Most carcinomas are prone to metastasize despite successful treatment of the
primary tumor. One way to address this clinical challenge may be targeted
therapy with α-emitting radionuclides such as astatine-211
(211At). Radioimmunotherapy utilizing α-particle
emitting radionuclides is considered especially suitable for the treatment
of small cell clusters and single cells, although lesions of different sizes
may also be present in the patient. The aim of this study was primarily to
evaluate the toxicity and secondarily in vivo efficacy of a
211At-labeled monoclonal antibody (mAb) directed against
colon carcinoma with tumor diameters of approximately 10 mm.
Methods
Eighteen rats with subperitoneal syngeneic colon carcinoma were allocated to
three groups of six animals together with three healthy rats in each group.
The groups were injected intravenously with either 150 μg of
unlabeled mAbs (controls) or 2.5 or 5 MBq 211At-mAbs
directed towards the Lewis Y antigen expressed on the cell membrane of
several carcinomas. Tumor volume, body weight, and blood cell counts were
monitored for 100 days after treatment.
Results
Local tumors were non-palpable in five out of six rats after treatment with
both activities of 211At-mAbs, compared to one out of six in the
control group. At the study end, half of the animals in each group given
211At-BR96 and one animal in the control group were free from
disease. Radioimmunotherapy resulted in dose-dependent, transient weight
loss and myelotoxicity. Survival was significantly better in the groups
receiving targeted alpha therapy than in those receiving unlabeled mAbs.
Conclusions
This study demonstrates the possibility of treating small, solid colon
carcinoma tumors with α-emitting radionuclides such as 211At
bound to mAbs, with tolerable toxicity.
The online version of this article (doi:10.1186/2191-219X-3-23) contains supplementary material, which is available to authorized users.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SEE designed and performed the in vivo studies, evaluated the
immunohistochemistry, performed the statistical analyses, and wrote the manuscript.
TB participated in planning the experiment, performed the dosimetric calculations,
and contributed to data interpretation and manuscript writing. EE participated in
planning and performing the in vivo studies. HJ developed and performed
production of 211At. SL developed and performed the radiolabeling, and
contributed to data interpretation and manuscript writing. JT and RN contributed to
the study design and analyses of data as well as writing of the manuscript. All
authors read and approved the final manuscript.
Background
Although the treatment of primary tumors is often successful, metastatic disease is
the major cause of cancer-related mortality [1]. There is thus a need for new treatment modalities targeting metastases
in order to improve the survival of patients suffering from malignant tumors.
During radioimmunotherapy, radionuclides are directed to tumor lesions by specific
targeting using monoclonal antibodies (mAbs) as carrier molecules. The concept has
resulted in two FDA-approved radioimmunoconjugates, 90Y-ibritumomab
tiuxetan and 131I-tositumomab, for the treatment of non-Hodgkin's
lymphoma. However, several studies have shown radioimmunotherapy to be less
effective in the treatment of larger solid tumors [2, 3], and the focus has therefore changed to treating small lesions including
metastases [4, 5]. Due to the relatively long path length of β-particles,
β-emitting radionuclides are generally considered unsuitable for targeting
microscopic tumors as much of the radiation will be deposited outside the tumor.
Alpha-emitting radionuclides have a much shorter particle path length (typically
<100 μm) resulting in less irradiation of healthy tissue [6]. One such α-emitting radionuclide is 211At, which has a
half-life of 7.2 h and a particle range in soft tissue of 55 to
80 μm. Promising results have been obtained in preclinical studies on
211At-labeled mAbs in models of, e.g., leukemia [7, 8] and ovarian [9‐11] and colon carcinoma [12], and in clinical studies of glioma and ovarian carcinoma [13, 14].
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Colon carcinoma is among the most common cancer diseases and is prone to metastasize [15]. In vivo studies on radioimmunotherapy with the β-emitting
radionuclide 177Lu (maximal range in soft tissue, 1.8 mm) with the
aim of treating colon carcinoma metastases resulted in prolonged survival [16, 17]. The primary aim of the present study was to evaluate the toxicity and
secondly the therapeutic efficacy of 211At-labeled mAb on small solid
tumors of colon carcinoma in a syngeneic immunocompetent rat model. To the best of
our knowledge, 211At-mAbs have not previously been evaluated in an
immunocompetent animal model. The same animal model and mAb have previously been
utilized for studies of radioimmunotherapy with the β-emitting radionuclides
177Lu and 90Y [18, 19].
