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ORIGINAL ARTICLE

Spatial dose distributions in solid tumors from 186Re

transported by liposomes using HS radiochromic media

Luis A. Medina & Beth Goins &

Mercedes Rodrguez-Villafuerte & Ande Bao &

Arnulfo Martnez-Davalos & Vibhudutta Awasthi &

Olga O. Galvn & Cristina Santoyo & William T. Phillips &

Mara-Ester Brandan

Received: 27 April 2006 / Accepted: 20 September 2006 / Published online: 8 February 2007# Springer-Verlag 2007

Abstract

Purpose A procedure for the measurement of spatial dose

rate distribution of beta particles emitted by 186Re-lipo-

somes in tumoral tissue, using HS GafChromic films, is

presented.

Methods HNSCC xenografts were intratumorally injected

with 3.7 or 11.1 MBq of 186Re-liposomes, and planar

gamma camera images were acquired to determine the

liposome retention in the tumor. After imaging, rats were

sacrificed and tumors were excised and processed in slices;

HS film sections were placed between slices and the tumor

lobe was reassembled. Tumors and films were kept in the

dark at 4C for 18 h. After irradiation, films were removed

and response was read using a transmission scanner. Films

were analyzed to determine two-dimensional spatial dose

rate distributions and cumulative dose volume histograms.

Dose rate distributions were quantified using a 60Co

calibration curve, the 186Re physical half-life, and a

perturbation factor that takes into account the effect of the

film protective layer.

Results Dose rate distributions are highly heterogeneous

with maximal dose rates about 0.4 Gy h1 in tumors

injected with 3.7 MBq and 1.3 Gy h1 in tumors injected

with 11.1 MBq. Dose volume histograms showed dose

distributed in more than 95% and 80% of the tumor when

injected with the lower and the higher activity, respectively.

Conclusion The described procedures and techniques have

shown the potential and utility of HS GafChromic film for

determination of dose rate distributions in solid tumors

injected intratumorally with 186Re-liposomes. The films

structure and the liposomes biodistribution must be taken

into account to obtain quantitative dose measurements.

Keywords Radionuclide therapy. Liposomes .186

Re .

HSGafchromicfilm . Intratumoraldosimetry. MCsimulation

Introduction

The interest in the use of liposomes as carriers of

therapeutic drugs and radionuclides has increased in recent

years [18]. Several investigators have reported the

advantages of using intravenously injected liposomes to

transport and specifically deliver therapeutic drugs intumors of hematopoietic origin [1, 5, 6, 9], and for the

treatment of solid tumors [3, 6, 10]. However, a major

limitation for the clinical application of chemotherapeutic

and radiotherapeutic liposomes in solid tumors has been

their insufficient penetration and, thus, their inhomoge-

neous distribution within the tumor tissue [2, 11, 12]. In the

case of radiotherapeutic liposomes, although the physical

characteristics of the radionuclide carried by the liposomes

could limit the level of variability in the spatial deposition

of the dose inside the tumor, the penetration and distribu-

Eur J Nucl Med Mol Imaging (2007) 34:10391049

DOI 10.1007/s00259-006-0297-x

L. A. Medina (*) : M. Rodrguez-Villafuerte :A. Martnez-Davalos : O. O. Galvn : M.-E. BrandanInstituto de Fisica, UNAM, A.P. 20-364,Mexico 01000 D.F., Mexico

e-mail: [email protected]

B. Goins : A. Bao : V. Awasthi : C. Santoyo : W. T. PhillipsDepartment of Radiology,

University of Texas Health Science Center at San Antonio,

San Antonio, TX, USA

A. Bao

Department of OtolaryngologyHead and Neck Surgery,

University of Texas Health Science Center at San Antonio,

San Antonio, TX, USA
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tion of the liposomes within the tumor still play an

important role in the spatial distribution of the dose.

An alternative option for an interstitial pressure-related

strategy for treatment of locally confined solid tumors, as in

the case of head and neck tumors, would be to inject the

liposomes directly into the tumor. This should increase

the internal pressure at the core of the tumor relative to the

surrounding tumor tissue, allowing the liposomes to spreadat the interior of the tumor [11]. In this manner a

radionuclide carried by the liposomes could gradually

irradiate the tumor depending only on the type and energy

spectrum of the emitted ionizing particles, producing a

better spatial dose deposition and a higher intratumoral

absorbed dose as compared with the intravenous injection

of the same kind of radiolabeled liposomes [13].

