Biological Assessment for micro-AMS Thyroid Cancer
Cells using Iodine-125
Daniel José Gonçalves Bidarra
[email protected]
Instituto Superior Técnico, Lisboa, Portugal
November 2014
Abstract
This project aims to find the best thyroid cells for biological sample preparation using iodine 125
(
125
I) in study of the symporter NIS. Besides that, apply these cells, in the future, with micro
accelerator mass spectrometry (micro-AMS) using iodine 129 (
The use of
129
I).
125
I will allow knowing which radioactive activities could be given to the cellular
cultures as well as understand the cellular uptake mechanisms. The
reference protocol for
129
I. When applying the
129
125
I uptake assay will be a
I AMS technique to biological samples, such as
the thyroid gland, there is the necessity of prepare specific standards and samples for a
posterior use in AMS facility. The use of
129
I in studies of thyroid cancer therapy, through the
implementation of high sensitivity analysis technique - AMS facility at CTN / IST.
Due to the AMS high sensitivity and the low specific activity of the iodine-129, there are
advantages in using it in the future in preclinical research for clinical verification concerning the
existence of an inability to capture iodine-131 as well as in evaluation of the residual disease.
The inovation of this project lays in the association among well established experimental
techniques related to cellular manipulation and radioactive uptake assays with the physical and
more experimental nature of
129
I-AMS technique applied to biomedical science. The
combination of these two distinct areas may improve the follow-up for patients with thyroid
cancer, especially in those whose therapy became inefficient and there was a relapse of the
disease. In addition it may also contribute to the development of safer methods for patients and
caregivers through the use of less harmful isotopes and minimal radioactive doses, thus
reducing all the concerns related with radiological protection and safety.
Key-words: Iodine, Thyroid, micro-AMS, Iodine-125
following of these diseases became an
1. Introduction
essential tool. Alternative therapies, new
The
multiple
thyroid
pathologies
biomarkers
are,
and
new
techniques
of
frequently, associated with subtle and non-
detection have been increasing to find new
specific clinical practice. The investigation
pathways for oncological cases without
to allow a more accurate diagnosis and
effective resolutions
[1]
3,29
.
The main function of thyroid gland is the
measurement
production
(thyroid) for determination of the isotopic
of
essential
hormones
for
energetic regulation, growth, development
ratios
45
of
biological
samples
127 129
I/
I, and by this way, determine
and maturation of several organs. For a
the amount of radioactive iodine present in
normal hormone production it is necessary
thyroid cells.
a normal development of the gland, an
Unfortunately, the measurement of thyroid
appropriate running and regulation of the
cells could not be performed because the
biosynthesis mechanism and a normal
µAMS system present in LATR-CTN/IST
iodine intake. The iodine is a halogen
was not operable for
129
129
I. Although, it was
element present in the human body, and
possible to prepare
I-biological samples
due to its great affinity, the radioactive
for AMS using human thyroid cells, an
isotopes are commonly used for diagnosis
innovative approach never applied.
29
and therapy of thyroid pathologies .
2. Motivation
Thyroid pathologies can be treated with
Most of the cases of thyroid cancer (≈ 80%)
antithyroid drugs, radioactive iodine or by
are successfully treated with radioactive
surgical removal of the thyroid glands. The
iodine (RAI) therapy, however for 20% of
administration of antithyroid drugs creates
the cases there is a regression of the
controversy due to immunosuppressive
disease and in these cases the therapeutic
effects in some patients, and the surgical
with
ablation is becoming less used. Finally, the
available. The incapability of tumor thyroid
radioiodine therapy is also a controversial
cells to incorporate iodine is a complex
option due to the radiation exposure of
process being the object of investigation, as
patients and all the risks associated with
well as, the recurrence of the disease after
this process, although, the therapy is more
therapy.
economical
the
It is crucial to study and understand the
with
cellular mechanisms activated in iodine
treatment of thyroid pathologies is due to
uptake to in the future find alternative
the cases of relapse or regression of
therapeutics.
disease where the therapeutic applied no
alternative therapy in these cases is crucial
longer make effect, so it is necessary to find
and object of investigation. To understand
current
and
easier.
problematic
alternatives
24,29,33
However,
associated
.