Methods
Monoclonal antibody
BR96 (Seattle Genetics Inc., Seattle, WA, USA) is a chimeric (mouse/human) IgG1
mAb recognizing the Lewis Y epitope. Lewis Y is expressed in several carcinomas,
including breast, gastrointestinal, pancreatic, non small cell lung, cervical,
and ovarian cancer, and in some melanomas. As with many tumor-associated
antigens, the epitope is also expressed in some normal tissues, including the
epithelial cells of the gastrointestinal tract in humans [20] and in the rat strain used in this study [19].
Radiochemistry
Astatine-211 was produced by irradiating stable bismuth using the
209Bi(α,2n)211At reaction at the Cyclotron and
PET Unit, Rigshospitalet, Copenhagen, Denmark. After irradiation, the target was
transported to the Department of Nuclear Medicine at Sahlgrenska University
Hospital, Gothenburg, Sweden, where the astatine was transformed into a
chemically useful form by dry distillation, as described previously [21].
211At labeling of BR96 was performed essentially as described
previously [22]. Briefly, the antibody was first reacted with the
N-succinimidyl-3-(trimethylstannyl) benzoate reagent to give the
ε-lysyl-3-(trimethylstannyl)benzamide BR96 immunoconjugate. After
30 min of incubation with the reagent, the BR96 conjugate was isolated in
0.1 M citrate buffer (pH 5.5) using a Sephadex NAP-5 column. From the
BR96 conjugate preparation, 800 μg was added to a vial containing
336 MBq of 211At oxidized by 15 μL
N-iodosuccinimide (NIS; 67 μM) in methanol with 1% acetic
acid during vigorous agitation. After 1 min, 3 μL NIS (1 mg/mL)
was added, and the reaction mixture was incubated for 1 min before
terminating the reaction with 5 μL sodium ascorbate (50 mg/mL). The
antibody fraction, 211At-BR96, was isolated using a Sephadex NAP-5
column. To protect the antibody from radiolysis, the 0.9-mL product volume was
eluted into a vial containing 0.1 mL phosphate buffered saline (PBS) with
10% bovine serum albumin. Unlabeled BR96 was added to adjust the mAb dose to
150 μg per animal.
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The antigen-binding properties (immunoreactivity) of 211At-BR96
relative to BR96 were analyzed by determination of the equilibrium binding
constant (Kd) by saturation binding curve analysis as previously described [18], using BN7005 cells as the target antigen. The immunoreactivity is
given by the ratio Kd(BR96)/Kd(211At-BR96).
Animal model
The BN7005-H1D2 cell line was originally established from a
colon carcinoma detected in a 1,2-dimethylhydrazine-treated Brown Norway rat.
The cell line expresses the Lewis Y antigen both in vitro and in
vivo. The survival fraction after external irradiation with 2 Gy
has been determined to be 0.5, which indicates moderate radiosensitivity
comparable to human colon carcinoma cell lines [23]. Cells were cultured in RPMI 1640 medium supplemented with 10% fetal
bovine serum, 1 mM sodium pyruvate, 10 mM Hepes buffer (all from PAA
Laboratories GmbH, Pasching, Austria), and 14 mg/L gentamicin (Gibco,
Invitrogen, Carlsbad, CA, USA) at 37°C in a humidified environment before
harvesting by trypsin treatment and suspension in supplemented medium prior to
inoculation.
Immunocompetent male Brown Norway rats (Harlan Laboratories, Horst, the
Netherlands), with a body weight of approximately 250 g, were inoculated
with 3 × 105 BN7005-H1D2 cells
in 50 μL culturing medium between the peritoneum and the abdominal muscle
under anesthesia with isoflurane (Abbott Scandinavia AB, Solna, Sweden). Tumor
growth was monitored by palpation and tumor measurement with a digital caliper.
The animals were housed under standard conditions and fed pellets and fresh
water ad libitum. The experiment was approved by the regional animal
ethics committee and followed Swedish legislation on animal welfare and
protection.