Our groups are interested in evaluating the therapeutic

use of a radionuclideliposome system directly injected in

solid tumors, based on direct dosimetric measurements.

Recently, a practical method for labeling preformed lipo-

somes by loading the therapeutic beta-emitting radionuclide186Re (T1/2=89 h; Emax=1.069, 0.932 MeV; Eave=0.359,

0.306 MeV; mean range 1.8 mm) [14] into the liposome

interior aqueous space was reported [15].

In the present work, we report first results describing a

dosimetric procedure based on the use of HS GafChromic

dosimetric media to determine the spatial distribution of

absorbed doses imparted by 186Re in tumor xenografts,

using an experimental model of head and neck cancer in

nude rats by means of intratumoral injection of 186Re-

liposomes. The features of high spatial resolution, weak

energy dependence, and good tissue equivalence of

GafChromic films have made this radiochromic material

useful for monitoring energy deposition in dosimetric

procedures with high dose-gradient radiation fields [16],

as is the case in radionuclide therapy with 186Re. HS films,

with an active radiochromic layer sandwiched between two

sheets of transparent 97-m-thick polyester, were designed

to increase dose response to high-energy photon beams

(>1 MeV) [17] in a dosimetric range of 0.540 Gy. HS film

contains the same active component as the previous MD-55

media, but has a higher sensitivity because of a thicker

active layer, 40 m in HS versus 15 or 30 m in MD-55-1

or MD-55-2, respectively [17; D. Lewis, 2006, personal

communication]. Thus, basic interactions between ionizing

radiation and the basic active components are the same for

both types of film, and the known dosimetric properties of

MD-55 films should apply to GafChromic HS media [18;

D. Lewis, 2006, personal communication]. Some inves-

tigators have reported the useful application of HS films in132Ir brachytherapy dosimetric procedures [19, 20]. Also,

GafChromic films have been used previously by Mayer et

al. [21] to determine beta-particle dose rate distributions in

tumor sections previously injected with 90Y. The present

report describes the dosimetric use of the high-sensitive HS

film to determine dose rate distributions in tumor sections

injected with 186Re-labeled liposomes.

Materials and methods

SCC-4 cell line

A human origin head and neck squamous cell carcinoma

(HNSCC) cell line, SCC-4, was originally obtained from

American Type Culture Collection (ATCC, Manassas, VA)

and provided by Martin Thornhill, PhD, of the University

o f Tex as Health S cience C en ter at S an An to nio

(UTHSCSA), School of Dentistry. SCC-4 cells were

cultured in Dulbeccos modified Eagles medium (Invitro-

gen, San Diego, CA) with 10% fetal bovine serum, 2 mM

L-glutamine, and antibiotics, penicillin, and streptomycin,

in 10020 mm2 Corning cell culture dishes (Corning Inc,

Corning, NY) at 37C in 5% CO2. When near confluence,cells were trypsinized and collected to determine the

number of cells and viability by trypan blue dye exclusion

assay. The appropriate volume of cell suspension was

transferred to a new tube and centrifuged in an Allegra 21R

Centrifuge (Beckman Coulter, Fullerton, CA) at 800 rpm at

4C for 5 min. Following aspiration of the supernatant, the

cell pellets were diluted with saline (Abbott Laboratories,

Abbott Park, IL) to a concentration of 5106 cells in 0.2 ml

saline. Aliquots of 0.2 ml cell suspension were drawn into

tuberculin syringes and used for subcutaneous inoculations

in nude rats.

HNSCC xenografts in nude rats

Animal experiments reported in the present paper were

performed according to the NIH Animal Use and Care

Guidelines and were approved by the UTHSCSA Institu-

tional Animal Care Committee. During all procedures, the

animals were anesthetized with 13% isoflurane in 100%

oxygen using an anesthesia inhalation machine (Bickford,

Wales Center, NY). Male rnu/rnu athymic nude rats

(Harlan, Indianapolis, IN) at 45 weeks of age (75100 g)

were inoculated subcutaneously with 5106 SCC-4 tumor

cells in 0.20 ml of saline on the dorsum at the level of the

scapulae. The animals were fed and housed following the

protocol for the nude rat living environment at the animal

facility at the UTHSCSA, and were checked daily after

tumor cell inoculation. When the growing tumor in each

animal was palpable and of sufficient size to be measured,

the tumor size was obtained via caliper by measuring the

length (l), width (w), and thickness (t) of each tumor. The

tumor volumes were subsequently calculated using

the ellipsoid volume formula, V =6 l w t [22].