radioactive
The
iodine
need
is
no
of
longer
find
an
why tumor thyroid cells stop to incorporate
The initial goal of this investigation is to
iodide, different tools are applied. In this
develop
that
work, the sample preparation is developed
permits the follow-up of thyroid cancer
in order to use µAMS technique in thyroid
patients by a radioactive method with high
cells for the first time. This technique is
sensitivity in detection of iodine present
capable to detect if iodide is present in a
inside the thyroid cells. The technique
cell or not, even in small quantities (1-2 mg)
selected
due to its high sensitivity.
an
was
alternative
micro
technique
accelerator
spectrometry (µAMS) using
mass
129
I to do the
[2]
The application of
129
I with µAMS in
In order to accomplish the previous goals it
biomedical approaches is recent, and there
is necessary to follow a work plan that
is only one case in Argentina, where it was
implies intermediate goals as:
used in measurement of
129
I of bovine
i.
Determination of the radioactive
thyroids. Furthermore, the µAMS is an
activity to be given to thyroid cells;
unique technique in Portugal only exists in
ii.
Lisbon in Campus Tecnológico e Nuclear
iii.
(CTN/IST).
for
Make a comparative study with
125
I;
Preparation of biological samples
125
I;
3. Goals
The preparation of biological samples for
The main goal of this investigation is to find
µAMS using
the best thyroid cells for µAMS analysis and
cultures with this technique was never been
125
I) in study of the NIS symporter.
performed previously. The only work that
Besides that, apply these cells, in the
can be comparable was made by Negri et
future, with accelerator mass spectrometry
(AMS) using iodine 129 (
The utilization of
129
129
al
I).
cellular
culture
standards
implement
and
manipulation
protocols
cells. The experiments with
in
mechanisms.
I. The
The
radioactive
I as
125
I give us the
activity
that
can
be
administrated to the cells, how they react, if
there is iodine uptake from cells and which
is the uptake percentage in normal and
125
I
cancer cells.
uptake assay will be a reference protocol
The main advantage of
129
I.
129
I is due to the fact
that µAMS is a technique with high
129
I has a large advantage
sensitivity that enables to know the amount
due to the fact that is a long-lived
of iodine absorbed by thyroid cells even in
radionuclide (15.7 million of years), which
lower sample concentrations.
allows the administration of low doses and
consent the manipulation of
125
knowledge necessary to know which is the
125
I will allow knowing which
Besides that, the
I in thyroids
access the amount of iodine present in
cellular cultures as well as understand the
for
129
isotope is largely used in cellular cultures to
standard
125
uptake
was
biological samples, due to the fact that this
radioactive activities could be given to the
cellular
where
reference method to radioiodine assays in
adapt
applied
radioiodide uptake assays is the
use of
Argentina,
reason, in this work, was used the
protocols for radioiodide therapy. One of
the
in
collected from bovine animals. For that
I µAMS in biological
performed previously. Due to this fact, it
necessary
(2012)
measured the amount of
samples as thyroid cells has never been
was
I is a very recent approach
and the use of thyroid cells from cellular
biological sample preparation using iodine125 (
129
There were made two experimental assays
129
I as a stable
to evaluate the
isotope with minimal safety and radiological
125
I uptake from K1, XTC-1
and PCCL3 thyroid cell lines. In the first
protection concerns.
assay were used K1 and XTC-1 cells, and
[3]
in the second assay the three cell lines
functioning of NIS and doesn’t interfere with
were applied. In both assays a NIS blocker
radioactive iodine (Na
were
used
–
Potassium
In the first assay was used only one
concentration of cold NaI. In the second
useful to calculate the NIS specific uptake
KClO4
I).
Perchlorate
(KClO4). The utilization of a blocker is
because
125
has
assay
competitive
were
applied
two
different
concentrations of cold NaI to access, which
interference with iodide in the entry into the
is the best concentration to give to the cells.
cell trough NIS. The KClO4 is added to
This difference between the two assays is
some cells functioning as a negative
justified due to the fact that, in literature
control.
were reported higher and lower values of
concentrations of cold NaI. This variability
4. Experimental
require test and assess which is the optimal
quantity
4.1 125I Uptake Assay
cold
NaI
radioactive activity of Na
4.1.1 Method of Preparation
An
125
I
uptake
125
for
a
specific
I.
assay
include
the
determination of radioactive activity present
The experimental setup was developed at
in thyroid cells by the measurement of
CTN/IST – UCQR in laboratories specifics
emitted gamma rays in a gamma-counter
for biological assays.