Dosimetry and estimation of maximum tolerable activity
The activities of 211At in this study were chosen with respect to the
absorbed dose to the bone marrow. In a review on the toxicity of α-emitting
radionuclides, Dahle et al. [24] presented values for maximal tolerable doses to the bone marrow
(MTDBM) ranging from 0.4 to 7.6 Gy for different
α-emitters. Intravenous (i.v.) administration of 211At-labeled
antibodies to mice indicated values of MTDBM from 2 to 4 Gy [25, 26]. A value of 4 Gy was chosen as the MTDBM in the
present study, and the absorbed dose to the bone marrow was estimated using bone
marrow uptake data from a previous biodistribution study of
177Lu-BR96 in the same animal model [27]. Using these 177Lu-BR96 uptake data (% injected activity/g
tissue), theoretical time-activity curves were obtained for
211At-BR96 and used to calculate the cumulated activity (i.e., the
total number of decays, Ã) of 211At. Assuming an
absorbed fraction of the α-particles, ϕα, to be 1 and including contributions only from α-particles, the
absorbed dose (D) was calculated using the following equation:
where m denotes tissue mass, and Δα is the mean
energy released per 211At decay (here assumed to be
1.09 × 10−12 J). The estimated mean
absorbed dose to the bone marrow for 211At-BR96 was found to be
0.8 Gy/MBq. Using the assumed MTDBM of 4 Gy, the
corresponding maximum tolerable activity that could be injected was
5 MBq.
Radioimmunotherapy
Two weeks (defined here as day 0) after inoculation, the tumor-bearing rats were
allocated to three groups (six rats per group) with similar distributions of
tumor size, see Table 1. In addition, three rats without
tumors were included in each group to monitor toxicity without influence from
tumor burden. The rats were injected with either 150 μg of unlabeled
BR96 in PBS (control group), 2.5 MBq 211At-BR96 corresponding to
9 MBq/kg body weight (2.5 MBq group), or 5 MBq
211At-BR96 corresponding to 19 MBq/kg body weight (5 MBq
group). The mAbs were administered via the tail vein under anesthesia, and the
injection volume was 0.4 mL.
Table 1
Group characteristics at day 0 and injected activities (average
values and ranges)
Treatment group
Body weight (g)
Tumor volume (mm
3)
Injected activity (MBq)
IA/kg body weight (MBq/kg)
Controls (unlabeled BR96)
Tumor (n = 6)
290 (282 to 300)
520 (282 to 640)
-
-
Non-tumor (n = 3)
289 (281 to 296)
-
-
-
2.5 MBq 211At-BR96
Tumor (n = 6)
272 (269 to 278)
475 (256 to 640)
2.5 (2.5 to 2.6)
9.3 (9.0 to 9.6)
Non-tumor (n = 3)
276 (271 to 278)
-
2.4 (2.4 to 2.4)
8.8 (8.7 to 8.9)
5 MBq 211At-BR96
Tumor (n = 6)
259 (247 to 266)
371 (176 to 520)
5.0 (4.9 to 5.1)
19.2 (18.3 to 19.9)
Non-tumor (n = 3)
264 (259 to 267)
-
4.8 (4.6 to 4.9)
18.0 (17.5 to 18.3)
IA, injected activity.
Monitoring after treatment
Tumor sizes were measured twice weekly after treatment. Tumor volumes were
calculated as tumor
length × width2 × 0.4. Tumors not
palpable for at least one consecutive week were classified as undetectable. Body
weight was also recorded twice per week. Bone marrow toxicity was monitored by
counting red blood cells, white blood cells, and platelets in arterial blood
samples with a Vet CA530 Medonic Cell Analyzer (Boule Medical, Stockholm,
Sweden) twice a week for the first 4 weeks, then once weekly until the end
of the study. Plasma was sampled from animals without tumors for the analysis of
liver and kidney function markers.
Animals were monitored up to 100 days after treatment as this was sufficient
for detection of metastases in previous work with animals followed up to
180 days post injection (p.i.) [19]. The rats were sacrificed with an overdose of isoflurane at the end
of the study or when the tumor exceeded 20 × 20 mm, body
weight decreased by >15%, or if the animal's general health was affected. At
the time of sacrifice, all animals were dissected systematically by the same
person, and the number and location of metastatic sites were noted. Tumor
findings detected at autopsy were fixed in 4% paraformaldehyde and embedded in
paraffin.