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Preparation of liposomes

Liposomes comprised distearoyl phosphatidylcholine

(DSPC) (Avanti Polar Lipids, Pelham, AL), cholesterol

(Calbiochem, San Diego, CA), and -tocopherol (Aldrich,

Milwaukee, WI) having a molar percentage of 55:44:1. A

dried film was formed by mixing the lipid ingredients in

chloroform and then removing the chloroform by rotaryevaporation and vacuum desiccation for 24 h. The dried

lipid film was rehydrated with 300 mmol/l sucrose (Sigma,

St. Louis, MO) in sterile water for injection (120 mmol/l total

lipid) and warmed to 55C and then lyophilized over-

night. The dried lipidsucrose mixture was rehydrated

with 200 mmol/l reduced glutathione (GSH) (Sigma) in

Dulbeccos phosphate-buffered saline (PBS) (pH 6.3).

Before extrusion, the lipid suspension was diluted to

40 mol/ml with 100 mM GSH in PBS containing

150 mM sucrose. The diluted lipid suspensions were then

extruded through a series of Whatman Nucleopore

polycarbonate filters (Florham Park, NJ) (two passes,2 m; two passes, 400 nm; two passes, 200 nm; five

passes, 100 nm) at 55C using Lipex Extruder (Northern

Lipids, Vancouver, Canada). The extruded lipid solution

was then stored in the refrigerator at 4C for up to 6

months until needed for radiolabeling studies. Following

manufacture, the liposome particle diameter was mea-

sured using a 488-nm laser light scattering instrument

(Brookhaven Instruments, Holtsville, NY). The measured

diameter was 122.15.5 nm prior to the radiolabeling

experiment.

Preparation of 186Re-BMEDA

An SNS pattern ligand, N,N-bis(2-mercaptoethyl)-N,N-

diethylethylenediamine (BMEDA) was synthesized from a

modification of the method by Corbin et al. [23] and the

chemical structures were verified using 1H/13C NMR.186Re-aluminum perrhenate [186Re-Al(ReO4)3] was

obtained from Missouri University Research Reactor

(Columbia, MO). Stannous chloride (Aldrich) was used as

the reductant and glucoheptonate (GH) (Sigma) was used as

an intermediate ligand to make 186Re-BMEDA as previ-

ously described by Bao et al. [15]. BMEDA (1.0 l;

1.1 mg) was pipetted into a new vial. Then, 0.50 ml of

0.17 mol/l GH-0.10 mol/l acetate solution, pH 5.0, was

added, followed by the addition of 60 l of stannous

chloride (15 mg/ml). The pH of the solution was adjusted to

5.0 with 1.0 MNaOH. After flushing the solution with N2gas, 1.3 GBq (35.1 mCi), 0.70 ml of 186Re-Al(ReO4)3(0.4 g Re per mCi 186Re) was added. The vial was sealed

and heated in an 80C water bath for 1 h. The resultant186Re-BMEDA was used directly for the following 186Re-

liposome labeling.

Radiolabeling of preformed liposomes by loading 186Re

into liposome aqueous space

Liposomes were prepared with 1.2 ml of GSH neutral

liposomes (60 mM of total lipids) first undergoing

separation via Sephadex G-25 column chromatography

(PD-10 column, Amersham Biosciences, Piscataway, NJ)

eluted with PBS, pH 7.4. The product was added to the186Re-BMEDA solution and incubated at 37C for 1 h.

Again, the 186Re-liposomes were separated from any free186Re-BMEDA via Sephadex G-25 column chromatogra-

phy eluted with PBS buffer, pH 7.4. The labeling efficiency

(40%) was determined from the186

Re activity before and

after separation using a Radix dose calibrator.