The
of
but also the quantification of the levels of
125
protein present in cells.
I uptake assay was performed with
three types of thyroid cells: K1, XTC-1 and
The assessment of protein concentration in
PCCL3. In the first assay were used K1 and
each well is important to normalize results.
XTC-1 cells, and in the second assay were
It allows us to be sure that difference
applied the three types of thyroid cells.
between
has the capability to incorporate iodide and
The protein quantification is based in
if NIS is present and functional in the
48
Bradford method . The levels of protein
cellular membrane. NIS is a glycoprotein
present in cell were achieved by a linear
-
that needs two Na and one I to have a
normal functioning. The quantity of Na
I uptake is due to more NIS
function and not by more quantity of cells.
The iodide uptake only is possible if the cell
+
125
regression obtained from NaOH standards.
+
All the process is described in specific
-
needs to be higher than the quantity of I .
elsewhere
48
.
In iodide uptake assay were used a
radioactive solution of Na
125
3.1.2 Materials
I and in order to
achieve the Na+ needed for a normal NIS
- Ionization Chamber for Dose Calibration
performance, a cold solution of NaI was
- Fume Cupboard
added. Although, the amount of cold NaI
- Multi-Crystal LB 2111 Gamma Counter
should be the sufficient to allow the
- Microplate UV-Vis Spectrophotometer
- Well Plates
[4]
- Micro Pipettes
uptake assay. The K1 cells were cultured in
- Beat and Gloves
a T-25 flask (25 cm ) in a RPMI medium.
2
- Freezer
The solutions used in this preparation are
acidified solutions and due to this, the
4.1.3 Protocol (General)
assay was performed in a fume hood.
1. Remove the cells medium from wells
The following reagents were prepared
and wash with Hank´s Balanced Salt
(Table 2.2): Pentan-3-one 4%, H2SO4,
Solution HBSS (37ºC);
H2O2, HNO3, KIO3 and AgNO3. In a
2. Add 990 µL of HBSS only to the positive
separation funnel with a total volume of 100
control wells and 990 µL HBSS plus
mL was mixed 20 mL of distilled water, 1
KClO4 (100 µM) to negative control
mL of Pentan-3-one 4%, 1 mL of H2O2 and
wells;
2 mL of H2SO4, in a total volume of 24 mL.
3. Add 10 µL of Na
125
I (approx. 15000
The other solutions were placed in sealed
cpm) to each well;
flasks.
4. Incubate for 45 minutes at 37ºC;
5. During
this
time,
prepare
Table
sodium
4.1:
AMS
Biological
Sample
Preparation Reagents
hydroxide (NaOH) solution and dial the
tubes;
Reagents
6. Remove cell buffer and wash with 500
K1
µL of ice-cold HBSS;
(Cells
Volume (mL)
+
20
Medium)
7. Cells lyse with 500 µL of NaOH (0.5 M);
Distilled Water
20
Pentan-3-one 4%
1
H2SO4
2
4.2 Method of Preparation – AMS
H2O2
1
Biological Sample (Thyroid)
HNO3
1
KIO3
2
AgNO3
0.4
8. Scrape off and counting each tube in a
gamma-counter;
4.2.1 Sample Preparation at CTN/IST
The preparation of the AMS biological
sample occurred in Campus Tecnológico e
4.2.2 Protocol:
Nuclear (ITN), and was based in the
-
protocols made by Negri et al.(2012) and
Cells lysed and placed in a 50 mL
sterile conical tube;
Marchetti et al (1997) for thyroid tissues.
-
Transferred the 20 mL of biological
In this work, thyroid cell lines were used
sample to four cellular tubes – 5 mL
instead of thyroid tissues, for this reason it
of cells in each tube;
was not necessary to oxidize the sample .
-
The thyroid cell line applied in this assay
was K1, one of the cell lines used in
Centrifugation at 10000 rpm for 10
minutes;
125
I
[5]
The centrifugation allows the separation
5. Results
between cells and supernatant.