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Immunohistochemistry
The tumor findings were sectioned and stained with hematoxylin and eosin for
histological evaluation or examined immunohistochemically to evaluate BR96
target antigen expression and proliferation. In short, sections were dehydrated,
and antigen retrieval was performed by heating the slides in citrate buffer,
pH 6. After washing in Tris-buffered saline with 0.25% Tween 20 and
blocking of endogenous peroxidases with Peroxidase Blocking Solution (Dako,
Glostrup, Denmark), the sections were incubated with 5 μg/mL BR96 in
Antibody Diluent (Dako) overnight or with Rabbit anti Ki67 (Clone SP6,
NeoMarkers, Fremont, CA, USA) for 2 h, all at room temperature in a moist
chamber. After washing, the secondary antibody donkey F(ab)2
anti-human IgG-HRP (BR96) or donkey anti-rabbit HRP (Ki67; both from Jackson
ImmunoResearch Laboratories, West Grove, PA, USA) in Antibody Diluent was added
to the sections and incubated for 3 h at room temperature. Finally,
diaminobenzidine (Dako) was added before dehydration and mounting. The target
antigen expression was evaluated in relation to tumor histology by estimating
the approximate percentage of viable tumor cells with complete cell membrane
staining.
Statistical analyses
Prism 5.04 (GraphPad Software Inc., La Jolla, CA, USA) was used for statistical
analyses. Weight loss and blood cell counts were analyzed with either one-way
analysis of variance (ANOVA) or two-way ANOVA with Bonferroni multiple
comparison post tests. Survival was analyzed with the log-rank Mantel-Cox
test.
Results
Radiochemistry
The radiochemical purity (RCP) was 97% according to methanol precipitation. The
non-decay-corrected radiochemical yield (RCY) was determined to be 78%, using
the relation
where Atot is the total activity eluted from the NAP-5 column, and
Aadded is the activity added to the reaction. The immunoreactivity,
expressed as the ratio of the Kd for BR96 and 211At-BR96, was 0.93 for both specific
activities. The Kd of 211At-BR96 was within the 95% confidence interval of
the Kd for BR96.
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Toxicity
The administration of 2.5 and 5 MBq 211At-BR96 resulted in
dose-dependent relative weight losses of 3% (range 1% to 6%) and 5% (range 4% to
8%), respectively, the nadir being seen on the first recording after treatment
(day 2 p.i.). This can be compared to a weight increase of 1% in the control
group (range −1% to 1%; p < 0.0001). The animals
in the 2.5 MBq group showed full recovery of weight within 1 week,
whereas the animals in the 5 MBq group showed a delayed progression in
weight compared to the control group (difference not statistically significant)
(Figure 1A).
×
The white blood cell counts decreased in a dose-dependent manner; the nadir being
seen on day 2 (first sampling occasion) (p < 0.0001).
Full recovery of white blood cell counts was observed on days 19 (median, range
15 to 22) and 33 (26 to 33) in the 2.5 and 5 MBq groups, respectively
(Figure 1B). On day 8, the platelet count had decreased to
approximately 50% of the baseline in the 2.5 MBq group and to 25% in the
5 MBq group (p < 0.0001) (Figure 1C). Platelet counts had recovered on day 19 (range 15 to 22) in the
2.5 MBq group and on day 22 (range 19 to 26) in the 5 MBq group. In
one rat in each group given 211At, the number of platelets started to
decrease after initial recovery and then remained at 25% to 50% of the initial
value until the end of the study. There was no statistically significant
variation in red blood cell counts between the three groups (data not shown).
Data from rats with and without inoculated tumors were included in the
statistical analyses.
The levels of alanine transaminase (ALAT) and gamma glutamyl transpeptidase
(γGT) in plasma were used as markers of liver function. ALAT remained
unaffected by treatment, whereas the level of γGT was elevated less than
2.5 times the upper limit of normal level (grade 1 according to the National
Cancer Institute Common Terminology for Adverse Events version 4.0) for
3 weeks after the administration of the higher activity of
211At-BR96. The plasma levels of creatinine did not reveal any kidney
toxicity after radioimmunotherapy with 211At-BR96.
Treatment outcome
Survival was significantly prolonged in groups given 211At-BR96
compared to the control group (p = 0.017) (Figure 2A). Undetectable tumors were recorded in five out of six
animals in both groups given 211At-BR96 and in one out of six animals
in the control group, see Table 2. Remaining tumors
continued to grow except in one animal in the 5 MBq group, which remained
stable until the end of the study (Figure 2B,C,D). One
recurrent local tumor was detected in each group given 211At-BR96, in
both cases after 3 weeks of non-palpable tumors. In all groups, smaller
tumors seemed to respond better to the therapy, as has been observed by others [28, 29].
