Treatment of implanted liver tumors with focused ultrasound

PII S0301-5629(98)00134-3

● Original Contribution

TREATMENT OF IMPLANTED LIVER TUMORS WITHFOCUSED ULTRASOUND

LILI CHEN, GAIL TER HAAR, C. R. HILL , SUZANNE A. ECCLES, and GARY BOX

*Joint Department of Physics, Institute of Cancer Research and Royal Marsden Hospital, Sutton, Surrey, U.K.

(Received9 February1998; in final form 5 August1998)

Abstract—This article reports treatment of implanted liver tumors (HSN fibrosarcoma) with focused ultrasound(FUS). Experiments were carried out on implanted liver tumors in vivo. In order to determine the optimumtreatment conditions, various combinations of exposure parameters were investigated. The results showed thatit is possible to achieve total destruction of tumor cells in the treatment volume using an FUS system with afrequency of 1.7 MHz, with in situ ISAL of 261 W/cm2, 5-s exposure duration, and 1.5-mm exposure separation,with an in situ ISAL of 266 W/cm2, 10-s duration, and 2-mm separation, or within situ ISAL of 213 W/cm2, 8-sduration, and 1.5-mm separation. Fifteen selected tumors were treated with these experimentally determined“optimum” exposure conditions. All the tumors were destroyed completely. Assessment of tumor viability in thetreated volume was performed using both histologic and tissue culture methods. The mechanism of tumordamage, the limitations of the tumor model, and the effect of exposure parameters and liver blood flow on thetreatment are discussed. © 1998 World Federation for Ultrasound in Medicine & Biology.

Key Words:Ultrasound surgery, Tissue ablation, Liver tumors, HSN fibrosarcoma, Histologic changes, Tissueculture, Cell survival, Local tumor control.

INTRODUCTION

Focused ultrasound (FUS) surgery currently is receivingincreased attention due to the potential capabilities ofmagnetic resonance imaging to provide guidance andmonitoring (Cline et al. 1993, 1994, 1995; Hynynen et al.1994, 1995, 1996). There have been a number of studiesdesigned to investigate the potential of FUS for nonin-vasive surgery (Chen 1993; Clarke and ter Haar 1997;Fry and Johnson 1978; Hill 1994; Hill et al. 1994;Hynynen 1990; Hynynen and Lulu 1990; Malcolm andter Haar 1996; Rivens 1992; Sibille et al. 1993; ter Haaret al. 1989, 1991a, 1991b; Vaughan et al. 1994; Watkinet al. 1995; Yang et al. 1991). The use of this techniquefor a range of clinical applications has been described byter Haar (1995) and Hill and ter Haar (1995). This articlereports the treatment of implanted rat liver tumors withFUS. In order to ablate bulky (.1 cm3) tumors usingFUS, it is necessary to use an array of ultrasound expo-sures. Because the ultrasound lesion is cigar shaped, it is

difficult to choose an appropriate value for the spatialseparation of the lesions with which the treatment vol-ume can be covered completely, while avoiding theeffect of an existing lesion on the formation of a subse-quent lesion, i.e., lesion-to-lesion interaction (LLI)(Chen et al. 1997). In a previous study of FUS treatmentof tumors (ter Haar et al. 1991b), a fixed exposureseparation of 2 mm was used. Although some tissue inthe treated volume may have remained untreated for lowexposure intensities with short exposure durations, insome cases no viable tumor cells were found within thetreated volume. This reduced effect of LLIin vivo isthought likely to be a consequence of “indirect ultra-sound damage” due to compromised blood flow (Chen etal. 1997; Rivens 1992). It therefore seems possible, withthe correct selection of ultrasonic treatment conditions,to achieve total killing of tumor cells within a largevolume using an array of lesions. A judicious choice ofexposure conditions can be made, for which the numberof unexposed cells within the treatment volume is kept toa minimum.

In order to determine optimum treatment conditions(i.e., to destroy all the tumor cells in the predeterminedtreatment volumes with minimum exposure dose andacceptable treatment time), the influence of exposure

Dr. Chen’s current address is Radiology Department, StanfordUniversity, Stanford, CA 94305.

Address correspondence to: Lili Chen, Ph.D., Radiological Sci-ences Laboratory, Stanford University, Lucas MRS Imaging Center,Stanford, CA 94305-5488, USA. E-mail: [email protected]

Ultrasound in Med. & Biol., Vol. 24, No. 9, pp. 1475–1488, 1998Copyright © 1998 World Federation for Ultrasound in Medicine & Biology

Printed in the USA. All rights reserved0301-5629/98/$–see front matter

1475

parameters, namely, focal peak intensity, focal depth intissue, exposure time (duration of a single exposure), andexposure separation (the distance between the centres ofindividual exposures), on rat tumor treatments were stud-ied. The purpose of these experiments was to investigatethe possibility of total destruction of all the tumor cellswithin a given treatment volume. Based on these exper-imentally determined treatment conditions, a group ofrats implanted with HSN fibrosarcoma cells were treated.Assessment of tumor viability in the treated volume wasperformed using both histologic and tissue culture meth-ods. The results of treatments with varying exposureparameters are reported. The mechanism of tumor dam-age, the limitations of the tumor model, and the effects ofexposure parameters and liver tumor blood flow on thetreatment are discussed.