The results from first and second assays
-
After
the
centrifugation,
the
cells
are different due to the fact that in the first
medium was aspired;
-
experiment was not used the PCCL3
A volume of 6 mL of the acidified
thyroid cell line. In terms of iodide uptake, in
solution was added to each tube;
-
both assays K1 and XTC-1 thyroid cells
Each tube was mixed, alternately, in
were analysed. The results from the two
vortex and in ultrasound bath for 5 to 10
assays were compared. The values of
minutes;
-
values
Initially, the expected results would be a
Formation
of
Silver
Iodate
higher
(AgIO3)
by scrape
-
I uptake of K1 cells in comparison
125
I uptake of XTC-1 cells.
The
results obtained are not according to the
Dry tubes in a oven for 2 hours and at
Remove precipitate
125
with the
literature as can be seen in the next tables
and graphs. The
and
125
I uptake is expressed in
cpm.
weighted in an analytic balance;
-
in
Incubate tubes during 18 hours in an
room temperature during 48 hours;
-
quantification
5.1. First Experimental Assay
precipitate
-
protein
absorbance (Abs).
humid incubator;
-
of
Second centrifugation at 10000 rpm for
5 minutes;
-
I
uptake are expressed in cpm and the
To each tube was added 0.5 mL of KIO3,
0.25 mL of HNO3 and 100 µL of AgNO3
-
125
125
Weight the same quantity of silver
Graph 5.1:
powder;
Cells with and without KClO4
I Uptake for XTC-1 Thyroid
Mix the precipitate with silver powder
and press it in a sample holder specific
for AMS targets;
The AgIO3 precipitate is showed in figure
4.1:
Figure 4.1: AgIO3 Precipitate
Graph 5.2:
125
I Uptake for K1 Thyroid Cells
with and without KClO4
[6]
In terms of protein quantification, the XTC-1
cell lines express more quantity of total
protein (1168,95 cpm) than K1 cell lines
(743,048 cpm) as illustrated in graphs 5.3
and 5.4 where is the amount of protein
present in each well, although, when was
calculated the specific uptake (the
125
I
uptake per quantity of protein), it was found
a higher value in K1 cells (2.545 cpm)
Graphs 5.3 and 5.4: Protein Concentration
instead
for K1 and XTC-1 (with and without KClO4)
of
XTC-1
(0,468)
as
initially
expected.
5.2 Second Experimental Assay
In the second assay were used the same
thyroid cells of first assay, K1 and XTC-1
cells, plus another subtype of thyroid cell
line, the PCCL3.
Graph 5.5 and 5.6: Quantity of 125I Uptake
per μg of Protein in PCCL3 (1mM NaI) with
and without KClO4
In the first experiment were analysed K1
and XTC-1 thyroid cells. The results reveal
low
125
I uptake values for the two types of
cells, 104,9 cpm (MS) for XTC-1 and 82.50
cpm (MS) for K1, respectively (see graphs
5.1 and 5.2). In the other side, XTC-1 cells
exhibit a higher
125
I uptake in the presence
of NIS blocker, with a mean value of 210,57
cpm (see graph 5.1) ,in opposition, K1 cells
have a mean uptake of 82,50 cpm (see
graph 5.2).
[7]
The
results
from
this
second
assay
membrane as well as the
corroborate the results achieved in the first
assay for K1 and XTC-1, a lower
125
I uptake is
higher in PCCL3 cells.
125
I uptake
of these two cell types. The use of these
6. Conclusions
cells for AMS is very difficult to implement,
6.1 125I – Uptake Assay
due to the fact that, an AMS analysis
require the use of small samples, and even
with
the
high
sensitivity
of
AMS
The utilization of thyroid cell linages to
to
study the NIS and the
determine the ratio of iodine present in cells
NIS expression varies between cell lines,
the cells or in cell cythoplasm.
as an example, PCCL3 cell line expresses
more NIS in comparison with K1 and XTC-1
125
I uptake values of PCCL3 are, as
cells
expected, considerably high in comparison
5000 cpm (see graphs 5.5 and 5.6). The
cancers,
125
I
incorporate
125
I uptake (almost the double)
that almost iodine entry into the thyroid cell.
