Relative Biologic Effectiveness and Linear Energy Transfer effects of low energy proton irradiations for T98G human glioblastoma cells

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Relative Biologic Effectiveness and Linear Energy Transfer effects of low

energy proton irradiations for T98G human glioblastoma cells

Vera Helene Tormodsrud

Thesis for the Degree of Master of Science

Department of Physics

Faculty of Mathematics and Natural Sciences UNIVERSITY OF OSLO

May 2019

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Acknowledgements

The work presented in this thesis was carried out at the Biophysics and Medical physics group in collaboration with the Nuclear physics group at the Department of Physics, University of Oslo.

I would like to thank my supervisors Nina F. Edin, Eirik Malinen and Sunniva Siem for their guidance throughout this thesis. A special thanks goes to Nina F. Edin for great support during experiments and writing. Anne Marit Rykkelid, although not a supervisor, deserves my gratitude for all discussions and guidance as an honorary supervisor.

I would also like to thank Joe A. Sandvik for his assistance and instructions in Laboratory techniques, and the engineers at Oslo Cyclotron Laboratory for their help in setting up the cyclotron for experiments.

In addition, I want to thank Hilde Skeie for her help in performing experiments when I was recovering from surgery and could not carry equipment. She has been the best lab assistant I could have asked for, and her company has been greatly appreciated during experiments. A thanks also to Emma Thingstad, my study buddy through 5 years of physics studies, for the great company and good friendship over all these years.

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Abstract

The study presented in this thesis investigates T98G human glioblastoma cells response to proton irradiation with different linear energy transfers (LETs). The main objective is to characterize clonogenic survival throughout the proton radiation track. Previous experiments performed in our group suggest a large range of relative biologic effectiveness (RBE) values.

As more experience with proton irradiation of cell dishes at Oslo Cyclotron Laboratory has been gained since the experiments were performed the first time, the aim was to obtain more precise data and to validate the previous findings.

Cell irradiations using a 13 MeV proton beam were performed at the Oslo Cyclotron Laboratory using two or five different positions throughout the proton track to obtain radiation with different LETs. 220keV X-ray irradiations were also performed to obtain reference data for the data obtained through proton irradiations. Colony assays were used to calculate the surviving fraction for different doses and create survival curves for high and low LET irradiations. The procedure for proton irradiations was adapted slightly during the project to achieve an optimalized setup.

The survival curves for proton irradiations were found to show higher survival for high doses than predicted with the LQ-model, especially evident in the high LET irradiations where the surviving fraction ended in a plateau where the survival level stayed the same even as the dose increased. The same problem was present at the previous experiments, where it was hypothesized to arise from the use of several layers of inhomogeneous parafilm as absorber to position the cells in the distal end of the Bragg peak. However, even though measures were taken to avoid this, the problem remained in this study. Therefore, there had to be another explanation, and one hypothesis to explain for the plateau of survival was that the T98G cells have a tendency to migrate. The cell dishes were irradiated vertically with the medium removed, but a drop of medium always remained in the bottom of the dish. This shielded for the protons and it was observed that a large number of cells survived in this area. Even if this region was not included when colonies were counted, the plateau in survival still remained.

We therefore hypothesized that these surviving cells were able to migrate, sending out satellite colonies. As a high number of cells were seeded for the dishes receiving large doses, this effect became dominant over the true surviving fraction. The effect would not be visible for low dose irradiations where fewer cells were seeded.

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V It was then decided only to analyse the data for 5 Gy and below. The effect that a variation in LET had on surviving fraction was examined, and RBE values were calculated as a function of LET with 220keV X-rays and gamma irradiation from a Co-60 source as reference radiations. The Co-60 irradiations had been previously performed in our laboratory. RBE values at 10% survival ranged from 1.0 for low LET proton irradiation, to 4.2 for high LET proton irradiation for both reference radiations, with an increase along the particle track.

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Radiation treatment with charged particles (CP) has an advantage over photon therapy due to the difference in dose distribution as the radiation beam travels through tissue. While photons deliver the maximum dose only a few centimetres into the tissue with a following gradual drop in dose, CPs deliver their maximum dose at the end of the particle track and falls drastically towards zero after this (Desouky & Zhou, 2016; J. H. LAWRENCE et al., 1965).

Some dose is given along the particle track on its way through the tissue but this deposit is significantly lower when compared to the dose distribution of the photon at the beginning of its track (Durante & Loeffler, 2009). The dose distribution curve for CPs is known as a Bragg curve, and the area where the dose deposition rises to its maximum and then falls down to zero is called the Bragg peak. This peak allows for radiation treatment with more precise margins than for photon therapy, where there is still a considerable amount of dose given behind the maximum dose deposition (Durante & Loeffler, 2009). There is also evidence to support that the cell cycle dependency on radiation sensitivity is reduced when using some CPs (Tobias et al., 1982). When using CP for radiation treatment, more healthy tissue will be spared from damage due to the low dose deposit at the beginning of the track and since the peak can be adjusted to strike the target volume and avoid healthy tissue. Due to its defined dose distribution the CP beam can avoid exposing organs at risk to high dose levels at a greater extent than photon beam can, successfully reducing the amount of damage to these vital organs (Hall, 2019).

The challenge with the use of CP therapy arises in the combination of the Bragg peak and the linear energy transfer, LET. LET is the average rate of energy loss per unit distance, and for CPs it increases rapidly at the end of the particle range. An increase in LET is associated with more biological damage (Mohan et al., 2017). When the Bragg peak has been reached and the dose deposition is on its way down towards zero, the LET is rapidly increasing. So, while the dose is reduced, the damages given by this dose are larger and more severe than the damages in the middle of the Bragg peak, where the dose is at its highest. This gives rise to a variable relative biological effectiveness, RBE, throughout the particle track. For proton therapy in cancer treatment, this variability is not accepted (Mohan et al., 2017). There is not enough data that maps the different RBE throughout the proton track, and the data that does exist varies a great deal. Therefore, a fixed value of RBE=1.1 is accepted and used in clinical proton therapy (Paganetti, 2003).

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It has been shown that RBE can vary a lot during the protons path through tissue. Accepting a static value for RBE can cause problems in cancer treatment. At the end of the particle track, LET is high, which consequently makes RBE high. The value of 1.1 is an underestimation in this area. Studies imply that RBE can vary from 1 in front of the Bragg peak, to 4 in the back of the Bragg peak (Guan et al., 2015), but there is a need for more data on this subject in order to determine definite values. The value in use in radiotherapy today, can lead to increased toxicity and suboptimal treatment of the cancer. Since the value is underestimated for the distal end of the Bragg peak, tissue will receive more biological damage than is accounted for when the treatment is planned in the conventional way used today (Mohan et al., 2017). If we were to exploit this increase in RBE in treatment planning, the dose to the tumour could be reduced while still keeping an acceptable level of cell death, therefore also reducing the dose to the normal tissue effectively sparing it from unnecessary damage.