MATERIALS AND METHODS

EquipmentThe equipment used for the experiments has been

described in detail by Chen et al. (1993a), ter Haar et al.(1989, 1991b), and Rivens (1992). A schematic diagramof the experimental arrangement used in this work wasshown in Chen et al. (1993a). The high-intensity 1.7-MHz ultrasound focused field is produced by a plane,10-cm diameter piezoelectric ceramic transducer (PZT4)and focused by a biconcave Perspex (Lucite) lens withfocal length of 14 cm. The acoustic field was mappedusing a calibrated polyvinylidene difluoride (PVDF)membrane hydrophone (GEC Marconi, Chelmsford. Es-sex, UK) (ter Haar et al. 1989). Varying the total acousticpower of the transducer from 12 to 15 W resulted inspatially averaged focal intensities of 221–276 W/cm2

(measured in degassed water). In this article, “intensity”values are given in terms ofin situ spatially averagedfocal intensity, as determined under linear conditions(ISAL) (Hill 1994), unless otherwise specified. Thein situspatially averaged focal intensity ISAL (in situ) was cal-culated from the free-field spatially averaged focal inten-sity in water ISAL (free field) by:

ISAL ~in situ) 5 ISAL (free field)e2md

wherem (0.19 Np/cm) is the ultrasound attenuation co-efficient in liver for the frequency used in this work (1.7MHz), derived from the published data (Kuc et al. 1976)andd is the focal depth. The focal region is cigar shapedand is about 21 mm long3 2.1 mm wide (full-width,half-pressure maximum). The relationship among beamproperties, temperature elevation, and lesion size in ratliver has been reported elsewhere (Hill et al. 1994; Riv-ens 1992). The transducer, lens, and animal to be treatedwere suspended in a bath of degassed water. A laser

pointer was used to position the area to be treated at thepoint of peak intensity on the beam axis.

AnimalsBoth adult female CBH/Cbi (Chester Beatty

Hooded) rats and 12-week-old athymic nude mice (nu/nu) were used in this study. The rat livers were implantedwith HSN fibrosarcoma cells (North et al. 1982) andsubsequently treated with FUS. The procedure of tumorimplantation has been described by Chen et al. (1993a).Mice were used for assessment of treated tumor cellviability by injection of cells cultured from the treatedvolume at predetermined times after treatment. Allinvivo experiments were performed under Home Officeapproval, subject to the Animals (Scientific Procedures)Act 1986 UK.

Treatment procedureOne week after tumor cell implantation, laparotomy

was performed under general anesthesia using intraperi-toneal diazepam (0.05 mL per 100 g) and intramuscularHypnorm (0.1 mL per 100 g). Anesthesia lasted up to3 h. Tumor dimensions were measured on both the frontand back faces of the liver lobe using vernier calipers.The rat to be treated was mounted vertically in a cylin-drical Perspex tank with a thin Mylar window of dimen-sions 5 cm3 9 cm facing the transducer. The tank wasfilled with degassed phosphate buffered saline (PBS) at37°C. The holder was placed in a large tank of degassedwater.

The tumor-bearing liver lobe was exposed to anarray of FUS exposures at intensities in the range of213–266 W/cm2 with durations of 5–10 s and generallywith a 5-s interval between individual exposures. Theultrasound beam was focused at a depth of 1.5–3 mmbelow the tumor surface. The separation between thecenters of individual exposures was in the range of 1–2.5mm, a value derived based on our previous experimentaldata (ter Haar et al. 1991b). A margin of normal tissueabout 2 mm wide around the visible tumor also wastreated. During the treatment, the skin behind the exte-riorized liver was protected from damage using a cush-ion. The cushion comprised a thin rubber glove fingercontaining degassed water wrapped in damp gauze. Theliver surface was flattened using a Mylar window. Aftertreatment, the liver was replaced into the abdomen. Theabdominal wound was closed (with a 4-0 chromic suture)and the animal was allowed to recover.

Histologic processingRats were sacrificed from 1–24 d after treatment.

The treated liver lobes were removed and fixed inmethacarn. Three-micrometer thick histologic cross-sec-tions, taken at intervals of 0.25–1.0 mm through the liver

1476 Ultrasound in Medicine and Biology Volume 24, Number 9, 1998

lobe, were stained with either hematoxylin and eosin orvan Gieson stain. The slides were examined microscop-ically. In histologic examinations, particular attentionwas given to evidence of cell integrity and mitosis (takenas indicative of cell viability). The effect of ultrasoundlesioning on blood vessels (and vessel walls) and tissuerepair has been reported elsewhere (Chen 1993; Chen etal. 1993a).

Tissue cultureTraditional histologic methods have limitations in

their assessment. As the histologic sections are taken atvarious intervals through the treated liver lobe, a treat-ment can be considered to be unsuccessful if there areviable tumor cells observed on any histologic sections. Ifthere are no viable tumor cells found on the sections,however, one still cannot rule out the existence of viabletumor cells within the whole treated volume. In order toinvestigate the possible survival of any tumor cellswithin the treated volume, a tissue culture method hasbeen adopted for this study.

For the experiments using this tissue culturemethod, rats were implanted with HSN fibrosarcomacells and treated with FUS. Selection of treatment pa-rameters was based on results of histologic examinationsfollowing ultrasound treatment. The liver tumors wereexposed to 1.7-MHz FUS with an intensity of 213W/cm2, exposure separation of 1.5 mm, and exposureduration of 8 s. The beam focus was set at 2 mm belowthe tumor surface. This was found to be optimum in thehistologic slices.

The rats were sacrificed 2, 10, 14, or 17 d aftertreatment and the livers were removed. Treated anduntreated tumors were extracted from different animals.The untreated tumor tissue was taken as a control. Inorder to evaluate the effects of cell chemical toxin ontumor growth, mixtures of treated tumor with untreatedtumor (from the same liver), and of treated tumor withuntreated tumor and normal liver (from the same liver)also were processed for cell culture.