concentrations,
them
became
iodide
lose
NIS
incapable
of
and
consequently,
131I
).
underexpression and to optimize the
the
125
I
uptake assay in different cell lines, so it can
become a valuable research tool to improve
cells with the minor cold NaI concentration
therapy. The thyroid cell line K1 and XTC-1
(100 μM) had the higher 125I uptake value
analysed in this study demonstrates that,
(≈ 8000 cpm) in opposition to the values of
even
the PCCL3 cells with cold NaI (1mM),
having
thyroid
cell
lines
with
expression of NIS, this is not sufficient to
approximately, 5500 cpm.
automatically
Finally, the protein quantification results
have
higher
levels
of
radioiodide uptake. A set of factors can
obtained by Bradford method, demonstrate
contribute for low levels of radioiodide
that PCCL3 cells have higher protein levels
uptake such as the cell culture, transport,
(195,28 cpm for 1 mM and 226.28 cpm for
incubation, manipulation, level of protein,
100 μM) in wells without NIS blocker. This
125
of
molecular mechanisms leading to NIS
results show (see graphs 5.5 and 5.6) that
indicates that the
NIS
Our main goal is to understand the
In terms of comparison between the two
NaI
,unpublished).
became resistance to therapy (
which indicates that is by NIS symporter
cold
C.
some
expression,
specific uptake is also higher than the non-
different
(Tavares,
expression is also variable in human thyroid
with K1 and XTC-1 cells with values above
specific
I uptake is
commonly implemented around the world.
is mandatory high levels of iodine inside of
The
125
among others.
I specific uptake in
these cells is raised, as predicted. The
Due to this, it was necessary to perform
amount of NIS protein is increased in
another experiment to evaluate the capacity
cellular membrane as in uptake values, in
of thyroid cells (healthy or carcinogenic) to
cells with cold NaI (100 μM). In comparison
incorporate radioiodide and, also, if NIS
with the two other thyroid cells types
transduction and dislocation into the cell
analysed,
can be manipulated and evaluated.
the
amount
of
protein
in
[8]
Due to this fact, a second assay was
use peroxydisulfate to oxide cells as thyroid
carried out using PCCL3 thyroid cells. In
tissues
this case, the
125
were
oxidized
Negri’s
in
I uptake had values higher
investigation, due to the fact that cells do
than the previous values obtained in the
not need to be oxidized because they were
first experiment. The quantity of NIS was
in culture with the conditions that mimics
125
I uptake was higher in
the in vivo characteristics. Also, potassium
cells without KClO4 than in cells with the
iodate was used instead of potassium
NIS blocker, as expected.
iodide as a test, due to the more self-life
also superior. The
and
Two different concentrations, 1mM and
NaI
in
125
I
uptake.
The
in
and
loss
of
climates
because
iodine vapors. The
131
I
therapy. Potassium iodate acts as a stable
I uptake due to the
iodine
I to
source
that
competes
with
radioactive iodine for the same receptors,
entry into the cell.
saturating
thyroid
before
radiation
exposure.
The thyroid cell line eventually applicable
for an AMS study is the PCCL3 due to the
6.3 Future Perspectives
125
I uptake values registered, where
the probability of have an iodide uptake is
upper even with a low dose of
humid
overexposure that could happens in
that the less value of NaI (100 µM) is better
high
in
effective thyroid blocker in radioactive
identical values of protein. This indicates
125
iodate
potassium iodate is also used as an
the verified with 1 mM, for the almost
fact that NaI compete with the Na
hot
hydrolytic
NaI (100 µM) the iodide uptake was over
125
potassium
though is more used but it is more instable
results
demonstrate that with a smaller value of
for a high value of
of
comparison with potassium iodide, even
100µM, were used to evaluate the influence
of
stability
The
µAMS
technique
could
be
an
125
I
alternative to measure the quantity of iodine
administered to the cells.
present inside de cell with sensitivity higher
6.2 AMS – Sample Preparation
than other existing technique even in small
The AMS biological sample preparation at
brings advantages because their long half-
UFA-UNL/CTN-IST using K1 thyroid cells
life (15.7 Million of years) which can be
was successful, achieving an AgIO3 as
consider an stable isotope and by this way
precipitate able to be analysed in an µAMS
all the radiological concerns are minimized,
laboratory. This was the first assay to
which allows the utilization of this isotope.
samples. Besides that, the utilization of
understand the protocol applied before by
Negri et al (2012)
43
129
I
The application of µAMS in this specific
and corroborate if it was
area is still under investigation but the
possible to do that using cells as biological
perspectives are positive and the work in
subtract.
biomedical
applications
using
mass
spectrometric techniques is increasing.