The motivation for the thesis is this uncertainty in the RBE value. We use a low energy proton beam in order to achieve higher LETs than obtained during proton treatment in the clinic, which gives us biologic effects of higher severity. The thesis is a continuation of Anne Marit Rykkelid’s master thesis from 2017. Rykkelid developed a method for proton irradiations at the Oslo Cyclotron Laboratory and performed clonogenic cell survival experiments with low LET and high LET protons. Together with her findings, we hope to more precisely

characterize the different RBE values and LET effects throughout the Bragg peak.

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2 Theory

2.1 Cell biology

This section is based on chapter 1,3 and 17 in “Molecular Biology of the Cell” (Alberts, 2002).

The DNA molecule is a storage for all genetic information inside the cell. It consists of two long polymer chains built up by the four nucleotides Adenine, Thymine, Cytosine, and Guanine. Sugar phosphates links the nucleotides together to form a sequence which encodes the genetic information: the DNA strand. An opposite DNA strand is complementary to the first, and these are linked together with hydrogen bonds between the nucleotide bases, thus creating the double-stranded DNA helix.

Figure 1 DNA structure and building blocks. The four nucleotides are linked together by sugar phosphates to form a DNA- strand. Two DNA-strands are bound together with hydrogen bonds between base pairs to form the double-stranded DNA and

the DNA double helix (Alberts, 2002).

Proteins have a very important job within the cell, they express the information stored within the DNA. Proteins are built up by a chain of amino acids, which are three bases in specific order, and different proteins are made with different arrangements of amino acids. The proteins have many assignments, for instance that they can bind to molecules to catalyse chemical reactions.

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2.1.1 The cell cycle

The cell cycle is a complex process that results in the duplication of the cell and all its genetic material. It is divided into four main phases: G1, S, G2 and M. G1 and G2 are known as gap- phases, while the replication of the DNA occurs in the S-phase, and the division in to two daughter cells occur in the M-phase.

The gap-phases allow for cell growth and necessary response of the condition of the cell, G1 is the most important of the gap-phases. If the cell enters the S-phase under suboptimal extracellular conditions, it can lead to mutations in the daughter cells. When conditions are not favourable, either G1 is prolonged or the cell enters a resting phase G0 where it remains until the conditions for replication are optimal or until the cell or organism dies. In the S- phase, the DNA synthesis phase, the chromosomes and chromatin proteins in the mother cell are duplicated. It is important that the DNA replication is accurate, every base must be read and copied correctly to avoid mutations. Once all the chromosomes have been duplicated, the cell enters the G2-phase where it has time to grow big enough to divide into two cells. After the G2-phase, the cell enters the M-phase where mitotic spindle separates the sister

chromatids to form two daughter cells.

2.1.2 Cell cycle control system

Before the cell-cycle moves from one phase to the next, the cell goes through checkpoints to make sure that the optimal conditions for duplication are met. The G1 checkpoint makes sure that the conditions for duplication are met, and the G2 checkpoint checks if all chromosomes are duplicated correctly. Both checkpoints also check if the environment is favourable. If the chromosomes are incorrectly duplicated, the cell will not enter the M-phase. In the M-phase, there is an M-checkpoint that ensures that the mitotic spindle has attached to all the

chromosomes so that the daughter nuclei are separated correctly.

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Figure 2 The cell cycle is a centrally controlled system, and essential processes are triggered at specific times, here marked with yellow boxes. (Alberts, 2002).

The cell-cycle is centrally controlled by proteins activated at different points in the cell-cycle to drive the system forward. The proteins are a family of cyclin-dependent kinases (Cdks) and are split into four groups depending on what part of the cell-cycle they control. Cyclins bind to Cdks and form a cyclin-Cdk complex. This complex trigger cell-cycle events depending on which cyclin the Cdks bind to. G1/S-cyclins bind to Cdks in late G1-phase and trigger

progression into S-phase. S-cyclin forms the S-Cdk in early S-phase and stimulate

chromosome duplication. The levels of S-Cdk remains high through G2 and to the beginning of M-phase. The M-cyclin and Cdk bind together in the G2-phase and levels rise as the cell enters M-phase, where the M-Cdk stimulates the mitosis process. All cyclin levels fall after they have activated their respective processes.

2.2 Radiation physics

The next section is based on “Introduction to Radiological Physics and Radiation Dosimetry”

(Attix, 1986).

2.2.1 Ionizing Radiation and Interaction with Matter

Radiation is characterized as ionizing when it has a kinetic energy high enough to cause an electron in a target atom to escape its orbit. The energy needed is 4-25 eV, so the radiation requires an energy above this magnitude to ionize atoms. In this study we focus on photons (X-rays) and protons as sources of radiation.

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Photons

The X-ray beam is made up of photons, which may be regarded as quantized electromagnetic wave packets. The photon has no electric charge. In vacuum, it moves at the speed of light.

We distinguish between five types of photon interactions with matter: Compton effect, Photoelectric effect, Pair production, Rayleigh scattering and Photonuclear interactions. We can exclude Rayleigh scattering as this interaction has no transfer of energy, hence there is no dose delivered to the matter. Also, the probability for Photonuclear interactions is so low that this can also be neglected in the big picture.

Compton scattering

An incoming photon interacts with an outer shell electron that is considered stationary in reference to the incoming photon and considered to be free due to weak binding energy.

Energy is transferred from the photon to the electron in the interaction and both the electron and the photon are then scattered at different angles. There is conservation of energy and momentum in this interaction, which means that the incident photon will depart with a lower energy and momentum. The energized electron will then travel through the matter and excite and ionize along its track.

The Compton effect is the most important interaction for mid-energy photons (100 keV – 10 MeV), and the probability of the interaction is predicted by the cross section, which is proportional to the atomic number Z.

Figure 3 Compton effect. An incoming photon with energy ℎ𝑣 strikes an unbound electron. The electron is scattered at an angle 𝜃 with kinetic energy 𝑇, and the photon is scattered at an angle 𝜙 with energy ℎ𝑣′. There is conservation of energy and

momentum in the interaction (Attix, 1986).

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9 Photoelectric Effect

If the photon interacts with a tightly bound atomic electron it can transfer all its energy in the collision to the electron and the atom. Figure 4 is an illustration of the process. The incoming photon collides with an electron with binding energy 𝐸_𝑏 and transfers kinetic energy 𝑇 = ℎ𝑣 − 𝐸_𝑏 to the electron. The photon is absorbed and the electron leaves at an angle 𝜃. The atom is deflected at an angle to conserve momentum and energy as the atom receives a momentum corresponding to the loss of momentum in the photon. Photoelectric effect is important in low-energy photon interactions with matter (< 100 keV).

Figure 4 Photoelectric effect. A photon of energy ℎ𝑣 strikes an atomic electron with binding energy 𝐸_𝑏. The photon is absorbed and the electron departs at an angle 𝜃 with kinetic energy 𝑇 = ℎ𝑣 − 𝐸_𝑏. The atom departs at an angle 𝜙 to

conserve momentum (Attix, 1986).