The tissue to be cultured was placed into sterilizedPetri dishes for tissue culture processing immediatelyafter the liver lobe was removed. The tissue was washedin PBS, minced, and enzymatically disaggregated with 1mg/mL DNASE I, 100 U/mL collagenase II, and 100U/mL collagenase IV for 1 h atroom temperature whilestirring. Cells were decanted from the resulting digestsolution, neutralised, and washed in 10% fetal calf se-rium (FCS) and then spun for 3 min at 80–100g. Thesmall pellet was suspended and washed in Dulbecco’smodified Eagle’s medium (DMEM) and spun again.Washing and spinning was repeated once more. Theresultant pellet was resuspended in 10 mL of DMEMcontaining 10% FCS and seeded into an 80-cm2 Nunc

tissue culture flask. The cells were incubated at 37°C inan atmosphere of 5% CO2 for at least 2 weeks. Themedium was changed after 7 d. The exhausted medium,containing floating debris, was spun for 3 min at 80–100g. The resultant pellet was resuspended in PBS con-taining 0.1% trypan blue. A cell viability count wasperformed under a microscope using a Neubauer count-ing chamber (supplied by Gallenkamp, B.S. 748). Thenuclei of dead cells stained blue.

Two weeks after incubation, the cells were fixedwith methanol at220°C for 20 min. The cells werewashed with PBS. The cells were stained with 10%Giemsa solution and examined microscopically. In somecases, regrowing cells were observed in the “treated”samples. The cells were identified morphologically asphagocytes and fibroblasts from the host, presumably inresponse to the ultrasonically induced damage in differ-ent stages after treatment. In order to investigate possiblepresence of any viable tumor cells among the phagocytesand fibroblasts, the samples were cultured for another 1month so as to obtain enough cells. The regrowing cellswere counted using a Neubauer counting chamber.

The cells then were injected into a nude mouse bysubcutaneous injection of these viable cells, suspended in0.1 mL of DMEM as described previously. Viable tumorcells from a metastasis (using the same amount of tumorand the same concentration) were injected into anothermouse as a control.

RESULTS

General observations

Tumor size.Fifty rats were implanted with HSNfibrosarcoma cells. Forty-six rats were treated with FUSand four rats were used as a control. For the controlgroup, one rat was untreated while the other three werepartially treated with FUS for cell chemical toxin effectstudy. Tumors grew in all the implanted livers. Paletumor nodules could be distinguished clearly from nor-mal liver tissue before treatment. Irregularly distributedperipheral blood vessels sometimes could be seen aroundthe edges of the tumors. Central necrosis was observed insome tumors, with apparent smaller thickness near thetumor center than that around the periphery regions,together with the presence of a concavity in the middle ofthe tumor where the tumor color was light yellow. Thesize of the tumors varied considerably: Three tumorswere between 3 and 5 mm in diameter, 43 tumors werebetween 5 and 10 mm in diameter, and eight tumors werebetween 10 and 15 mm. The average tumor diameter forthe 46 rats treated was 9.5 mm.

Among the 46 rats due for treatment with FUS, 22had visible tumor on the front and back faces of the liver.For these cases, the tumor thickness could be measured

Ultrasound treatment of liver tumors● L. CHEN et al. 1477

accurately using Vernier calipers. The tumor thicknessvaried between 3.5 and 9 mm; the average tumor thick-ness was 5.8 mm.

Number of tumor nodules.The number of tumornodules ranged from one to six for the 46 rats treatedwith FUS. Twenty rats had a single tumor. Ten rats hadtwo tumors. The rest had three to six tumors (threetumors in six rats; four tumors in six rats; five tumors inthree rats; and six tumors in one rat).

Tumor location.For convenience of description, thetumor location was simply divided into two sites. Onesite was considered to be the area around the edges of theliver lobe. Tumors growing in this area were described asedge tumors (ETs). The other site was the area at least 3mm away from the edges of the liver lobe. Tumorsgrowing in this area were described as central tumors(CTs). Among the 50 rats implanted with HSN fibrosar-coma cells, 38 rats had CTs and 12 had ETs. Both sitescould have either a single tumor or multiple tumors.

It was found that, in general, the host respondedquickly to the invasion of the implanted tumors, itsgreater omentum with dilated blood vessels envelopedwith the tumor surface. The extent to which greateromentum is in contact with the tumor depends upon thetumor position in the liver. In general, tumors growing inthe inferior liver lobes were well wrapped by the greateromentum.

Histologic observationsThe histologic changes in treated rat liver tumor

have been described in detail (Chen et al. 1993a). It hasbeen demonstrated previously that tumor damage can beidentified 1 d after treatment, and small islands of mor-phologically intact tumor cells with mitosis were foundwithin the treatment volume from 1–14 d after treatment

in the results reported in Chen et al. (1993a). Cell divi-sion was taken as evidence of tumor survival in theassessment. The histologic changes reported in this arti-cle are mainly of interest as an aid to the treatmentplanning, i.e., whether or not tumor cells can be damaged(morphologically) completely within a given treatmentvolume using experimentally determined treatment con-ditions. In the histologic examinations, any intact tumorcells seen within the treated volume are considered asevidence of a failed treatment.

We first report the histologic results of the treatmentof 31 rat liver tumors using different combinations ofultrasound parameters.

First treatment group.Table 1 gives the histologicresults of 10 liver tumors treated with free-field 276W/cm2 (ISAL) and 5-s exposure duration. The exposureseparation varied from 1.5–2.5 mm. The focal peak wasset at 0–3.5 mm below the tumor surface. The rats weresacrificed from 1–19 d after the ultrasound treatment.

For convenience, the tumor size has been dividedinto three grades depending on its diameter. Tumors ofdiameters between 3 and 5 mm are described as “smalltumors” (S). Tumors of diameters between 5 and 10 mmare “medium tumors” (M). For “large tumors” (L), thetumor diameter is from 10 to 15 mm. In order to inves-tigate whether the tumor location affects the result of thetreatment, the tumor location also is listed in Table 1.The tumor location has been divided into two types asdescribed previously.

From Table 1 it clearly can be seen that only tumorsthat grew near the central portion of the liver lobe couldbe treated successfully; intact tumor cells were found inthe histologic sections of all the ETs although the sameultrasound parameters were used for both CTs and ETs.This may be explained by the excessive heat loss into the

Table 1. Histologic results of ultrasound treatment of ten rat liver tumors.