×
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Table 2
Tumor response after treatment with211At-BR96
Treatment group
Undetectable tumors
Stable tumors
Progressing tumors
Recurrence
Metastases at autopsy
Controls (unlabeled BR96)
1/6
0/6
5/6
0/1
5/6
2.5 MBq 211At-BR96
5/6
0/6
1/6
1/5
3/6
5 MBq 211At-BR96
5/6
1/6
0/6
1/5
2/6
Metastases were found in all treatment groups, most commonly in the lymph nodes,
but also in the liver and spread throughout the abdomen. Metastases were
detected at autopsy in five animals in the control group that were sacrificed
between days 16 and 35 due to high primary tumor burden. In the 2.5 MBq
group, one animal with a remaining primary tumor sacrificed on day 33 due to
signs of metastatic disease and one that was sacrificed on day 82 due to the
burden of the recurrent tumor showed metastases at autopsy. Another rat in this
group, sacrificed at the end of the study without any primary or recurrent
tumor, was found to have a lymph node metastasis. In the 5 MBq group, one
rat with recurrent disease sacrificed due to tumor burden at the end of the
study was found to have two lymph node metastases, while one rat with stable
disease was free from detectable metastases. An additional rat in this group,
without a primary tumor, was found to have a lymph node metastasis at the end of
the study.
All tumor findings contained viable and proliferating tumor cells. Both primary
tumors and detected metastases in the control group showed high expression of
the BR96 target antigen, with complete membrane staining of at least >50% of
all tumor cells; in half of the samples, the number of stained cells was >90%
(Figure 3A). The target antigen was detected in more than
50% of the cells in half of the specimens from the two astatine-treated groups
(Figure 3B,C,D). No tumor finding lacked expression of the
target antigen; however, the number of positive cells was less than 10% in
one-third of the tissues. The antigen was commonly detected in larger areas
rather than on single cells surrounded by negative cells. Areas with weak
staining were observed in several of the tissue sections. There were no trends
regarding number of antigen-expressing tumor cells regarding the metastatic
site. The two recurred primary tumors had lower expression compared to the
stable and progressing tumors.
×
Discussion
In this animal study, the concept of targeted alpha therapy was shown to be both
tolerable and efficient in the treatment of established colon carcinoma. The
activities administered were based on an estimate of the maximal tolerable dose to
bone marrow, using historical 177Lu-BR96 biodistribution data for the
theoretical calculation of the absorbed dose resulting from treatment with
211At-BR96 [27]. Both administered activities, 2.5 and 5 MBq/animal (9 and
19 MBq/kg body weight), were tolerated with regard to myelotoxicity, kidney,
liver, and general toxicity. The minor tendency towards increased hematologic
toxicity and the delayed weight progression in the group given 5 MBq indicate
that this activity was probably close to the maximal tolerable activity.
The treatment resulted in undetectable tumors in five out of six rats in both groups
treated with 211At-BR96. Survival was prolonged compared to the control
group given unlabeled mAb. The increased survival in the group given 5 MBq
211At-BR96 was not significantly different from that in the group
given 2.5 MBq. Due to the efficacy of the administration of 2.5 MBq, it is
difficult to motivate the use of higher activity.
Assuming a heterogeneous tumor uptake of the antibody, as has recently been found for
177Lu-BR96 [30], due to physiological factors such as the distribution of functional
blood vessels and high interstitial fluid pressure [31], eradication of all viable tumor cells by short-range α-particles
could be impaired. The limited range may be compensated to some extent by the
bystander effect [32]. Irradiation of the blood vessels resulting in compromised nutrition may
also enhance the antitumor effect [33‐35].
Targeted alpha therapy is generally regarded as being more appropriate for
loco-regional treatment since the short physical half-life and the kinetics after
i.v. administration of intact mAbs could mean that most decays have occurred before
binding to the target antigen on tumor cells [36]. This also applies to our animal model, in which the maximal tumor uptake
was observed 24 h p.i. [30]. Therefore, the strong antitumor response, resulting in undetectable
tumors in five out of six animals in both groups given 211At-mAb seen in
the present study, was not expected. Recent mouse studies on i.v. therapy of small
solid tumors using 211At-labeled antibodies [37] indicated that mean absorbed tumor doses above 10 Gy were required
for the eradication of small solid tumors. No experimental data on tumor uptake of
211At-BR96 were available for the estimation of the absorbed dose to
the tumors in the present study. However, using tumor uptake data for
177Lu-BR96, the estimated mean absorbed dose to the tumors would be
approximately 9.6 and 4.8 Gy, for activities of 5 and 2.5 MBq,
respectively.