Pair Production

When a photon passes through the Coulomb force field near an atomic nucleus, its energy can be converted into and electron and a positron. This process is dominant for high-energy photons (>10 MeV), as the energy needed to create the electron-positron pair has a threshold value of above 2𝑚₀𝑐², where 𝑚₀ is the mass of the electron and positron.

Figure 5 Pair Production. A photon with energy ℎ𝑣 enters the Coulomb force field of an atomic nucleus and disappears. A positron-electron pair is created to conserve energy and momentum. (Attix, 1986).

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The total likelihood of energy transfer (to secondary electrons) per mass during irradiation can be described with the Mass Energy-Transfer Coefficient 𝜇_𝑡𝑟/𝜌 in equation 1.

𝜇_𝑡𝑟 𝜌 =𝜏_𝑡𝑟

𝜌 +𝜎_𝑡𝑟 𝜌 +𝜅_𝑡𝑟

𝜌 (1)

Where 𝜏_𝑡𝑟/𝜌 is the contribution from the photoelectric effect, 𝜎_𝑡𝑟/𝜌 is the contribution from the Compton effect and 𝜅_𝑡𝑟/𝜌 is the contribution from pair production. By considering that some secondary-electron energy is lost in the production of bremsstrahlung, this equation can be used to find the Mass Energy-Absorption Coefficient 𝜇_𝑒𝑛/𝜌, given in equation 2.

𝜇_𝑒𝑛 𝜌 =𝜇_𝑡𝑟

𝜌 (1 − 𝑔) (2)

Here, g represents the fraction of energy that is lost in the production of bremsstrahlung.

Protons

The proton is a positively charged particle. Charged particles have an electric field which mediates interactions with electrons in every atom they pass along their path, only losing a small fraction of energy for each interaction. As they interact with many electrons through their track, the energy loss is gradual, referred to as a “continuous slowing-down

approximation”, CSDA. The interactions can be divided into three different types depending on the impact parameter: soft collisions, hard collisions and interactions with the atomic nuclei. The impact parameter is the distance from the atomic nucleus to the point in the charged particles trajectory that is closest to the atom. The atomic nuclei interactions are only relevant for heavy charged particles, as they require a lot of energy. We will not be operating with energies of this magnitude (>100 MeV), so this interaction can be neglected in our experiments.

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Figure 6 Schematic presentation of charged-particle interactions: atomic radius a and impact parameter b (Attix, 1986).

Soft Collisions (𝑏 ≫ 𝑎)

The particle passes the atom at a large distance, with the impact parameter b being larger than the radius a of the atom. The entire atom is excited to a higher energy level. Valence electrons can be ejected, resulting in ionization of the atom. These soft collisions are the most common charged particle interaction since large impact parameters are more probable.

Hard Collisions (𝑏~𝑎)

In hard collisions the particle passes close to the atom with the impact parameter being in the order of the dimensions of the atom. It will most likely interact with one of the atomic electrons, knocking it out of its shell creating a ray of delta electrons which can then go through Coulomb-force interactions on its own. If an inner-shell electron is ejected from the atom instead of an outer-shell electron, the atom will emit characteristic X-rays and/or Auger electrons as in a photon interaction (section 2.2.2).

Hard collisions are much less frequent than soft collisions, but the exchange in energy from the charged particle to the atomic electron is much higher in hard collisions than the energy exchanged from the charged particle to the electron in the soft collision, thus the fraction of energy in these two interactions are of comparable size.

All interactions will give rise to a radiation dose received by the matter. The dose from charged particles is basically reflected in the stopping power; the energy loss per unit length.

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The total collision stopping power (per mass) for the hard and soft collisions taking place in the matter is given by (equation 3):

(𝑑𝑇 𝜌𝑑𝑥)

𝑐

= (𝑑𝑇_𝑠 𝜌𝑑𝑥)

𝑐

+ (𝑑𝑇_ℎ 𝜌𝑑𝑥)

𝑐

(𝑀𝑒𝑉 𝑐𝑚⁄ ) (3)

Here, (^𝑑𝑇

𝜌𝑑𝑥)

𝑐

is the mass collision stopping power, and (^𝑑𝑇^𝑠

𝜌𝑑𝑥)

𝑐

and (^𝑑𝑇^ℎ

𝜌𝑑𝑥)

𝑐

are the mass

collision stopping power contributes from soft and hard collisions respectively. By combining quantum mechanical considerations for soft collisions and the Rutherford cross section for hard collisions, the total equation stopping power becomes (equation 4):

𝑐

= 𝑘 [ln (2𝑚₀𝑐²𝛽²𝑇^′_𝑚𝑎𝑥

𝐼²(1 − 𝛽²) ) − 2𝛽²] (4)

Where k is a constant, 𝑚₀ is the electron mass, 𝛽 = 𝑣/𝑐 and 𝐼 is the mean excitation potential of the matter. Here, 𝑇′_𝑚𝑎𝑥 is the maximum energy that can be transferred in a hard collision.

An expression for this is presented in equation 5.

𝑇^′_𝑚𝑎𝑥 ≃ 2𝑚₀𝑐²( 𝛽²

1 − 𝛽²) = 1.022 ( 𝛽²

1 − 𝛽²) 𝑀𝑒𝑉 (5)

When we insert this into the equation for the total mass collision stopping power, we get a simplified equation for the mass collision stopping power, which can be seen in equation 6.

𝑐

= 0.3071𝑍𝑧²

𝐴𝛽²[13.8373 + ln ( 𝛽²

1 − 𝛽²) − 𝛽²− ln 𝐼 −𝐶

𝑍] (6)

Here, 𝑍 is the atomic number of the matter, z is the charge of the particle, A is the mass number of the matter, and 𝐶/𝑁 is a shell correction factor for the matter. We can now obtain an equation for the absorbed dose in the medium by simply taking the product of the fluence 𝜑 of the radiation and the mass collision stopping power. The final equation for dose is then

𝐷 = 𝜑 × (𝑑𝑇

𝜌𝑑𝑥) [𝐽 𝐾𝑔⁄ ] (7)

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13 Depth dose curves

The interactions in matter leads to energy deposits through the object. These can be visualized with depth-dose-curves (figure 7). X-rays and electrons deposit most of their energy close to the surface, while protons deposit most of its energy further into the matter. Right after the main energy-deposit, the dose delivered to the tissue falls drastically towards zero, both for protons and electrons. The X-ray beam has an exponentially decreasing energy deposit

through the matter, still giving a significant dose throughout the tissue. The main advantage of protons is this way of depositing energy. When used in radiation therapy, the protons will spare more healthy tissue than X-rays since this peak in energy is at the end of the track instead of at the beginning, and little to no dose will be given behind the peak. The peak in the depth-dose-curve for protons is known as a Bragg-peak.