Rat no. Tumor location Tumor size

ISAL

in situ(W/cm2)

Exposureseparation (mm)

Focal depthin tissue

(mm)Days aftertreatment

Remaining intacttumor cells

1 CT M 266 1.5 2.0 1 22 CT M 261 1.5 3.0 6 23 ET M 261 1.5 3.0 19 14 CT M 258 2.0 3.5 1 25 CT M 261 2.0 3.0 1 16 ET M 261 2.0 3.0 1 17 ET M 261 2.0 3.0 1 18 ET M 258 2.0 3.5 2 19 ET M 271 2.0 1.0 1 1

10 ET L 258/271 2.5/1.0 3.5/0 1 1

The free-field intensity was 276 W/cm2 and the exposure duration was 5 s. “1” 5 intact tumor cells are found on the histologicsections of the treated tumor; “2” 5 no intact tumor cells are found. CT5 central tumor; ET5 edge tumor; L5 tumor of large size;M 5 tumor of medium size; S5 tumor of small size. See text for further details.


water due to the immediate contact of the liver edgeswith the water bath. Although tumor 10 was treated usingtwo sets of parameters ([1] for the tumor portion at oraround the edges, thein situ intensity was 271 W/cm2,the exposure separation was 1.0 mm, and the beam focuswas set at the liver surface; and [2] for the tumor portionaway from the edges, the intensity was 258 W/cm2, theexposure separation was 2.5 mm, and the focal depth was3.5 mm in tumor), the treatment was still unsuccessful.

Tumors 4 and 5 both grew in the central portion ofthe liver lobe and were treated using similar ultrasoundparameters. The locations of the viable tumor cells foundonly on the histologic section taken at 0.5 mm below thefront face of tumor 5 corresponded to the gaps betweenthe individual exposures, which were not covered com-pletely by the lesion array, as observed on the day of thetreatment. The explanation of the total destruction of thetumor cells in the treated volume of tumor 4 is thought tobe due to the effect of the indirect damage (see Discus-sions in Chen et al. 1993a, 1997, and following).

There appeared to be no indication that the slightvariation in in situ ultrasound intensity (due to the vari-ation of focal depth of the ultrasound beam) had affectedthe results of the treatment.

Second treatment group.Table 2 gives the histo-logic results of eight liver tumors treated with free-field276 W/cm2 (ISAL) and 10-s exposure duration. The ex-posure separation varied from 1.0–2.5 mm. The focalpeak was set at 2–3 mm below the liver surface. The ratswere sacrificed from 1–3 d after the ultrasound treatment.Tumor 12 was treated with two different exposure sep-arations: 1.5 mm for tumor near the center, and 1.0 fortumor around the edges.

Here, all the CTs have been treated successfullyusing an exposure separation#2 mm, except for tumor17, which was treated with separation of 2.5 mm. Nointact tumor cells were found within the treated volumeon any of histologic sections for the successfully treatedtumors. For the ETs, intact tumor cells (some of them

were in the state of cell division at the time of sacrifice)were found only in the areas around the edges of thehistologic sections, which correspond to the tumor edges,whereas the tumor cells away from the liver edges havebeen destroyed completely. Again, the survival of thetumor cells in the areas around the liver edges may beattributable to the excessive heat loss to the bath watersurrounding the liver edges.

The treatment of Tumor 17 (a CT) was unsuccess-ful. An obvious reason for the failure is that the exposureseparation (2.5 mm) may have been too large for theultrasound intensity and exposure duration used. Theintact tumor cells (including dividing cells) only arefound on the first histologic section (about 0.5 mm belowthe front liver surface), and their positions correspond tothe gaps between the evident lesions observed on the dayof the treatment. It also was found that, using 1.5-mmexposure separation within situ ISAL 5 266 W/cm2 and10-s exposure duration, the tissue has been overtreated(some tissue began to fall off immediately). Although thetumor cells away from the liver edges were destroyedtotally, excessive tissue tearing and more “implosioncysts” (Chen et al. 1993a) were observed.

Third treatment group.Table 3 gives the histologicresults of 13 liver Tumors treated with 221 W/cm2 (ISAL)and 8-s exposure duration. The exposure separation var-ied from 1.5–2.5 mm. The focal peak was set 2 mmbelow the tumor surface. The rats were sacrificed from1–24 d after the ultrasound treatment.

For tumors located near the central portion of theliver lobe, there are no intact tumor cells found within thetreatment volume for all the tumors treated with 1.5-mmexposure separation at this intensity except for rat 26,presumably because the tumor was too thick (9 mmthick) to be treated at only one focal depth. Based on ourexperimental resultsin vitro, a “double-depth” treatmentis necessary for tumors of thickness.6 mm. The focalspot first should be placed at sufficient depth to damagethe back of the tumor, and then at a smaller depth to

Table 2. Histologic results of ultrasound treatment of eight rat liver tumors.


ISAL

in situ(W/cm2)

Exposureseparation

(mm)


(mm) Days after treatmentRemaining intact

tumor cells

11 CT M 266 1.5 2 2 212 ET M 261 1.5/1.0 3 1 113 CT M 261 2.0 3 3 214 CT M 266 2.0 2 1 215 CT M 266 2.0 2 1 216 ET M 266 2.0 2 1 117 CT M 261 2.5 3 2 118 ET M 266 2.5 2 1 1

The free-field intensity was 276 W/cm2 and the exposure duration was 10 s. Abbreviations as in Table 1.


damage the rest of the tumor up to the front surface. Inthis way, the creation of the second lesion would not beaffected by formation of the first lesion at the large focaldepth. Rats 30 and 31 failed to be treated successfully,presumably because the 2-mm exposure separation wastoo large at anin situ ISAL of 213 W/cm2. It is confirmedagain, by the results in Table 3, that the tumor locationaffects the tumor treatment; all the CTs,6 mm thicktreated with 1.5-mm exposure separation were destroyed“completely” (as seen from the histologic sections),whereas the only ET treated with the same exposureconditions showed some intact tumor cells at the areas ator around the edges of the liver lobe. Figure 1 shows theedge of the tumor from rat 28 taken 15 d after treatment.The tumor cells away from edge (a) have been damaged,

whereas those around the edges survived and developedinto a bulky tumor (b). Also, a small metastasis (M) canbe seen in the top left corner of the photograph.