In our preparation we used thyroid cell lines
– K1. For this reason, was not necessary to
[9]
The use of thyroid cell lines to test the
dissertação. Um agradecimento também à
viability of preparation of biological samples
Prof. Patricia Carvalho pela orientação e
for µAMS held as expected and is the first
porque sem ela este trabalho também não
step to in the future apply this technique to
teria sido possível.
tumor thyroid cells (in vitro), in vivo studies
Queria também agradecer ao Hugo Silva e
with the measurement of the blood and
urine
samples,
and
at
the
end
à Cátia Santos por todo o companheirismo
the
em dias e dias no CTN e também a toda a
applicability in humans. This investigation
ajuda por eles prestada.
also confirms that PCCL3 thyroid cell line is
the most appropriate type of thyroid cell to
Um agradecimento à Catarina Tavares,
use with µAMS due to its high iodide uptake
investigadora do IPATIMUP, porque sem
between the cell lines investigated.
ela este trabalho também não era possível,
pois foi uma peça fundamental nesta
The measurement of biological sample
investigação e que também tanto me
prepared in CTN-IST in an AMS laboratory
ajudou. O meu muito obrigado à Dra.
as the National Accelerator Center (CAN) in
Lurdes Gano, ao Hélio Luis, ao Prof. João
Seville, Spain is one of the plans for the
Cruz, à Prof. Adelaide Jesus, à Rute e à
future.
Catarina por todas as ajudas.
In the future the objective will be the
O meu agradecimento à minha amiga
application of this technique routinely to
Leticia e companhia por todos os bons
evaluate the presence of iodine in remissive
thyroid
cancers
always
concerned
momentos e almoços, e também à Nadine
in
por toda a ajuda e disponibilidade.E por
radiological safety of patients and improve
sensitivity
and
efficiency
of
último, mas talvez o mais importante, quero
these
agradecer à Inês pelo seu enorme apoio e
techniques.
compreensão, por toda a força que me deu
e por ser a mulher que é que eu admiro
7. Acknowledgements
tanto e que sem ela isto não era mesmo
É sempre complicado agradecer algo a
possível.
quem temos tanto a agradecer e no final de
8. References
contas não sabemos bem como fazê-lo
mas aqui vai.
1. Alfimov, V., & H., S. (2010 ). 129I AMS at 0.5
MV tandem accelerator. Nuclear Instruments
and Methods in Physics Research B , 769–
772.
Primeiro que tudo, quero agradecer à
minha familia, pais e irmãos por toda a
força e incentivo que me deram para que
2. Alvarez, L. (s.d.). The Early Days of
Accelerator Mass Spectrometry. Berkeley,
California : Lawrence Berkeley Laboatory University of California .
continuasse o meu trabalho e desse o meu
melhor.
agradecer
Em
à
segundo
minha
lugar
orientadora,
queria
Dra.
3. American Cancer Society. (2014). Thyroid
Cancer. Retrieved Feb 20, 2014, from
Cancer
Web
site:
Micaela Fonseca, por todo o apoio e
compreensão e ajuda dada durante esta
[10]
http://www.cancer.org/acs/groups/cid/docum
ents/webcontent/003144-pdf.pdf
15. Feng, F. et al. (2012). Re-induction of cell
differentiation
and
131I
uptake
in
dedifferentiated FTC-133 cell line by TSHR
gene transfection. Nuclear Medicine and
Biology, 1261–1265.
4. Borman, S. (12 de Jan de 1998). A Brief
History
of
Mass
Spectrometry
Instrumentation. Chemistry Crystallizes Into
Modern Science, pp. 39-75.
16. Ferlay, J. et al. (2013). GLOBOCAN 2012
v1.0, Cancer Incidence and Mortality
Worldwide: IARC CancerBase No. 11. Lyon:
International Agency for Research on
Cancer.
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