Figure 7 Depth-dose-curves for X-rays, electrons and protons in human tissue (Paul, 2009)

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2.2.2 Radiation generators

X-ray Tube

X-ray radiation is produced with an X-ray tube. It consists of, amongst others, a negatively charged cathode and a positively charged anode. The entire system is placed in vacuum, and there is a difference in voltage from the cathode to the anode. At the edge of the cathode, there is a filament which serves as an electron source. Since the electrons have negative charge and reside in a strong cathode-anode electric field, they will be accelerated towards the anode. The electrons then collide with the anode and deposit their energy in the target. Most of the energy is lost in excitations and ionizations, deposited mainly as heat, but

approximately 1% is deposited as characteristic x-rays and bremsstrahlung. Due to the high heat the target is exposed to, the target material must withstand this. Tungsten is usually chosen as the target material, as it has a high melting point and can convert a large fraction of the electron energy to bremsstrahlung due to its high atomic number.

Figure 8 X-ray tube. Electrons are released from the cathode by thermionic emission and are accelerated towards the anode.

When the electrons strike the target on the anode, Bremsstrahlung is produced (Hinrichs & Urone, 2018)

The spectrum from bremsstrahlung is known as the continuous slowing down spectra, while the spectrum from characteristic x-rays are shown as peaks known as K-lines and L-lines at distinct energies. The characteristic x-rays are produced from excitations of atomic electrons moving from one shell to a shell with a higher energy level, and the subsequent de-

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15 excitations. The difference in binding energy from the two energy levels are released as

photons and give rise to the peaks in the characteristic x-ray spectrum. Bremsstrahlung is created when the electron is decelerated in a Coulomb force field.

Figure 9 X-ray spectrum from 100 keV electrons on a tungsten target. The solid curve is the photon spectrum and the dashed curve is the exposure spectrum (Attix, 1986)

Cyclotron

A cyclotron was used in this study to provide us with a low energy proton beam to irradiate plated cancer cells. The cyclotron uses a circular trajectory to accelerate charged particles. It consists of two D-shaped electrodes placed back-to-back with a small gap between them. A schematic presentation is in figure 10. The D’s have a static magnetic field which causes the particles to bend in a circular trajectory as illustrated in the figure.

In the gap between the D’s an alternating voltage that accelerates the particles is applied. As the particle enters this gap from two different directions depending on which D it is coming from, the voltage has to alternate. If we did not alternate the voltage, the particle would be decelerated every other time it entered the gap. Each time a particle reaches the gap, it gains velocity, while the magnetic field keeps the particle in its circular trajectory with no

additional acceleration.

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Figure 10 Principle of the Cyclotron. Two D-shaped electrodes back-to-back. Voltage is applied in the gap between the dees which accelerates the particle. The dees keep the particle on a circular track (E. O. Lawrence, 1934)

The magnetic force on the particle is given by Lorenz force law, which together with the particle charge and mass will determine the time it takes for the particle to travel though one D. These considerations are elaborated in equations (8-10).

𝐹 = 𝑞𝑣𝐵 =𝑚𝑣²

𝑟 (8)

𝑣 =𝑟𝑞𝐵

𝑚 (9)

𝑡 =𝜋𝑟 𝑣 =𝜋𝑚

𝑞𝐵 (10)

Where F is the magnetic force, 𝑞 and 𝑣 is the charge and velocity of the particle, B is the strength of the applied magnetic field, r is the radius of the circular trajectory and m is the particle mass.

Each time the particle is accelerated, the radius of the circular trajectory increases, but the time it takes to travel through one D stays the same. The frequency 𝑓 of the alternating voltage is thus only dependent on particle mass and particle charge as shown in equation 11 where t is the time it takes the particle to circulate once.

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17 𝑓 = 1

2𝑡 = 𝑞𝐵

2𝜋𝑚 (11)

When the particle reaches the desired energy and velocity it will move on to an evacuated beam tube and the beam is steered with electromagnetic fields to keep it in the correct path.

2.2.3 Dosimetry

Ionization chamber

An ionization chamber is a gas-filled dosimeter, which is used to determine the dose through ionizations. It consists of a gas-filled cavity with a central electrode that is surrounded by an outer electrode. When ionizing radiation strikes the detector, the gas is ionized and charged particles will move from the outer electrode and gather at the central electrode, creating a current that can be measured with an electrometer. The current is proportional to the absorbed dose, which can be found through calibrations (Attix, 1986).

The primary calibration of an ionization chamber is done by a nationally certified laboratory, by exposing the chamber to a known source of radiation. It is usually done with a Cobalt 60 source as this emits gamma rays at two distinct energies, making it easy and reproducible to calibrate with. We can then relate the absorbed dose to the ionization charge produced by using equation 12.

𝐷_𝑔 = 𝑄 𝑚(𝑊̅

𝑒)

𝑔

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Where 𝐷_𝑔 is the absorbed dose in the gas, 𝑄 is the charge produced in the mass 𝑚 of gas and (^𝑊^̅

𝑒)

𝑔 is the mean energy spent per unit charge produced in the gas (Attix, 1986).

Gafchromic EBT3 Radiochromic Dosimetry Films

The Gafchromic EBT3 films can measure absorbed dose of ionizing radiation. The film has an active layer containing amongst others, an active component and a marker dye. The active layer is situated between two matte-polyester substrates (GAFCHROMIC). The construction of the film can be seen in figure 11.

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Figure 11 Construction of EBT3 Dosimetry Film. Active layer surrounded by matte polyester on each side (GAFCHROMIC).

When the film is exposed to radiation, the active component forms a colour that varies in strength depending on the intensity of the radiation. The film can then be scanned in a flatbed colour scanner to obtain its optical density. With calibration of the film following function 13, we can achieve a dose response curve for the specific radiation.

𝑑_𝑥(𝐷) = 𝑎 + 𝑏

𝐷 − 𝑐 (13)

Where 𝑑_𝑥(𝐷) is the optical density of the film, dependent on the dose D, and a, b and c are equation parameters that have to be fitted (GAFCHROMIC).

Since the active layer is protected by polyester on both sides, it can be irradiated from both sides and give the same optical density. This means that the films used for dosimetry and the films used for calibration do not have to be irradiated from the same side to obtain

comparable results. Even so, it is important to keep the same orientation in space during exposure and scanning to avoid orientational differences in optical density (Borca et al., 2013).

2.3 Radiobiology

The following section is based on “Radiobiology for the Radiobiologist” (Hall, 2019).

2.3.1 Direct and Indirect Action of Radiation

Radiation from charged and uncharged particles can damage cells through two different processes: direct and indirect action of radiation. The direct action is the main process with high-LET radiation like protons or 𝛼-particles, and it occurs when primary or secondary particles interact directly with the DNA-molecule in the cell. This leads to ionization or excitation of the atoms in the target and may cause biologic change. The indirect action of

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19 radiation is when the particles interact with the molecules surrounding the DNA, creating free radicals that are highly chemically reactive. The indirect effect is mainly caused by low-LET radiation like X-rays and 𝛾-rays.

80% of the cell is made up of water molecules, so most of the radicals formed in the indirect action of radiation will be water radicals. In the water radical, there will be one unpaired electron in the outer orbit that causes the chemical instability. The reaction can be expressed as 𝐻₂𝑂 → 𝐻₂𝑂⁺+ 𝑒⁻. Here, 𝐻₂𝑂⁺ is an ion radical, an electrically charged water radical. It can react with another water molecule 𝐻₂𝑂⁺+ 𝐻₂𝑂 → 𝐻₃𝑂⁺+ 𝑂𝐻 · and create a highly reactive hydroxyl radical (𝑂𝐻·) that can diffuse twice the width of the DNA double helix and cause damages.