As shown in Table 3, two CTs (24 and 25) weretreated successfully with FUS, as these tumors wereextracted 24 d after treatment. However, extensive me-tastases were found not only in the lungs, diaphragm,mesentery, intestines, and abdominal walls, but also inthe treated liver lobe (rat 24) outside the treated volume.Figure 2 shows the section of the treated live lobe for rat24. Area (A) shows that the original tumor has beensuccessfully treated, but in area (B) a new metastasisappears. These results demonstrated the limitations of thetumor model; investigations of long-term local controlcould not be carried out due to rapid onset of metastases.

From these histologic results it can be concluded

Table 3. Histologic results of ultrasound treatment of thirteen rat liver tumors.


ISAL

in situ(W/cm2)

Exposureseparation

(mm)


(mm) Days after treatmentRemaining intact

tumor cells

19 CT M 213 1.5 2.0 2 220 CT M 213 1.5 2.0 2 221 CT M 213 1.5 2.0 2 222 CT M 213 1.5 2.0 1 223 CT M 213 1.5 2.0 1 224 CT M 213 1.5 2.0 24 225 CT S 213 1.5 2.0 24 226 CT M 213 1.5 2.0 15 127 ET M 213 1.5 2.0 24 128 ET M 213 1.5 2.0 15 129 ET M 213 1.5 2.0 1 130 CT M 213 2.0 2.0 2 131 CT M 213 2.0 2.0 2 1

The free-field intensity was 221 W/cm2 and the exposure duration was 8 s. Abbreviations as in Table 1.

Fig. 1. Section of “edge tumor” 15 d after treatment, taken fromrat 28 (see Table 3). The tumor cells away from the edge (a)have been damaged, whereas those around the edges survivedand developed into a bulky tumor (b). A small metastasis (M)can be seen in the top left corner of the photograph (hematox-

ylin and eosin).

Fig. 2. Section of “central tumor” 24 d after treatment, takenfrom rat 24 (see Table 3). The original tumor has been suc-cessfully treated in area (A), but in the area (B) a new metas-

tasis appeared (hematoxylin and eosin).


that it is possible to damage all the tumor cells within thetreatment volume with judicious choice of exposure con-ditions. For CTs of thickness,6 mm, for instance, thiscan be achieved using anin situ ISAL in the range of261–266 W/cm2 with 5-s exposure duration and 1.5-mmexposure separation, orin situ ISAL ranging from 261–266 W/cm2 with 10-s exposure duration and 2-mm sep-aration, or anin situ ISAL of 213 W/cm2 with 8-s expo-sure duration and 1.5-mm separation.

Tissue cultureIn order to verify the experimentally determined

“optimum” treatment conditions reported in the previoussection, 15 CTs were selected and treated using either213 W/cm2 (in situ ISAL) with 1.5-mm exposure separa-tion and 8-s duration, or 266 W/cm2 with 1.5 mm-separation and 6-s duration, or 266 W/cm2 with 2.0-mmseparation and 10-s duration. Table 4 summarizes thetissue culture results of 19 liver tumors, 15 treated underthe “optimum” treatment conditions and four untreatedand or partially treated as a control. The focal peak wasset 2 mm below the tumor surface. The rats were sacri-ficed 2–17 d after the ultrasound treatment.

As can be seen from Table 4, all the 15 selected CTshave been treated successfully with the “optimum” treat-ment parameters based on the results of histologic ex-aminations. This is confirmed by both tissue culture andhistologic examinations. Intact tumor cells were found inall samples of the control group, both in the “pure” tumor

samples and in the “mixed” tumor samples, i.e., samplesof untreated tumor with treated tumor, and samples ofuntreated tumor with treated tumor and treated normalliver tissue, both taken from partially treated tumor-bearing liver lobes. There was no significant differencein cell survival between the “pure” tumor samples andthe “mixtures.”

For the treated tumors extracted 2 d after treatment,there were no tumor cells found in the cultured tissuesamples taken from the treated volume. Instead, thereappeared some phagocytes and young fibroblasts in thetissue samples (Fig. 3a). These cells are thought to haveinfiltrated from the untreated area due to the acute in-flammatory response to the ultrasound treatment. Thecells are different morphologically from the tumor cellsin the control samples (Fig. 3b). From 10–17 d aftertreatment, there were no tumor cells found within thetreated tissue samples (Fig. 4), although the fibroblastsbecame dominant and mature.

In order to investigate the possible existence of anytumor cells among the fibroblasts, the cells were culturedfor an additional 1 month. There were 63 103 viablecells (i.e., total cell number from one 80-cm2 flask)counted using a chamber as described previously. Thecells were suspended in 0.1 mL of DMEM and thenimplanted into one athymic nude mouse by subcutaneousinjection. The same number of viable tumor cells fromthe control group (using the same concentration) alsowere injected into another mouse as a control. Two

Table 4. Tissue culture results of ultrasound treatment of fifteen rat liver tumors and four control tumors.