Intratumoral administration of186

Re-liposomes

Two groups of nude rats (n=3/group) aged 45 weeks (75

100 g) and bearing a HNSCC xenograft with an average

volume of 2.00.4 cm3 were injected with 0.20.05 ml of3.7 MBq (0.1 mCi) (3.7-MBq group), or 0.60.05 ml of

11.1 MBq (0.3 mCi) (11.1-MBq group) of186Re-liposomes,

which contained 4.00.7 mg of DSPC and cholesterol. The186Re-liposome doses were equally divided and delivered to

several separate locations of the tumors, trying to cover the

central core of the tumor via intratumoral injections.

Image acquisition and biodistributions

Static planar images at baseline and 3 h after injection were

acquired using a micro-SPECT/CT scanner in a 565656

image matrix, with a pixel size of 2 mm (XSPECT, Gamma

Medica, Northridge CA). Images of the 186Re-liposomes

were acquired in a 186Re gamma-ray window (137 keV

15%) using a high-resolution, small field of view, parallel-

hole collimator (resolution

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the baseline image to determine the number of counts

detected in the body image (N0). Proportionality between

injected activity in the whole body (A0) and the number of

counts detected in the body image (N0) is assumed. The

percentage number of counts at some specific time t

accumulated in the ROI (%N(t)) was calculated as the ratio

of the counts in the ROI (NROI(t)) and the total number of

counts detected in the whole body at baseline, multiplied by100 (%N(t)=100 NROI(t)/N0).

Tumor sectioning and irradiation

After imaging, the rats were euthanized by cervical disloca-

tion under anesthesia. Tumor lobes were excised, washed in

saline solution, weighed and measured for volume determi-

nation, and embedded in agarose (3%) (USB, Cleveland,

OH) solution in TBE buffer concentrate (10) (USB,

Cleveland OH) to form a rectangular solid matrix with

33 cm2 transverse section. The agarose matrix helped to

provide support during the sectioning of the tumors into3-mm-thick slices. Dosimetric HS GafChromic film squares

(33 cm2) (ISP Technologies Inc, Wayne NJ; Lot

M0525HS) were placed between slices, and the tumor lobe

was carefully reassembled to its original shape, maintaining

the match between tumor slices. When necessary, thin ink

marks were drawn on the dosimetric film to provide

registration of the tissue outline. Tumors and films were

kept in the dark at 4C for 18 h. This time was selected as

practical for the performance evaluation of the HS Gaf-

Chromic film dosimetric procedure proposed in this work.

Dosimetry media handling and readout

GafChromic films were handled according to recommended

procedures [16] aimed at obtaining reproducible results of

the highest sensitivity. In particular: the films were kept in a

dark environment and, during handling, efforts were made

to reduce exposure to fluorescent light; films were cut 24 h

prior to irradiation in order to stabilize their response after

mechanical stress; calibration was performed using a

uniform field delivering dose in the range of interest;

readout took place 11 days after irradiation (sufficient to

reach color stabilization); and a film scanning procedure

was followed [24] to assure reproducibility.

After removal from the tumors, each film was washed

with water using cotton swabs to remove blood or

radioactive residuals. After washing, films were inspected

to assure that there was no sign of damage in their surface

or presence of radioactive residuals. No signs of surface

changes due to the direct contact with tissue and agarose

were observed in the films.

Film response was read 11 days after the end of

irradiation employing an Agfa DuoScan T1200 scanner in

transmission mode using a 36-bit RGB (12 bits per color)

and saved as tagged image file format. The optical density

range of the scanner was set to a maximum with all filters

and image enhancement options turned off. Two scanning

resolutions were used, 200 dpi for films uniformly

irradiated (calibration) and 300 dpi for films irradiated with

a highly non-uniform source (tumor irradiation). Every film

was scanned using an opaque frame to minimize lightcontributions from areas other than the film. Images were

analyzed by an in-house-written image manipulation

routine using MatLab 7.0 (Math Works, Natick, MA) to

obtain the red, blue, and green components of the image.

The film response, R, was quantified as:

R log10Cni

Ci

where Cni and Ci are the measured color levels of the

background (non-irradiated) and irradiated films, respectively

[24].

Film calibration

Film calibration between 0 and 40 Gy was performed using60Co gamma rays under conditions of charged-particle

equilibrium. Calibration readout took place 11 days after

exposure to match the conditions in the tumor irradiation

study.

