spacing series using ultrasound alone. BCNU non-irradiated shams received all procedures that the BCNU plus 2 mm spacing ultrasound group received, except that the ultrasound was not turned on. There was one secondary tumor in the 10 animals in the BCNU plus 2 mm spacing ultrasound group. Secondary tumors were present in 2 of the 14 BCNU non-irradiated shams. Locations of secondary tumors for all groups are given in Table 2. This study shows that secondary tumor sites can evolve when ultrasound or ultrasound in combination with BCNU is used. These secondaries appear in body sites seen in other research and clinical studies.

Table 1. Solid tumors in hamster flank Treatment Sham Control Ultrasound 4 nun spacing 2 nun spacing BCNU non-irradiated sham BCNU + Ultrasound 2 mm spacing

Mean survival" Sig?

Percent cures

Sig.c

Percent 3econdaries

32.3 27.!

IqSe

0 0

NS

0 0

38.6 52.0 43.1

NS >0.05

0 29.4 0

>0.5 0.026

44.4 17.6 14.3

40.5

NS

40.0

0.020

10.0

"Days. bDouble-tailed t-test. ~2x 2 contingencytable (Finncy et al., 1%3). dNS = no significantdifference.

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F. J. Fay and L. K. Joast'~;~ Table 2. Location of secondary tumors Treatment Ultrasound 4 nun spacing

Animal number

Location of secondary tumor

4 14 17 26 31 35 38

L lung; L axiilary region Lung L axillary region; R lobe of lung R axillary region; R neck L axillary region L axillary region Peripheral to primary tumor

BCNU non-/rrad/ated sham

77

BCNU + Ultrasound

83 70

Peripheral to primary tumor; soft mass Peripheral to primary tumor Center of chest (non-invasive);

2 nun spacing I

L axillary region ~Two animals had neck nodules that resressed spontaneously.

Two BCNU non-irradiated shams developed tumors, apparently unconnected with but peripheral to the primary tumor. Five cured animals were re-innoculated as a potential test for any specific immune response activity. Four animals grew tumors and died within the normal time period seen for first innoculation shams. The animal with the longest time period between first innoculation and re-innoculation after cure did not develop a tumor. This animal was re-innoculated a second time without a tumor take. CONCLUSION

for non-cured treated animals and for BCNU non-irradiated shams is equivalent. Curative ultrasound (2mm spacing) alone yields a mean survival time in non-cured animals which is 58% longer than shams and 21% longer than BCNU non-irradiated shams. It is now important to study ultrasound regimes leading to a higher cure rate and, hopefully, less or no generation of secondary tumor sites. Also, ultrasound beam configurations and/or tumors in larger animals should be studied to avoid opening the overlying skin. Other combination therapies should be tried to study synergistic effects •leading, hopefully, to reduced dosages of the individual therapeutic modalities, and elimination of side effects, particularly secondary tumor sites. It is interesting that repeated re-innoculations would not produce tumor growth in one cured animal. This event needs further sutdy in order to define the conditions surrounding such a response.

Solid tumor irradiation in vivo with intense focused ultrasound at 907 W/cm z spatial peak intensity for 7 sec duration can lead to direct tumor tissue destruction. Using a 2 mm beam center spacing in a matrix designed to completely cover the solid tumor mass produced tumor extinction in 29% of the animals. Presumably a larger percentage would go to extinction if the tumor mass was more evenly irradiated than this spacing dis- AcknowledgementmThis research was supported by National Science Foundation Grant Number APR75tance provides. 14487. Approximately 18% of the animals treated with the regime producing 29% cure ~ E S developed a secondary tumor subsequent to Ballantine, H. T., Jr., Hueter, T. F., Nauta, W. J. H. and Sosa, D. M. (1956) Focal destruction of nervous tissue irradiation. When a considerably less than by focused ultrasound: Biophysical factors influencing optimal ultrasound irradiation regime leading its application. J. Exp. Me& I N , 337-360. to a mean survival time similar to shams was Bell, E. (1957) The action of ultrasound on the mouse liver. J. Cellular Comp. Physiol. S@, 83-104. used, 44% of the animals developed secondary tumors. For this tumor type, and pos- BiCNU Carmustine (BCNU) New Product Monograph (1977) Bristol Laboratories, Division of Bristol-Myers sibly for others, it appears important to select • Co., Syracuse, New York. the appropriate dosage and delivery regime. Bun~et, F. M. 0970) Immunological Survieilance. Pergamon Press, Sydney. When a single dose BCNU regime is apBurov, A. K. (1956) High intensity ultrasonic oscillations plied 24 hr after irradiation, the cure rate is for treatment of malignant tumors in animals and man. Doldady Aka& Nauk. SSSR I@6, 239-241. 40%. Under this regime, mean survival time

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