Rat no. Tumor size

ISAL

in situ(W/cm2)

Exposureduration (s)

Exposureseparation (mm)

Focal depth(mm)

Daysafter

treatmentIntact

tumor cells

32 M 213 8 1.5 2 2 233 M 213 8 1.5 2 2 234 M 213 8 1.5 2 2 235 S 213 8 1.5 2 2 236 M 213 8 1.5 2 10 237 M 213 8 1.5 2 10 238 M 213 8 1.5 2 14 239 M 213 8 1.5 2 14 240 M 213 8 1.5 2 17 241 M 213 8 1.5 2 17 242 M 213 8 1.5 2 17 243 M 266 6 1.5 2 10 244 M 266 10 2.0 2 14 245 M 266 10 2.0 2 14 246 M 266 10 2.0 2 14 247 M Control (Untreated) 2 148 M Control (Partially treated) 2 149 M Control (Partially treated) 10 150 M Control (Partially treated) 10 1

Only “central tumors” (CT) were selected and treated with experimentally determined “optimum” treatment conditions. “1” 5 intacttumor cells are found using both the histologic and tissue culture methods; “2” 5 no intact tumor cells are found. Other abbreviationsas in Table 1.


weeks after injection, the mouse that had been injectedwith viable tumor cells had a tumor 4.8 mm3 6.8 mm.The other mouse, which was injected with the fibroblastscultured from the treated tomor sample, did not show anysign of tumor. Twenty-three days after injection, thetumor reached 10 mm in diameter in the mouse that wasinjected with viable tumor cells (mouse A in Fig. 5),whereas there was still no sign of tumor in the mouse thatwas injected with cells from the treated samples (mouseB).

DISCUSSION AND SUPPLEMENTARYOBSERVATION

Effect of exposure parameters“Optimum” ultrasound treatment is defined here as

the exposure that led to total destruction of tumor cells inthe treatment volume using a minimum ultrasonic doseand a short treatment time. The former ensures that aslittle damage as possible to the surrounding normal tissue

is caused during and after the treatment. The treatmenttime includes not only the exposure time but also thetime required to move the ultrasound source between theindividual exposures. The separation between the singleexposures varies with intensity and exposure duration. Inthis study, it was found that, for CTs, total destruction oftumor cells in the volume covered by the exposure arraycan be achieved using anin situ ISAL of 213 W/cm2 for8-s duration and 1.5-mm separation. For the same sepa-ration, this also can be obtained using slightly higherintensity and shorter duration, i.e., 261–266 W/cm2 insitu ISAL for 5-s duration. Also, using 261–266 W/cm2 insitu ISAL and a slightly longer duration of 10 s, theexposure separation can be increased to 2 mm to achievethe same treatment results.

During treatment, it was found that, for intensitieshigher than 266 W/cm2, holes always formed in thelesion center. More holes were observed in the normalliver tissue surrounding the tumor than in the tumortissue for the same exposure conditions. Using an expo-sure duration of 5–10 s within situ ISAL lower than 213W/cm2, the size of a single lesion is much reduced. Theexposure separation must be decreased (to ensure that thelesion array covers the whole treatment volume), and thelesion length can no longer cover a tumor with thick-ness.6 mm. Thus, the total treatment time can increasesubstantially for a given tumor volume. A compromisemust be made between “high intensity with short dura-tion” and “low intensity with long duration.” Accordingto the results of this study, within situ ISAL of 213W/cm2, 8-s exposure duration, and 1.5-mm exposureseparation, total destruction of tumor cells in the treat-ment volume can be achieved while avoiding tissueovertreatment (i.e., tissue “dropout”). The overall treat-ment time is also acceptable (see following).

Fig. 4. Photograph of a tissue culture sample. Fibroblasts be-come dominant and mature 2 weeks after focused ultrasound

treatment (magnification3193).

Fig. 3. Photographs of tissue culture samples. (a) Phagocytesand young fibroblasts in the treated tumor tissue. (b) Tumor

cells in the control tumor tissue (magnification3193).


It has been shown that the temperature in the tumorreaches about 79°C at the focal peak for intensities of213 W/cm and 5-s exposure duration. This was measuredusing a thermocouple, and lesions formed in the tissuevolume for which the temperature exceeded 60°C, basedon the histologic studies (Chen 1993). To ensure therequired tissue damage, 8-s exposure duration is chosenso that the high temperatures can be maintained for atleast 3 s. The 1.5-mm exposure separation was deter-mined from the results of histologic examinations. Ascan be seen from Table 3, total destruction of tumor cellsin the treatment volume has been achieved for 1.5-mmexposure separations, but not for 2-mm exposure sepa-rations; for a given combination of intensity and expo-sure duration, a 0.5-mm difference in exposure separa-tion can change the outcome of the treatment. It wasfound, however, that the treatment was not affected bychanging the focal depth within the range of 1.0–1.5 mmfor the given intensity and exposure duration for tumorthickness between 3.5 and 6 mm.

It should be mentioned that, whenever a statementsuch as “no viable tumor cells in the treatment volume.”“total destruction of tumor cells in the treatment vol-ume,” or “a successful tumor treatment” is given basedon the histologic examinations, it does not necessarilymean that there were no tumor cells surviving in thetreated volume. The results are only for a series ofsections taken from the treated volume and the cellviability is sometimes difficult to assess morphologi-cally.

Assessment of tumor viabilitySatisfactory assessment of the extent of long-term

local control by the ultrasound treatment has not beenpossible with the animal tumor model used in this study,which exhibits rapid and extensive onset of metastases.Tumor nodules in the lungs can be seen 1 week after thetumor implantation as discussed previously (Chen et al.1993a), and evidence of tumor metastasis in the treatedliver lobes is demonstrated in Figs. 1 and 2. There havebeen reports on direct entry of cancer cells into the veinsor their tributaries, although entry is usually via themicrovasculature. Thus, for these cells and those enter-ing the systemic veins via the lymphatics, apart fromrapid transit through the heart, the first major organsencountered are the lungs (Weiss 1985). In this investi-gation, both histologic and tissue culture methods havebeen used for cell viability assessment. The two methodsare complementary. The histologic method gives thewhole picture of morphological changes during tissuerepair after the ultrasound treatment. The tissue culturemethod can overcome the limitations of the histologicmethod in terms of investigating the possible survival ofany tumor cells in the treatment volume. However, itcannot be used to study tissue repair at different stagesafter the treatment.