Figure 12 Direct and Indirect Action of Radiation on a DNA-molecule. (Desouky and Zhou (2016) (accessed 3. Sep 2018))

2.3.2 Radiation Damage and Repair

When cells are struck by radiation, multiple types of damages can be induced, including DSBs: breaks in both strands of the DNA, SSBs: breaks in one strand of the DNA, and base damage. Base damages and SSBs are easily repaired using the undamaged strand as a template to replace the damaged site with the correct, corresponding bases. When repaired

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correctly these damages will not give rise to any mutations. They are therefore not of high importance in cell inactivation and cell death. The goal when irradiating cancer cells is to inactivate the cell so that it can not proliferate, and eventually die.

To inactivate the cell, we need to induce double strand breaks in the DNA helix. DSBs can be induced in two ways, either by one particle inducing breaks in both of the DNA strands, or by two separate particles inducing SSB in each of the DNA strands. If the two separate SSBs are within a few base pairs of each other, they can turn into a DSB as long as the first induced SSB has no time to repair itself before the second SSB occurs.

When a DSB occurs, the damaged site experiences a loss of genetic material. There are two main processes for DSB repair depending on where in the cell cycle the break occurs. If the break is induced after all the genetic material has been copied, but before the cell is separated into two daughter cells, in late S/G2, the loss of genetic material can be repaired through homologous recombination repair, HR. The damaged chromatid can invade the undamaged sister chromatid and use it as a template to fill in the lost base sequence, giving perfect repair.

If the damage occurs before the DNA has been fully sequenced or if the repair is urgent it is repaired through the faster repair pathway, nonhomologous end-joining, NHEJ. In NHEJ, the ends of the separated strands are joined back together regardless of the loss of base sequence.

Often, a few more bases are cleaved off to create symmetric ends that can be joined together.

This leads to loss of genetic information and can cause mutations and even cell death. Most of the DSBs occur in non-coding areas of the DNA strand, known as introns, since the majority of the DNA strand is non-coding. The loss of bases in the introns is of little importance for the genetic code, but when this loss occurs in the coding areas of the DNA it will have

consequences. An illustration of the timing for both repairs can be seen in figure 13 along with a schematic figure of how the HR repair works.

Figure 13 Left: timing for NHEJ and HR processes during the cell cycle. Right: DSB repair through HR, the damaged chromatid invades the undamaged sister chromatid and the lost base sequence is replaced by a perfect copy (Hall, 2019).

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21 DSBs resulting from irradiation can behave in three ways. One of them is repair, as described above. The second option is that the break does not rejoin, which creates an aberration and can lead to loss of genetic material when the cell enters mitosis. The last option is that the broken ends rejoin with other broken ends, and not to the original break-site (Hall, 2019). The behaviour which is most important for cell death is option number two, aberrations. We have two kinds of aberrations, chromosome and chromatid aberrations. If the break occurs before duplication a single stranded chromatin will separate from the rest and replicate the break, this creates lethal dicentric chromosomes and rings. When the break occurs after duplication, we get chromatid aberrations, if this happens in both chromatids of the chromosome, the ends of the breaks can rejoin incorrectly and create a lethal anaphase bridge. An image of the lethal aberrations can be seen in figure 14.

Figure 14 Lethal aberrations. Left: Dicentric chromosome, sticky ends from two chromosomes join together. Chromosome ends up with two centromeres, making it dicentric. Middle: Overlapping rings, break in each arm of one chromosome, sticky ends are joined together to form a ring. Right: Anaphase bridge, break in both chromatids in one chromosome, incorrect re-

joining, one centromere moves to each pole during anaphase (Hall, 2019).

Radiation damage can be categorized into lethal damage, potentially lethal damage (PLD) and sublethal damage (SLD). Lethal damage leads to cell death, with no chance of survival. PLD does not kill the cell immediately but will turn into lethal damage unless the cellular

environment is manipulated to allow time for repair. A way to manipulate the environment is to let the cells grow to confluency, meaning they have no room to grow. The cell cycle will then slow down, and mitosis is delayed, giving time to repair PLD. SLD is damage that is not lethal in itself but can interact with other SLDs and become lethal. If a radiation dose is given in two fractions instead of one, we will have an increased survival as we allow enough time for repair of SLD.

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2.3.3 Cell death

When a lethal aberration has taken place in a cell, the cell will lose its function and eventually die. It can be physically present in the environment, but if it has lost its ability to divide it can not form a large colony and is therefore considered dead. There are five types of cell death:

The Bystander Effect, Apoptotic death, Mitotic death, Autophagic cell death and Senescence.

The Bystander Effect and Apoptotic and Mitotic death are of higher importance in irradiation of cancer cells than Autophagy and Senescence, so we will focus on the three that are relevant for irradiation.

The Bystander Effect

Radiation can induce cell damage such as aberrations, mutations and death in proximal cells, even though the proximal cells were not directly hit with radiation. This effect is increased if the cells are in gap junction with the irradiated cell, killing up to 30% of the cells surrounding the irradiated cell. If the cells are a few hundred micrometers apart from each other, signals secreted by the irradiated cells can induce cell death in unirradiated cells. A study done with a heavy-ion beam reported a 10⁴ reduction in cell survival than estimated, when only 0.0001%

to 0.002% cells in a confluent cell culture was irradiated (Harada et al., 2009).

Apoptotic Death

Apoptosis is a form of programmed cell death that occurs when tissues become obsolete, but it can also be induced by radiation. The dying cell stops communicating with its neighboring cells and detaches from them. Then, the chromatin and cytoplasm condensates, shrinking the cell, and eventually separating it into cytoplastic or nuclear fragments that phagocytic cells can remove before the fragments cause any damage. These phagocytic cells can induce an immune response that protects the host from the specific cell in the future (Alberts, 2002).

Mitotic Death

The most common way for a cell to die from irradiation is through mitotic death. The

chromosomes are damaged from DSBs which can lead to cell death. The cell may divide for a few generations with damage after irradiation before death occurs. There has been observed a strong correlation between the number of lethal aberrations and cell killing for cell lines where mitotic death is the norm, and no apoptotic death is observed.

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2.3.4 Cell survival curve

The cells that survive irradiation can keep growing and duplicate to form larger colonies of cells. The survival of these cells after irradiation can be visualized through in vitro

experiments. We can extract cells from tissue and seed them as single-cell suspension in culture dishes. These dishes can then be irradiated with different doses, and survival curves can be obtained by counting proliferative colonies, defined to contain above 50 cells, and plotting this survival against the given dose. The survival after irradiation is compared to a control group that does not receive any dose.

A known number of cells are seeded out in each dish. The number varies depending on which dose the dish is going to receive so that there is a countable number of colonies after

irradiation. If the number is too low the statistics will be poor, and if the number is too high the colonies might grow into each other, making the dish difficult to count.

