Irradiator Commissioning and Dosimetry for Assessment of LQ α and β Parameters, Radiation Dosing Schema, and in vivo Dose Deposition

Any steps of this protocol involving the use of laboratory animals, including handling and procedures, were approved by the Institutional Animal Care and Use Committee at West Virginia University in Morgantown, West Virginia (Protocol number: 1604001894).

1. Determination of dose output

1. Use this protocol, based on the “In-phantom Method” protocol of the American Association of Physicists in Medicine Task Group (AAPM TG) 61¹⁰ and similar to the commissioning protocol set by Xstrahl, to determine beam output of the small animal irradiator with respect to particular geometry under the following setup conditions.
   1. Set the irradiator to deliver radiation at 220 kVp and 13 mA, with an open field (17 cm by 17 cm) positioned at isocenter, or 35 cm from the source. Additionally, filter the beam with a 0.15 mm Cu filter with a broad focus. Some cellular irradiators contain only a radioactive source, this protocol can only be used for x-ray irradiators.
   2. Align the solid water phantoms in the following order: 1 cm slab, 2 cm slab with ionization chamber slot, 2 cm slab, 1 cm slab. Stacking the solid water phantoms in this order positions the ionization chamber at a depth of 2 cm, allowing 4 cm as well for backscatter. See Figure 1 for a graphical portrayal of the dosimetry setup.
      NOTE: To accommodate the large, fairly heavy stack of solid water, the authors recommend the acquisition of a custom 3D printed couch with variable support to ensure the phantom stack is level and at the correct distance from the source across the surface of the material, not just at the center.
2. Use the measurement equipment (i.e., ADCL calibrated ionization chamber, electrometer) and an explanation of correction factors used can be found in Table of Materials and Table 1 respectively.
   NOTE: That the ADCL provides values of N_k at a couple of points for different Half Value Layers (HVL, measure of beam quality). The value of N_k to be used in the protocol should be based on an interpolation of the ADCL values for the unit’s measured HVL. The manufacturer measured the HVL of our unit and we used that in our dose rate output determination.
3. Set up the phantom stack and insert the ionization chamber into the phantom as specified in step 1.1.2.
   1. Adjust the phantom stack such that the source to surface distance (SSD), or the distance from the radiation source to the first surface, is 33 cm when appropriately levelled.
      NOTE: The authors suggest creating a custom, 3D printed couch, large enough to support the dimensions of the solid water slabs. Additionally the one, utilized in this protocol has an adjustable component for levelling the phantom stack.
4. Take the average of three separate x-ray exposures, one minute readings with the electrometer bias voltage set at 300 V. The result will be termed M⁺.
   NOTE: Irradiations are performed with the instrument set to deliver radiation at 220 kVp and 13 mA. This is the same for the next two steps (steps 1.5-1.6). For user safety, ensure the doors remain closed during treatments.
5. Perform another set of three separate x-ray exposures, 1 min readings with the electrometer bias voltage set at -150 V. The result will be termed M_L.
6. Perform another set of three separate x-ray exposures, 1 min readings with the electrometer bias voltage set at -300 V. The result will be termed M_H, or also M^–.
7. Calculate P_pol and P_ion using Equation 1 and Equation 2 respectively as described below:
   (Equation 1)
   (Equation 2)
8. Measure the temperature, in Celsius, and the pressure, in kPa, inside the irradiator using a calibrated digital thermometer and barometer. Then, calculate P_TP as indicated below in Equation 3.
   NOTE: This calculation assumes that the ADCL used standard temperature and pressure values of 22 °C and 101.33 kPa when stating their value for the air kerma calibration factor.
   (Equation 3)
9. Calculate the corrected chamber reading, M, by multiplying the raw chamber reading, M_H, by P_elec, P_pol, P_ion, and P_TP. This equation can be found below in Equation 4.
   NOTE: This calculation assumes that the ADCL performed their calibration with the bias voltage set to -300V, which is a fairly common practice.
   (Equation 4).
10. Further multiply the corrected chamber reading by N_k, [(µ_jp/p)^w_air]_water, P_{Q, cham}, and P_sheath. P_sheath is only needed for measurements obtained in water. Therefore, for this protocol P­_sheath is just 1.
    NOTE: Using the conditions in this protocol, the latter three items give a value of 1.0731. This value depends on the beam quality, so the HVL must be known to determine it. The value of 1.0731 is specific to our unit and is given as an example. To determine the values of P_Q,cham and [(µ_jp/p)^w_air]_water specific to your unit, use the measured HVL and interpolate from Table VII, and Table VIII, and correct for the reference field size according to Figure 3 and Figure 4 from the AAPM TG61 protocol¹⁰. In our case, multiplying N_k by 1.0731 provides the dose to water, D_w, in Gy for a nominal time of 1 min, assuming the ADCL N_k value is given in Gy/Coulumbs.
11. Determine the end effect of the irradiator being used. When the x-rays are first generated, the output rises to its full value over some finite time. Similarly, when the x-ray source is turned off, the output decreases to zero over some finite time.
    1. Account for the time for this transition, or the end effect. This can be done by taking the average of three readings with the electrometer bias voltage set at -300 V, for a variety of time settings. Do this for 6, 12, 18, 24, 30, and 60 seconds.
    2. Plot the electrometer readings against time and find the best straight line. The total time, t, for a 1 minute treatment can be calculated by equation 5:
       (Equation 5).
12. Calculate the dose rate for a given irradiator by equation 6:
    (Equation 6)