Effect of tumor blood flow on focused ultrasoundtreatment

The effect of blood flow on the ablation of rat liverwith FUS and on temperature measurement in rat liver

Fig. 5. Tumor grows in the mouse (A; lower) injected with regrowing tumor cells. There is no sign of tumor growth inthe mouse (B; upper) injected with the cells cultured from the treated tumor sample (23 d after injection).


exposed to FUS has been reported quantitatively (Chen1993; Chen et al. 1993b). The results have shown thatblood perfusion affects the lesion size and temperaturerise in rat liver exposed to a beam of FUS. According tothe results of blood flow measurement (Chen 1993), theperfusion in any part of a liver tumor of any size is lessthan that in the surrounding normal liver tissue. Thismeans that the effects of blood flow on the lesion size inhepatic metastases should be much smaller than that innormal liver tissue. The “optimum” ultrasound parame-ters chosen for the rat liver treatment in this study werebased on the histologic results, which inherently includethe corrections for the effect of heat loss due to bloodflow.

Other factors affecting tumor treatmentAs shown in the previous section, there were always

intact tumor cells around the edges of the treated liverdespite the use of a variety of combinations of ultrasoundparameters. The reason for the survival of the tumor cellsaround the liver edges is thought to be due to the limi-tations of the experimental model used. During the ul-trasound treatment, the tumor-bearing liver was exteri-orized and the liver edges were in contact with the waterbath. As a result, the temperature at the tumor edgescould not be elevated to the treatment level. Furtherexperiments with higher intensities and shorter exposuredurations were carried out to elevate the temperature tothe threshold of tissue damage in order to destroy theseETs. The treatment was performed at higher intensity of434 W/cm2 (ISAL) and exposure duration of 5 s. Thefocal depth was 1 mm in the tumor tissue. It was ob-served that the damaged edge tissue dropped off near theexposure centers and the liver edge became saw shapedafter the treatment. Although no intact tumor cells werefound on the histologic slides (the rat was sacrificed 2 dafter treatment), it seemed that such exposure conditionsfor treatment ofin vivo hepatic tumors are not appropri-ate.

It has been discussed that tissue damage can arisethrough two different mechanisms: there is “direct,” pri-marily thermal, or mechanical (cavitation, acousticstreaming) damage and “indirect” damage resulting fromcompromised blood supply (Chen et al. 1993a). It isinteresting to note that, under the same exposure condi-tions, intact tumor cells can be seen around the liveredges for all the samples, whereas no viable normal livercells around the edges of the liver lobe have survived theultrasound treatment. Therefore, there must be someways the tumor cells that survived the direct thermal ormechanical damage can survive under conditions ofcompromised blood supply.

In order to investigate whether “indirect” ultrasounddamage has the same effect on both tumor cells and

normal liver cells, a separate experiment was carried out.A tumor-bearing liver lobe was treated with FUS of 418W/cm2, 10-s exposure duration, and 2-mm exposureseparations. The damaged area was produced in a spe-cially designed way. A narrow band of liver and tumortissue was treated with FUS in order to block the bloodsupply from the hepatic artery and portal vein. The ratwas sacrificed 5 d after the treatment and the liver wasremoved for histologic examinations. The results showedthat not only the tissue (both normal liver and livertumor) in the treated area, but also the tissue below thetreated area, was damaged (Chen et al. 1997). However,there were still some viable tumor cells observed aroundthe edges of the liver lobe. Clearly, these tumor cellsmust have received blood supply from other sourcesbecause both the hepatic artery and portal vein had beencut off completely.

A possible explanation is that the blood supply fromthe greater omentum played an important role in tumorsurvival around the edges of the liver lobe. It was foundthat the host had responded to the FUS treatment only 2 hafter the treatment; close contact of the greater omentumto the treated area began to form. Most of the tumor cellsthat had survived the “direct” ultrasound damage diedshortly after the treatment, due to lack of blood supply.These necrotic cells easily could be recognized morpho-logically in the untreated areas on the histologic sections.However, some tumor cells survived because they re-ceived enough nutrition from the capillaries of thegreater omentum after treatment. The edges of the liverlobe were in better positions to make contact with thegreater omentum. Thus, if some tumor cells around theliver edges survived the “direct” ultrasound damage, theycould survive with the support of the greater omentum,even if the blood supply from the hepatic artery andportal vein had been interrupted. Furthermore, it wasfound from histologic study that, 3 d after treatment,young granulation tissue generally has reached the edgesof the liver lobe (Chen et al. 1993a). Therefore, tumorcells that are nourished mainly by greater omentum afterthe treatment were further rescued by young fibroblastswith new capillaries. It appears that tumor cells must bemore tolerant towards a poor blood supply than normalliver cells, at least during the first few hours after treat-ment.

In order to study the role of the greater omentum intumor survival, a series of specially designed experi-ments were carried out. Six rats were used for theseexperiments. Tumors were implanted at the edges of theliver lobe and then treated with FUS at intensity of 261W/cm2, 10-s exposure duration, and 2-mm exposureseparations. After treatment, either the greater omentumwas cut or the treated area was covered using a thinrubber glove finger to prevent the treated area from


coming in contact with the greater omentum. The ratswere sacrificed either immediately or 6, 14, or 24 h afterthe treatment, and the tumors were processed for histo-logic examination. The results showed that some tumorcells looked intact in the tumors extracted immediatelyand 6 h after the treatment (Fig. 6). For the tumorsextracted 14 and 24 h after the treatment, however, onlynecrotic tumor cells could be seen throughout the sec-tions (Fig. 7). These experiments confirm that the livertumor blood supply is from not only the hepatic arteryand portal vein but also the greater omentum once goodcontact between tumor tissue and the greater omentumhas been established. It also seemed to be true that someHSN fibrosarcoma cells could survive for at least 6 hwithout proper blood supply.