When seeding cells, not all of them will survive the seeding process due to trypsinization, human errors or suboptimal growth conditions, to mention a few. Plating efficiency (PE) is a measure of the fraction of cells that survive the process and can form colonies, given from equation 14. Plating efficiency is ideally between 50-90% in experiments.

𝑃𝐸 =𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑜𝑙𝑜𝑛𝑖𝑒𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑

𝑁𝑢𝑚𝑏𝑒𝑟 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑠𝑒𝑒𝑑𝑒𝑑 × 100 (14)

The surviving fraction after irradiation is given by equation 15 and is the fraction of cells surviving irradiation compared to the original number of cells seeded when taking plating efficiency into account.

𝑆𝑢𝑟𝑣𝑖𝑣𝑖𝑛𝑔 𝑓𝑟𝑎𝑐𝑡𝑖𝑜𝑛 = 𝐶𝑜𝑙𝑜𝑛𝑖𝑒𝑠 𝑐𝑜𝑢𝑛𝑡𝑒𝑑

𝐶𝑒𝑙𝑙𝑠 𝑠𝑒𝑒𝑑𝑒𝑑 × (𝑃𝐸 100⁄ ) (15)

An example of a surviving curve for two types of radiation can be seen in figure 15. The shape of the curve varies between radiation types and cell lines, but they share a few

characteristics. The curve begins straight on the log-linear plot and starts bending down when the dose increases. At very high doses the curve approaches a straight line. When the type of radiation changes from a sparsely to a densely ionizing radiation, the shoulder of the curve gets smaller and the curve approaches a straight line earlier (figure 15). The shoulder of the

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survival curve is also dependent on the repair of SLD, where a large shoulder indicates more repair of SLD than a small shoulder.

Figure 15 Survival curves after irradiation for mammalian cells using the LQ-model. The survival is plotted on a log-linear scale. The X-ray curve has a distinct shoulder and becomes linear at higher doses. The neutron curve is linear for all doses

(Hall, 2019).

The linear-quadratic (LQ) model is the most common model in explaining the surviving fraction and the shape of the survival curve. The expression for the cell survival curve is given by equation 16.

𝑆 = 𝑒^{−𝛼𝐷−𝛽𝐷}² (16)

Where D is the dose, S is the surviving fraction and 𝛼 and 𝛽 are constants. It is based on the theory that exchange-type chromosomal aberrations can be created in two different ways. The linear term is linked to the creation of a DSB through one radiation particle, and the quadratic term is linked to DSB created through two separate interactions, two SSB (section 2.3.2). The former is proportional to the dose and can be related to the linear term in the surviving curve, and the latter is proportional to the square of the dose and can be related to the quadratic term in the survival curve.

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2.3.5 Dose-Rate Effect

Dose rate is linked to the biologic effect of a given dose, meaning that not only does the amount of dose delivered impact the biological system, but also the rate at which the dose is delivered. A reduced dose rate over a longer time reduces the biologic effect of the dose as we allow enough time for SLD to be repaired during irradiation before a new SLD occurs and it turns into irreversible damage.

2.3.6 LET and RBE

Linear energy transfer (LET) was defined by the International Commission on Radiological Units in 1962 as:

“The LET (L) of charged particles in medium is the quotient of 𝑑𝐸/𝑑𝑙, where 𝑑𝐸 is the average energy locally imparted to the medium by a charged particle of specified energy in traversing a distance of 𝑑𝑙. That is, 𝐿 = 𝑑𝐸/𝑑𝑙”(Hall 2019, p.101) It states that the LET is average energy transferred to a medium per unit length with unit 𝑘𝑒𝑉/𝜇𝑚. Since LET is an average energy, it can differ a lot from the reality due to the fact that the energy deposition in the track varies a lot over the distance in question. The average does not give a realistic view of the deposition of energy, and there is question of if the

quantity is meaningful or not. It can however give an indication of the quality of the radiation, as different types of radiation have different LET. The LET is usually calculated as a track- or energy average, splitting the track into equal lengths and averaging the energy or into equal energy intervals and averaging the track lengths. A visualisation of this can be seen in figure 16.

Figure 16 LET track average and energy average (Hall, 2019).

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When the energy of a particle increases, the LET decreases as the particle travels through the matter with fewer interactions and depositions, therefore lowering the biological

effectiveness. Typical LET values are listed in table 1. For x-rays and monoenergetic proton beams, the track averaged- and energy averaged LET is approximately the same.

Table 1 Typical track averaged LET Values. The proton beams are monoenergetic (Hall, 2019).

Radiation LET [𝒌𝒆𝑽/𝝁𝒎]

250-kV x-rays 2.0 10-MeV protons 4.7 150-MeV protons 0.5

The difference in the effectiveness on the absorber between two different sources of radiation is given by relative biologic effectiveness (RBE). Usually, x-rays are used as a reference source and are compared to the radiation in question to obtain a measure of biologic effect.

RBE is simply a ratio of the doses where the two radiation types have the same survival level, taken from survival curves of the two radiation types, as seen in equation 17 where D is the dose.

𝑅𝐵𝐸 =𝐷𝑟𝑒𝑓𝑒𝑟𝑒𝑛𝑐𝑒 𝑟𝑎𝑑𝑖𝑎𝑡𝑜𝑛

𝐷_{𝑟𝑎𝑑𝑖𝑎𝑡𝑖𝑜𝑛} (17)

Since different kinds of radiation can have differently shaped survival curves depending on LET and cell line, RBE varies greatly depending on the survival level chosen for the

comparison. A lower dose will result in a larger RBE, as the survival curves usually diverge from each other, increasing the difference between the two doses giving the same effect. The reason why RBE is depending on the cell line, is that different cell lines have different rate of repair of SLD, resulting in different initial shoulders in the survival curves, as mentioned in section 2.3.4. The slope of the curve is dependent on the repair of PLD. LET affects the RBE as a higher LET gives a steeper survival curve and a smaller shoulder, indicating less repair of SLD and PLD.

The differences in RBE due to LET can be seen by plotting RBE as a function of LET.

Graphs of this type for three different survival levels is shown in figure 17. We can clearly see that when the LET is higher than 10 𝑘𝑒𝑉/𝜇𝑚 RBE increases drastically for all survival levels, until it reaches a maximum at 100 𝑘𝑒𝑉/𝜇𝑚. Beyond this, the RBE decreases rapidly.

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27 The point of maximum RBE at 100 𝑘𝑒𝑉/𝜇𝑚 represents the most effective LET value for inducing DSBs. At this LET, the ionizing events in the radiation are separated by 2𝑛𝑚, which is the same distance as the width of the DNA double helix, meaning that this energy is enough to cause ionizing events in both strands of the DNA. A higher LET than this will give

ionizations that are closer together, but these events are wasted as they cause no additional damage.

Figure 17 RBE as a function of LET for three different survival levels (Hall, 2019).