It is known [16] that for photon energies in the range

0.11.33 MeV, and for secondary electrons from 0.1 to

1.0 MeV, photon mass energy absorption coefficients and

electron mass stopping powers for the active element in the

radiochromic films are within 2% of those for water. Monte

Carlo (MC) estimates (see Fig. 1) for the slowing-down

electron spectrum on the film active layer for a typical

arrangement used in this experiment show that15% of the

electrons have energies below 0.1 MeV. At these low

energies the electron mass stopping powers for the film are

within 4% of those for water, and this energy dependence

should have a minor effect on the water equivalence of the

film.

With respect to the use of 60Co gamma rays to calibrate

the response of the slowing-down beta spectrum, we have

based our technique on the analysis of Fig. 6 in reference

[16]. This graph shows the experimental energy depen-

dence in the response of MD-55-2 films to photons below

0.1 MeV, and the good data description by the normalized

mass energy absorption coefficient ratio (film sensitive

material to water). This agreement implies that the observed

dependence is totally accounted for by the initial photon

interactions. Therefore, one can conclude that a possible

energy dependence of the film response to the secondary

electrons, within the energy interval from about 15 to

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1,250 keV, is negligible. This has led us to assume that

calibration with 1.25-MeV gamma rays should be appro-

priate for the electron spectrum generated in the medium by

the 186Re beta particles (an MC estimate indicates that 98%

of the secondary electrons reaching the film active layer

have energies above 15 keV).

Effect of the HS film design on the dose measurements

The dosimetric medium studied in this work has a very

simple structure, as compared with other radiochromic

films [16]. However, its use for a continuous electron

spectrum, as in this case, requires an analysis of the

possible perturbation caused by the detector features on its

response as a dosimeter. The 49-m-thick sensitive layer,

where the deposited energy induces the visible change in

color, is inserted between two 97-m inert protective

layers, where no effect is induced. Figure 1 shows MC

results for the transport of beta particles from 186Re diluted

in a 3-mm-thick water layer (representing the tumor). The

geometry simulates the situation of a hypothetical central

detection plane (dashed line) between two tumor slices.

Figure 1 displays the slowing-down electron spectra

incident on A) a central plane in direct contact with both

water layers, B) the central plane in a 49-m-thick

radiochromic film without protective layers, and C) the

central plane in a realistic HS film. As seen in the

spectra, the main effect of adding material to the ideal

plane is to reduce the number of electrons without a

substantial change in the spectral shape. Calculation of

the absorbed dose at the sensitive layer indicates that,

because of the protective layers, the HS film detects 68%

of the dose that would be recorded in the hypothetical

situation depicted in Fig. 1a. The perturbation factor

(0.68)1 will be used as a correction to the dose measured

by the film. This effect has no influence at calibration

owing to the low absorption of the 60Co gamma rays.

Calibration curves were used to transform film response

into integrated dose received by the film during the 18 h of

dosimetric measurement. Doses were transformed into

initial dose rates at the tumor slice plane, assuming a fixed

liposome distribution inside the block, and taking into

account the 186Re physical half-life and the protective layer

perturbation factor.

Energy (MeV)

0.0 0.2 0.4 0.6 0.8 1.0

Intensity(a.u.)

0

500

1000

1500

2000

2500

a) 3 mm water - 3mmwater

c) 3 mm water - HS film - 3 mm water

b) 3 mm water - active layer - 3 mm water

Capa activa

Active layer

Water

Water

HS film

a

b

c

186Re

Fig. 1 MC simulations (106 histories each) of the electron spectra at the central plane of the three arrangements indicated in the right-hand

diagrams, as explained in the text

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Results

Figure 2 shows planar gamma camera images of two tumor-

bearing rats acquired at baseline and 3 h after intratumoral

injection with 3.7 or 11.1 MBq (0.1 and 0.3 mCi) of 186Re-

liposomes. The image analysis indicates that, at baseline,

506% of the injected material is retained in the tumor; 3 h

later, the liposome retention in tumor is similar, 456%. Interms of retained activity at 3 h, these values correspond to

1.7 MBq (0.045 mCi) and 5.0 MBq (0.135 mCi) of the186Re activity for the 3.7-MBq (0.1-mCi) and 11.1-MBq

(0.3-mCi) groups, respectively. By biodistribution calcula-

tions we have estimated the percentage injected activity per

organ as 483 and 4312, for the 3.7- and 11.1-MBq

groups, respectively, and the percentage injected activity

per gram as 153 and 2411, respectively, for the same

groups. The rest of the 186Re-liposomes move into blood

circulation and other organs (see Table 1). These observa-

tions are consistent with recent animal studies using

intratumoral injection of99mTc-liposomes of the same lipidformulation, showing that the amount of liposomes which

stays in the tumor after the immediate release remains in the

tumor for prolonged times [13]. Bao et al. [13] reported

approximately 44% retention of liposomes in the tumor 3 h

after injection, which was similar to our value. This

agreement is to be expected since biodistribution results

should depend only on the liposome formulation and not on

the carried radionuclide.