The antitumor response could have been enhanced by the mAb itself due to the
initiation of antibody-dependent cell-mediated cytotoxicity and/or
complement-dependent cytotoxicity [38, 39]. The presence of such effects was demonstrated in the control group, in
which one tumor disappeared completely and four other tumors showed a small initial
response, as has been seen previously in our animal model [30]. An advantage of syngeneic immunocompetent animal models is the
possibility to study the synergistic effects of radioimmunotherapy and the adaptive
immune system.
Metastases were detected in five out of six rats treated with unlabeled BR96, despite
the shorter survival time, and in approximately half of the animals in the groups
given 211At-BR96. All metastases demonstrated antigen expression, but to
a much lower extent in the groups given 211At-BR96. This may be the
result of the elimination of tumor cells with sustained antigen expression and
repopulation by clones of cells lacking the target antigen, thereby reducing the
possibility of eradicating metastases by repeating the treatment. It is currently
not known whether tumor cells are disseminated and spread before or during
radioimmunotherapy in our animal model since the untreated animals are often
sacrificed before any metastases can be detected due to heavy primary tumor
burden.
The targeted alpha therapy using 211At-labeled mAbs in this study resulted
in a comparable rate of CR in tumor-bearing rats to that found when using
177Lu-labeled BR96 in the same rat tumor model [18]. The proportion of animals with detectable disseminated disease was
around 50% after both α and β radioimmunotherapy. Beta-emitting
radionuclides are generally regarded as being better suited for the treatment of
solid tumors due to their longer particle range than α-emitting radionuclides,
reducing the effect of heterogeneous tumor distribution. However, this property
limits the effectiveness against small cell clusters and singe cells [40] as most of the energy is deposited outside the tumor. One therapeutic
strategy could be to combine α- and β-emitting radionuclides to utilize
their different properties. It would also be relevant to develop an orthotopic
metastasis model with our syngeneic system, with smaller tumor lesions in the liver
and/or lungs instead of the comparably larger solid tumors used in the present
study. Such a study on microscopic metastases in the liver has been performed with
177Lu-labeled mAbs in a model of colon cancer [16]. The results of that study showed increased survival and delayed tumor
growth in animals treated with radioimmunotherapy, but the cure rate was not
affected. A comparison of α and β radioimmunotherapy in such a model would
be useful in providing evidence of the suitability of the different radionuclides
regarding their physical properties in the treatment of metastases.
Conclusions
Treatment with 211At-mAbs was tolerable with respect to toxicity at
activity levels resulting in undetectable tumors in a syngeneic rat colon carcinoma
model. The results demonstrate that radioimmunotherapy with 211At can be
an effective treatment modality for well-established and well-vascularized tumors up
to a size of 10 to 15 mm, but did not affect the development of metastatic
disease.
Acknowledgements
The authors would like to thank Dr. Peter Senter (Seattle Genetics, Inc.) for the
kind gift of the BR96 monoclonal antibodies, and Anna Ebbesson (Department of
Oncology, Lund University) for her excellent technical assistance. This work was
funded by grants from the Swedish Cancer Society, Mrs. Berta Kamprad's
Foundation, Gunnar Nilsson's Foundation, the Swedish Research Council, the
Crafoord Foundation, King Gustaf V's Jubilee Foundation, Governmental Funding
for Clinical Research within the National Health Service, the Lund University
Medical Faculty Foundation, and The Lund University Hospital Fund.
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 2.0 International License (https://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Competing interests
The authors declare that they have no competing interests.
Authors’ contributions
SEE designed and performed the in vivo studies, evaluated the
immunohistochemistry, performed the statistical analyses, and wrote the manuscript.
TB participated in planning the experiment, performed the dosimetric calculations,
and contributed to data interpretation and manuscript writing. EE participated in
planning and performing the in vivo studies. HJ developed and performed
production of 211At. SL developed and performed the radiolabeling, and
contributed to data interpretation and manuscript writing. JT and RN contributed to
the study design and analyses of data as well as writing of the manuscript. All
authors read and approved the final manuscript.