2.3.7 𝜸-H2AX

H2AX is a protein involved in the histone structure that the DNA strand coils around to create the chromatin structure (Pinto & Flaus, 2010). When the DNA experience DSB from

radiation exposure, H2AX is phosphorylated, known as 𝛾-H2AX, and is included in the repair pathways (section 2.3.2) in order to stabilize DNA repair proteins (Redon, Dickey, Bonner, &

Sedelnikova, 2009). The number of 𝛾-H2AX foci present in the cell has been shown to be directly proportional to the number of DSBs induced in the cell, one 𝛾-H2AX represents one DSB (Rothkamm & Löbrich, 2003), and the number of 𝛾-H2AX foci is at a maximum 30 minutes after irradiation and decreases beyond this due to repair processes (Rogakou, Boon, Redon, & Bonner, 1999).

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2.3.8 Radiative treatment of cancer

High energy X-rays are most commonly used for radiation therapy today, but treatment with charged particles like protons and carbon ions are used more and more due to the sparing effect on healthy tissue. As mentioned in section 2.2.1, X-rays deposit most of their dose close to the surface of the irradiated matter, and then the deposit decreases slowly as the beam travels further into the tissue. This leads to a dose deposition can present a risk for healthy tissue and important organs. Charged particle irradiation on the other hand, has its maximum dose deposition deeper into the tissue, and behind this the dose quickly decreases toward zero.

Charged particles, such as protons, are more suited for sparing of healthy tissue and organs at risk, OAR, as the beam can be used for irradiation of tumours close to OARs without

exposing them to radiation due to the rapid dose cut-off behind the Bragg-peak.

Since the Bragg peak from a proton beam is narrow compared to a tumour volume, a spread- out Bragg peak, SOBP, is used in therapy to achieve a uniform dose over a larger area, as the SOBP has a broadened peak. By using absorbers of different thicknesses on the beam, we can obtain several different Bragg peaks of varying intensity, that together form one large,

broadened Bragg peak with uniform dose.

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3 Materials and Methods

3.1 Dosimetry

3.1.1 Ionization chamber dosimetry: X-ray

A Kerma calibrated ionization chamber IBA FC65-G was placed in a steel container with lid used for cell irradiations in the center of the X-ray beam. The X-ray unit used was the

PANTAK PMC 1000 at the Roentgen lab, UiO. The ionization chamber was connected to a Standard Imaging electrometer, MAX-4000. The ionization chamber was moved between four different positions around the edge of the chamber, and one in the middle of the steel chamber. This was done to obtain an average dose for the area where dishes are placed during the experiments. For some experiments, we placed the steel chamber on a table as close to the beam exit as possible to increase the dose rate of the irradiation as much as possible to reduce the irradiation time. Figure 18 is an image of the setup. The X-ray beam was filtrated with a 1mm Be internal filter and a 0.5mm Cu filter placed below the X-ray tube exit window.

The ionization chamber was irradiated three times in each of the five positions for 30 seconds at a time, and an electrometer reading was done for each irradiation. The readings were then averaged, and the dose to water given in 30 seconds was calculated through equation 19.

𝑀_𝑢 = 𝑀 (273,2 + 𝑇 273,2 + 𝑇₀

𝑝₀

𝑝) (18)

𝐷_𝑤 = 𝑀_𝑢𝑁_𝑘𝑘_𝑢(𝜇̅_𝑒𝑛 𝜌 )

𝑤,𝑎𝑖𝑟

𝑝_𝑢 (19)

where 𝑀_𝑢 is the chamber reading measurement corrected for pressure and temperature, 𝑁_𝑘 is the calibration factor for standard ambient condition, 𝑘_𝑢 accounts for the spectral distribution changes when moving from air to water, (^𝜇^̅^𝑒𝑛

𝜌 )

𝑤,𝑎𝑖𝑟

is the mass energy absorption coefficient ratio between water and air, and 𝑝_𝑢 is the perturbation factor. This dose rate was translated into Gy/min. Constants for the calculations are listed in table 2.

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Table 2 Constants for dosimetry at the X-ray unit.

𝑁_{𝑘,𝑎𝑖𝑟} 𝑘_𝑢 (𝜇̅_𝑒𝑛⁄ )𝜌 _{𝑤,𝑎𝑖𝑟} 𝑝_𝑢 𝑘_𝑇𝑝

43.77 ± 0.39 ≈ 1.0 1.07 1.023 ± 0.001 0.9945

Figure 18 Setup during dose calibrations with ionization chamber in the X-ray unit. Beam exit is at the top of the image, over the 0.5mm Cu filter.

3.1.2 Ionization chamber dosimetry: Protons

The proton experiments were performed with a Cyclotron Scanditronix MC-35 at Oslo Cyclotron Laboratory. The setup requires separate rounds of dosimetry every day of the experiments as the beam varies in energy and intensity between each day. We used a PTW Monitor Chamber Type 34014 connected to the Standard Imaging electrometer, together with a dose to water calibrated PTW Advanced Markus ionization chamber connected to a

UNIDOSE E electrometer to achieve accurate dose measurements. The monitor chamber was placed 35cm from the beam exit window, and the ionization chamber was placed behind this.

The ionization chamber was moved to different distances from the beam exit window to locate the Bragg peak, usually placed between 80 cm and 95 cm in air. A parafilm was used instead of the dish lid for cell irradiations in front of the Bragg peak. For dosimetry a parafilm

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31 was therefore placed in front of the ionization chamber to find positions in front of the Bragg peak, and a vent dish lid is used as an absorber in front of the ionization chamber to find positions in the top and back of the Bragg peak. A picture of the setup can be seen in figure 19. Since the beam intensity varied during irradiation, the monitor chamber (MC) was used to measure the fluence and thereby control the dose delivered. During initial dosimetry

measurements, the ionization chamber (IC) reading and the monitor chamber reading were divided by each other to obtain a calibration factor. This factor was used to calculate which monitor chamber units we needed to give to obtain the wanted doses in the different positions.

𝐷(𝑥) = 𝐹_𝑐(𝑥) ∗ 𝑁_𝐷 ∗ 𝑈_𝑀𝐶 (20)

Equation 20 relates MC units with the dose, where 𝐷(𝑥) is the dose given in position x, 𝐹_𝑐(𝑥) is the calibration factor ratio between the IC and the MC in position x, 𝑁_𝐷 = 1.411 𝑚𝐺𝑦/𝑛𝐶 is the dose calibration constant for the ionization chamber and 𝑈_𝑀𝐶 is the monitor chamber reading.

Figure 19 Setup during Ion chamber dosimetry. Ion chamber to the left and monitor chamber to the right. Beam exit window is to the far right.

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3.1.3 Gafchromic EBT3 film dosimetry

Before each day of proton irradiations, the Gafchromic EBT3 dosimetry film was attached to the beam exit window and exposed to radiation. Since the film changes color when exposed to the protons, we used this to check the shape of the beam and its direction out of the beam exit window. If the beam was not round and centered in the beam exit window, adjustments had to be done before proceeding with calibrations. The Gafchromic EBT3 film was also used to measure the homogeneity of the beam by placing it in front of the ionization chamber.