Figure 3 shows the calibration response curve of the HS

film as a function of absorbed dose in water. The response

of the film is shown in the three colours of the RGB

mode. Blue and green components show a linear

behavior throughout the dose range, suggesting thepossibility of extending the dynamic range of response

to doses higher than the nominal 40-Gy limit suggested

by the manufacturer (ISP Technologies Inc.). The red

component displays the most sensitive response, with

linear behavior at doses

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Figure 5 shows the two-dimensional spatial dose rate

distribution at excision time in six tumor slices from a rat of

the group injected with 11.1 MBq. These images are

representative of the distribution encountered in the other

animals. Dose rate values in each isodose (in this case, iso-

dose-rate) contour is denoted by a color as indicated by the

scale next to the graph. In both experimental groups it was

observed that the two-dimensional spatial distributions are

highly heterogeneous, with maximal dose rates (in some

hot spots) of about 0.4 Gy h1 in those tumors injected with

3.7 MBq and about 1.3 Gy h1 in tumors injected with 11.1

MBq. The lowest heterogeneity is observed in the central

slices (3 and 4).

In Fig. 6, a one-dimensional scan in horizontal and

vertical directions across the center of the two-dimensional

dose rate distributions in a central 11.1-MBq tumor slice

(slice 4 in Fig. 5) is presented. The figure shows relative

dose rate percentages along a single line through the center

of the horizontal and vertical directions. From representa-

tive scans in both groups of tumors it is possible to

conclude that the dose distributions are highly non-uniform

and non-symmetrical with respect to the center of the

tumor, reaching the highest doses in several hot spots

within the tumor volume, possibly associated with the site

of injection. Significant dose gradients within distances

smaller than 2 mm could be identified in the films.

Comparing dose distributions among the different tumors,

significant differences (larger than 30%) in the areas

covered by the iso-dose-rate curves of 0.08 Gy h1 were

observed.

In order to display the heterogeneity in the dose

distribution, Fig. 7 shows a cumulative dose volume

histogram for a tumor from each group. The tumor dose

was calculated from initial dose rate values in Fig. 5 (at

the time of sacrifice), integrated from baseline to 89 h (one186Re physical half-life), and assuming a constant liposome

retention in the tumor (equal to that at 3 h), as anapproximation to Baos measurements [13]. The histogram

for the 3.7-MBq group shows doses between 0 and

17 Gy heterogeneously distributed in more than 95% of

the tumor, and doses between 17 and 32 Gy in less than

5% of the volume. For the 11.1-MBq group, the

histogram shows doses between 0 and 17 Gy heteroge-

neously distributed in more than 80% of the tumor, and

doses from 17 to 32 Gy in less than 20% of the tumor

volume. Similar behavior was observed in the rest of the

tumors of these groups.

Dose (Gy)

0 10 20 30 40

Re

sponse

0.0

0.2

0.4

0.6

0.8

1.0

1.2

Red

Green

Blue

GafChromic HS

Fig. 3 Calibration curve response of the HS GafChromic film as a

function of dose. Calibration was performed with a source of 60Co

under charged-particle equilibrium conditions

Table 1 Biodistribution results

Values represent the

mean sem (n=3)

Organ 11.1-MBq group 3.7-MBq group t

% Inj. activity/organ % Inj. activity/g % Inj. activity/organ % Inj. activity/g t