Mechanisms of tissue damage with focused ultrasoundThe mechanisms of the tumor damage with FUS are

not yet fully understood. It is believed that thermaldamage is the predominant factor as temperature is ele-vated to 80–100°C in the focal peak in the rat livers andtumors (Chen et al. 1993a; Reilly et al. 1991).

Host immunity may play a role in the tissue dam-age. It was observed that there appeared to be morelymphocytes in the residual tumor area than in the un-treated tumors (Fig. 8). The concept of immunostimula-tion by tumor breakdown products has been advocatedby several investigators, and experiments have been per-formed using electrocoagulation (Ziegler et al. 1980),

radiation (Topalian and Ziegler 1987), laser (McCormicket al. 1989) in the treatment of C1300 neuroblastoma.Both tumor-specific and tumor-nonspecific immune re-sponses may be involved in the immunostimulation pro-cess. There also has been a report of significant reductionin tumor growth following a second tumor challenge inpreviously FUS-cured animals (Wagai and Kaketa1970). After FUS treatment of Horie’s sarcoma (1000W/cm2), the cured rats developed a high resistance tosubsequent tumor challenge.

It is believed that “indirect” ultrasound damage mayhave been involved in the treated tissue volume. It hasbeen demonstrated that, with the existing therapeuticsystem, no ideal lesion separation value has been foundin excised porcine liver (Chen et al. 1997). Because theultrasound lesion is cigar shaped, it is difficult to find aproper value for exposure separation with which thetreatment volume can be covered completely and at thesame time LLI can be avoided. In this study, however,the results show that it is possible to destroy all tumorcells within a given treatment volume. The reduced LLIeffect in in vivo tumor treatment may be attributed to“indirect” damage due to lack of blood supply after theFUS treatment and subject to the distribution of the localblood vessels.

There are also suggestions of other possible non-thermal damage, such as that due to the effects on cellmembranes reported by ter Haar (1980) and Loverock etal. (1991).

Fig. 6. Intact tumor cells (T) in the regions around the edges of the treated liver lobe (E). The liver was extracted 6 hafter focused ultrasound treatment. The greater omentum was removed after treatment to prevent the treated liver from

being in contact with the greater omentum (hematoxylin and eosin, magnification3150).


Does focused ultrasound promote metastases?Fry and Johnson (1978) have suggested that FUS

could promote metastases. They reported that those an-imals irradiated with ultrasound developed metastases,whereas those not receiving ultrasound exposures didnot. However, no evidence was found in this study tosupport their result. It was observed that the number oflung metastasis nodules in all the ultrasound-treated an-imals were considerably fewer than in the control ani-mals. This is consistent with the results reported by Gossand Fry (1984) and Yang et al. (1991).

Future workThe results of this study are of significance to the

clinical application of the technique. However, the ex-perimentally derived optimal exposure parameters can-not be applied directly to humans. Human liver tumorsgenerally lie deeper and are considerably larger than theexteriorized rodent tumors treated in this work. This wasnot truly a noninvasive treatment. Determination of op-timum exposure conditions for noninvasive treatment ofhuman liver tumors should be the subject of furtherstudies using large animals, such as pigs, before beingused for clinical trials.

SUMMARY

Treatment of implanted rat liver tumors with FUShas been carried out. The purpose of these experiments

was to investigate the possibility of total destruction ofall tumor cells within a treatment volume.

In order to determine the optimum treatment con-ditions, various combinations of exposure parameterswere investigated. The results show that it is possible toachieve total destruction of tumor cells in the treatmentvolume using anin situ ISAL of about 261 W/cm2 with5-s exposure duration and 1.5-mm exposure separation,or anin situ ISAL of about 266 W/cm2 with 10-s exposureduration and 2-mm separation, or anin situ ISAL of about213 W/cm2 with 8-s exposure duration and 1.5-mmseparation. To verify these experimentally determinedtreatment parameters, 15 selected tumors were treatedwith “optimum” exposure conditions. All the tumorswere destroyed completely.

Both histologic and tissue culture methods wereused in this study for assessment of tumor viability afterFUS treatment. The histologic method gives the wholepicture of the morphological changes and, therefore,information about tissue repair. The tissue culturemethod can overcome the limitations of the histologicmethod in terms of investigating the possible survival ofany tumor cells in the treatment volume.

The limitations of this treatment model were dis-cussed. Due to the rapid onset of development of metas-tases, long-term local control with the FUS treatmentcould not be assessed. Because FUS treatment was car-ried out with the tumor-bearing liver exteriorized, the

Fig. 7. ”Indirect” tumor damage (I) in the areas around the edges of the liver lobe (E) 14 h after focused ultrasoundtreatment. The greater omentum was removed after treatment to prevent the treated liver from being in contact with the

greater omentum (hematoxylin and eosin, magnification3240).


temperature rise in the regions around the edges of theliver lobe were lower than the threshold temperature fortissue damage. Therefore, some tumor cells may survivethe intense ultrasound exposures during the treatment.

The greater omentum may play an important role inthe survival of tumor cells in the regions around the liveredges, as the capillaries of the greater omentum providenecessary blood supply after the treatment. These sur-viving tumor cells are further rescued by the youngfibroblasts with new capillaries that developed in thetreated volume. It appears possible that the tumor cellsare more tolerant towards the hostile living conditionsthan are normal liver cells.

Acknowledgements—This work was supported by program fundingjointly from the UK Cancer Research Campaign and the MedicalResearch Council. UK Home Office approval was sought and grantedfor the in vivo work reported here. We thank Dr. J. P. M. Bensted forhistologic advice and Dr. I. H. Rivens for valuable comments on themanuscript. We are grateful to Sue Clinton for her expert technicalsupport.

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Treatment of implanted liver tumors with focused ultrasound