Approximately 24 hours after the film was irradiated, it was scanned in an Epson Perfection V850 Pro flatbed scanner in transparency mode. The scans were then analyzed to determine the optical density of the film which could be converted into the average dose given to the film (section 2.2.3). The optical density (OD) was found through the intensity of light (I) that was able to pass through the film during the scan. The relation between these two can be seen in equation 21.

𝑂𝐷 = log₁₀𝐼 (21)

3.2 Cell Survival Experiment

Clonogenic assays were done to acquire statistics of surviving cells after in vitro irradiations with X-rays and protons.

3.2.1 The cell lines

The cell lines we were interested in working with was T98G and A549. T98G is a cell line of human glioblastoma multiforme tumour from a 61-year-old Caucasian male. A549 is a cell line of human epithelial lung carcinoma from a 58-year-old Caucasian male. Both cell lines used were bought from the American Type Culture Collection, ATCC.

Since cancer cells lack the ability to control cell division they can divide indefinitely as long as they have access to the correct nutrients and growth factors. In the laboratory, we can provide all factors that the cells need to obtain exponential growth, which makes it possible to use the same type of cells in all the experiments. The main method of achieving this is by subculturing.

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3.2.2 Equipment

To avoid contamination of the cells, we had to work in a sterile, uncontaminated environment.

All cell work was therefore conducted in a Laminar Air Flow bench (LAF bench) that protects the cells from contaminations through air. The bench was cleaned with 70% ethanol before and after use. Sterile gloves and a sterile coat were used when working with the cells, since the open vent dishes were very susceptible to contamination. The cells were kept in a standard CO2 incubator that kept a temperature of 37℃ and 5% CO2 for optimal cell growth conditions.

The T98G cells were grown in a sterile RPMI 1640 medium. EDTA Trypsin was used to detach the cells from their dishes. When fixating the cells, we rinsed the dishes with Phosphate-buffered saline, PBS, fixated them with technical ethanol and dyed them with Methylene blue.

25cm²sterile flasks were used for subculturing of the cells, and 60𝑚𝑚 × 15𝑚𝑚 sterile vent dishes were used for experiments. During X-ray irradiation, a parafilm strip (Parafilm M Laboratory film) was used to seal the gap between the lid and dish bottom to prevent contamination. During proton irradiation, a parafilm strip was used to seal the lid during irradiations in the back of the Bragg peak, and a round parafilm piece was used to act as a lid for the vent dish bottom during irradiations in the front of the Bragg peak. All parafilm pieces were sterilized in ethanol and dried for 24 hours prior to use.

Sterile disposable pipettes attached to a Pipetus were used to handle all chemicals and cell suspensions, for the 2mL pipettes we used a manual Pipetus Junior or a balloon, and for the 5mL-25mL pipettes we used an electrical Pipetus-akku. A Bürker chamber was used for cell counting to obtain the correct cell density.

Three different centrifuges were used to centrifuge the cells into pellets: a Beckman GS-15 Centrifuge, a Hettich Rotofix 32 and a VWR Mega Star 600R. A Nikon TMS microscope was used to track cell growth and count cells.

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3.2.3 Subculturing

The cell lines are cultivated in 25cm²sterile flasks and will regularly run out of space to keep dividing as they grow and divide to fill the bottom surface of the flask. When this happens, the cells have to be subcultured to new flasks to avoid cell mutations and cell death. When subculturing, a small fraction of the cells in the old flask are transferred to a new flask with fresh medium, where they have enough space and nutrients to achieve exponential growth.

This subculturing was performed on Mondays and Fridays, and in addition, medium was changed on Wednesdays.

The old medium was removed and 2mL trypsin was added and removed twice to rinse the cells and detach them from the flask surface. The cells were put in the incubator, and after approximately 3 minutes the cells had detached. Then we added medium to the flask to inactivate the trypsin and resuspended the cells by using a pipette and pumping the solution.

The amount of medium added varied depending on the growth of the cells. The amount of solution needed for further culturing, 0.5mL, was then extracted and added to a new flask containing 4.5mL fresh medium.

3.2.4 Seeding for colony assay

First, we prepared the vent dishes needed by adding 4mL medium and putting them in the incubator for temperature- and CO2 equilibration. For X-ray experiments we had five dishes for each dose, ten for control and one for multiplicity counting that it used to correct for cell division, and for proton experiments we had four for each dose or position, eight for control and one for multiplicity counting. The dishes for X-ray experiments were prepared in the cell lab in the chemistry building at UiO, and the dishes for the proton experiments were prepared in the cell lab at OCL. Then we prepared two tubes, one containing 3mL medium and the other containing 4.5mL medium.

Next, we removed the medium from the 25cm² flask of subcultured cells and rinsed it with 1.5mL trypsin. Then we added 3mL trypsin and put the flask into an incubator for 5 minutes.

When the cells had detached from the flask, we used a 10mL needle and 2mm syringe and gently pumped the solution 3-6 times to achieve single cell suspension. The suspension was then transferred to the tube containing 3mL medium and the tube was centrifuged for 4 minutes at 1000 rpm to create a pellet of cells. Next, we removed the supernatant, added 3mL

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35 fresh medium to the tube and resuspended the pellet thoroughly. 0.5mL of this solution was added to the tube containing 4.5mL medium, diluting the solution 1:10 to make cell counting easier. A drop of this was counted in the Bürker chamber to find the cell density of the solution.

Lastly, the correct dilutions for the different doses were calculated, prepared and added to the previously prepared vent dishes. The number of seeded cells for each experiment can be seen in Appendix A. 10 000 cells were added in the vent dish for multiplicity counting. All dishes were incubated overnight to allow enough time for cells to attach to the vent dish bottom, but not enough time to allow for too much cell division. For X-ray experiments, an incubation time of 16-20 hours was used, while for proton experiments an incubation time between 12- 16 hours was used due to uncertainties in the beam stability.

3.2.5 X-ray irradiation

Overview

In total, I performed 9 clonogenic experiments with X-ray irradiation on the T98G cell line.

Vent lid dishes were used in all experiments to achieve comparable results to the proton irradiations. Small adjustments were made underway to eliminate mistakes and improve the results. Three clonogenic survival experiments were also done with the A549 cell line.

Setup

The setup during X-ray irradiation was similar to the setup during ionization chamber calibrations, as seen in figure 18. The steel chamber containing 5 vent lid dishes with cells placed in a circle around the center of the chamber, was placed inside the X-ray unit on a table. The table was centered under the X-ray tube exit window to obtain a dose distribution as even as possible over the area covering the steel chamber. During the standard X-ray experiments where we irradiated with doses ranging from 0.5 Gy to 10 Gy, the table was lower. When we had to do some experiments with higher doses, up to 13 Gy, we built the table seen in figure 18 higher to reduce the irradiation time.

For the majority of the X-ray experiments, the cells were irradiated with cell medium in the dish. In one of the experiments we used three different methods for X-ray irradiation with 10

Relative Biologic Effectiveness and Linear Energy Transfer effects of low energy proton irradiations for T98G human glioblastoma cells