Blood 20.64.14 1.50.34 21.03.25 1.50.21 tSkin 1.60.18 0.050.01 1.40.08 0.040.01 tHeart 0.10.01 0.10.02 0.10.005 0.10.01 tLiver 8.81.02 0.90.12 8.30.68 0.80.05 tSpleen 4.71.08 7.32.86 3.80.29 4.31.24 tKidney 0.90.09 0.90.10 0.80.03 0.80.03 tBowel 2.40.34 0.20.03 1.90.17 0.20.01 tBladder 1.20.20 1.80.42 0.60.16 1.20.17 tTestes 0.030.001 0.020.001 0.030.005 0.020.01 tSkin of neck 7.21.88 3.00.85 2.00.46 0.90.14 tBone 3.50.52 0.10.02 2.20.35 0.10.01 tMuscle 1.30.18 0.010.001 1.30.13 0.010.01 tStomach 0.60.12 0.10.01 0.60.01 0.10.02 tCecum 1.70.24 0.30.04 1.50.05 0.20.01 tLung 0.40.11 0.30.08 0.40.04 0.30.02 t

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Discussion

Success of radionuclide therapy for solid tumors with the

use of radiolabeled carrier systems, such as 186Re-lipo-

somes, is determined by an efficient delivery of cytotoxic

levels of absorbed dose throughout all neoplastic cells

within the tumor, with minimal irradiation of the surround-

ing normal tissues [4, 25]. However, factors such as the

limited tumoral penetration of the radiolabeled carrier

system, non-uniform activity distribution inside the tumor,

and the physical characteristics of the transported radionu-

clide lead to subcytotoxic absorbed doses of radiation to

neoplastic cells in the central region of the tumor, which

could result in sublethal damage repair [26, 27].

y(cm)

-1.0

-0.5

0.0

0.5

1.0

y(cm)

-1.0

-0.5

0.0

0.5

1.0

x (cm)

-1.0 -0.5 0.0 0.5 1.0

y(cm)

-1.0

-0.5

0.0

0.5

1.0

y(cm)

-1.0

-0.5

0.0

0.5

1.0

y(cm)

-1.0

-0.5

0.0

0.5

1.0

1

2

3

4

5

0.44

0.881.32

0.18

0.26

0.09

Dose rate

(Gy/h)

x (cm)

-1.0 -0.5 0.0 0.5 1.0

y(cm)

-1.0

-0.5

0.0

0.5

1.0

6

Fig. 5 HS films showing six

spatial dose distributions of

tumor slices from a tumor lobe

injected with 11.1 MBq of186Re-liposomes, and the

two-dimensional dose rate dis-

tributions calculated from the

digitalization of the HS films, as

described. Each iso-dose-rate

contour is denoted by a color as

indicated by the color-dose rate

scale. Films were irradiated

between tumor slices for 18 h

Fig. 4 Photographs of a) an

excised tumor, b) the agarose

matrix where the tumor was

embedded with the HS films, c)

the same agarose matrix viewed

from the bottom, and d) tumor

slices and films after removal

from the agarose matrix

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In order to achieve a better intratumoral penetration that

could result in a more homogeneous dose deposition inside

the tumor, intratumoral injection of the radionuclide system

could offer an alternative treatment option. However, a

better understanding of the dose deposition after intra-

tumoral injection of a radiolabeled carrier is necessary. A

goal of this study has been to describe and evaluate spatial

dose distributions in solid tumors from the direct measure-ment of dose rate in explanted tumoral sections of HNSCC

xenografts injected with 186Re-liposomes, using the recent-

ly available HS GafChromic film and digital image analysis

to evaluate the spatial dose distributions.

Image analysis during the first 3 h after186

Re-liposome

injection (Fig. 2 and ref. [13]) suggested that about 55% of

the injected activity was removed very quickly from the

tumor. It is known that pressure in the interstitial tissue of

solid tumors is uniformly elevated except in the outermost

rim, where it drops precipitously [11]. An intratumoral

injection of186Re-liposomes in the core of the tumor would

increase the pressure at that location, relative to thesurrounding tumor tissue, spreading the 186Re-liposomes

along the induced pressure gradient formed by convection

from the surrounding region to the periphery. In the

experiments reported here, HNSCC xenografts with vol-

umes of 2.030.37 cm3 and mass of 2.90.6 g were in-

jected with either 3.7 MBq/0.2 ml or 11.1 MBq/0.6 ml (i.e.,

18.5 MBq/ml) of 186Re-liposomes in several locations of

the tumor, aiming at producing a uniform distribution of

the injected activity. More than likely the increment in the

interstitial pressure of the tumor, due to the volume of the

injected 186Re-liposomes, promoted a high pressure gradi-

ent that induced a rapid removal of the liposomes from the

tumor into the blood circulation. However, the liposomes

that were not initially removed from the tumor (45%)

stayed within the volume and produced heterogeneous and

non-symmetrical dose distributions. The analysis of the

films also showed that the degree of heterogeneity was

lower in the central slices. Similar behavior was observed in

all tumors studied.

In general, the results showed that average absorbed

doses were higher at the central core of the tumor and lower

at the surface of the tumor. This result is significant when

compared with intratumoral absorbed dose distribution

when intravenous injection of the radionuclide carrier is

performed. A previous theoretical work that considered

intravenous injection of high-energy beta-emitters [28] has

reported that higher radiation doses are achieved at the

surface than in the core of the tumor, as a function of the

tumor diameter and the energy of the beta particle. In

the case of the intratumoral injection of 186Re-liposomes,

the size of the tumor, the volume and activity of the186Re-liposomes injected, the number and locations of the

sites of injection, and the retention time of the radionuclide

Dose (Gy)

0 10 20 30

V

olume(%)

0

20

40

60

80

100

3.7 MBq

11.1 MBq

Fig. 7 Cumulative (integral) dose volume histogram for a tumor from

each group. Doses were calculated using the two-dimensional iso-

dose-rate distributions during one 186Re half-life, as explained in the

text

Slice 4, Group 11.1 MBq

Horzontal axis (x/dx)

-1.0 -0.5 0.0 0.5 1.0

Relativedoserate(%)

Relativedoserate

(%)

0

10

20

30

40

50

Vertical axis (y/dy)

-1.0 -0.5 0.0 0.5 1.0

0

10

20

30

40

50

Fig. 6 One-dimensional scan, in horizontal and vertical directions

(dx=dy=2 cm), across two-dimensional dose rate distributions for the11.1-MBq group (slice 4 in Fig. 5). Arrows mark the physical edges of

the tumor

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inside the tumor will determine variations in the levels of

dose depositions between the core of the tumor and its

surface as well as the surrounding normal tissue. In terms of

dosimetric evaluations, a more detailed study that may

consider these parameters should be pursued.

Our method permits the estimation of the dose rate

spatial distribution at the moment of sacrifice. Complemen-

tary information, such as that provided by biodistributionstudies, e.g., the radionuclide effective half-life, might

permit the evaluation of the integrated total dose (and its

inhomogeneity) received by a tumor after the radionuclide

injection. Finally, the excellent spatial resolution associated

with radiochromic films may permit the use of the general

concept of this technique in smaller lesions, too.

We have presented the use of a commercial radiochromic

film, originally designed for penetrating gamma rays, in the

dosimetry of a distributed source of a beta emitter. We have

evaluated the effect of the particular design parameters in

its performance. In spite of the perturbation effect caused

by the convenient protective layer, we have shown itspotential use as a high-resolution image receptor. These

results could lead to improvements in the development of

new radiochromic media in the future.

Conclusion

This work has shown that the use of HS Gafchomic film

provid es experimental information that could help to

improve models and computational calculations of intra-

tumoral dose distribution in radionuclide therapy with high

energy beta-emitters. The use of the HS Gafchromic film

with the procedures and techniques described in the present

work is of value in the determination of dose rate

distributions and their degree of heterogeneity and symme-

try and in the evaluation of significant dose gradients in

small areas. Accurate use of these films under electron

irradiation requires specific consideration of the interaction

between the film structural design and the physi cal

properties of the radiation field.

Acknowledgements Luis A. Medina acknowledges the National

Institute of Cancerology (Mexico) for hospitality during the develop-

ment of this study, and the Instituto de Ciencias Nucleares, UNAM,

for the calibration of the films. The authors would like to thank Dr.Mohan Natarajan and Xiangpeng Zheng for help with SCC-4 cell

culture, Anuradha Soundararajan, Cristina Zavaleta and Maxwell

Amurao for help during the image acquisition, and Jos-Manuel

Lrraga for assistance with the Monte Carlo simulations. The authors

would also like to thank Dr. Cathy Cutler at the University of Missouri

Research Reactor for sponsoring our research under the Reactor

Sharing Grant (US Department of Energy grant DE-FG07-

02ID14380). This work was also supported in part by DGAPA-

UNAM grants IN-108906 and IN-110204.

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