2. Creating a radiochromic film calibration curve

1. For a list of necessary materials, see Table of Materials.
2. Using a near identical set up as the previous protocol, place the film at a 2 cm depth in the solid water phantom stack. The order of solid water phantoms is insignificant so long as there are 2 cm of solid water above and 4 cm of solid water below for buildup and backscatter effects.
3. Using the determined dose commissioned in protocol 1, determine the treatment times for the doses listed in Table 2 using equation 7:
   (Equation 7)
4. Prepare several pieces of film ensuring that each film is of the same size and remains in the same orientation from treatment through scan acquisition. This can be done by placing a small diagonal cut in the lower left corner. Each film from this point forward must be from the same batch of film.
   NOTE: Prepare 3 separate replicates for each dose point to be assessed.
5. Scan the cut pieces using a 48-bit color photo scanner with all corrections turned off. Ensure that each film is placed in the exact center of the scanning bed. The values obtained are the pre-exposure scans used for determining the unexposed optical density^11,12. Save all images in the .Tiff file format to avoid compression of key data.
   NOTE: The authors recommend scanning the films three times and using the obtained average as the single value for a given film.
6. Begin the irradiation of the films by placing a piece of film on top of 4 cm of solid water and positioned the remaining 2 cm of solid water above, as described previously in this section.
7. Adjust the phantom set so that the film is the same distance from the source as the ionization chamber was when determining the dose output. This is the isocentric point of the irradiator.
8. Program the treatment time calculated in step 2.3 above for one prescribed dose.
9. Repeat treatment for each of the doses listed in Table 2.
10. Allow films to rest for 24 hours protected from light.
11. Acquire the post-exposure film scans in the same manner as above.
12. Import images to ImageJ analysis software and perform all measurements on the red channel.
    1. Drag the image in .Tiff file format into ImageJ.
    2. Click the Image drop down menu. Select Color from Image drop down menu. Select split channels from Color option.
    3. Using only the red image channel, draw a region of interest using the rectangle tool. Press Ctrl+M. Transcribe mean value from the results window.
    4. Repeat steps 2.12.1-2.12.4 for all scanned films.
13. Obtain the pixel value in a centrally located 1 cm by 1 cm square for both the unexposed and exposed films. These values will be denoted as PV_U(D) and PV_(D) respectively, and can be used to calculate the net optical density as described in Equation 8.
    (Equation 8)¹³
14. Repeat step 2.13 for each pair of film images, both pre-exposure and post-exposure.
15. Plot a graph of the dose versus the net optical density and fit the curve to a cubic polynomial in the format of y = ax³ + bx² + cx + d. An example can be found in Figure 2B.

3. Determination of α/β value for specific cancer cell lines via clonogenic assay

NOTE: The following protocol is a modified version of the methods described by Franken et al¹⁴ and can be seen in Figure 3.

1. Grow cells to ~80% confluency. Avoid using over-confluent sources of cells for this experiment, as it is necessary that the cells are in the log-phase of cell growth. For the representative clonogenic assay results displayed in Figure 3C, brain-tropic MDA-MB-231 breast cancer cells were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum and penicillin/streptomycin and were incubated at 37 °C and 5% carbon dioxide in a humidified incubator.
2. Seed the cells at the desired density for the colony assay. Accurate dilutions during the seeding are crucial to determining the assay’s plating efficiency. Be sure to plate multiple replicates.
3. Proceed with this step if radiation treatment will precede cell plating (Figure 3A). Alternatively, proceed to step 3.4 if cell plating will precede radiation treatment.
   1. Perform desired radiation treatment on culture flasks. Any additional treatments (i.e. drug treatments) may be performed at any point before or after this. For the representative results in Figure 3C, radiation treatment occurred after plating cells, detailed in step 3.4.
   2. Extract the cells using preferred trypsinization method and create a single cell suspension. Remove culture media and add recombinant enzyme (e.g., TrypLE Express) to detach cells from the flask. Incubate cells with the enzyme for approximately 3 minutes until cells were detached as detected using a light microscope. Neutralize the enzyme using an equal volume of cell culture media. Centrifuge cells at 300 x g for 10 min and resuspend to the desired concentration in culture medium.
   3. Plate the cells at desired densities in multiple replicates.
   4. Replace with fresh media after the first 24 h.
   5. Continue to replace media every 2-3 days.
   6. Continue culturing cells until control colonies exceed 50 cells per colony, ~9-14 days. Control colonies are those treatment groups that receive no radiation doses. For experiments using drug treatments as well, another control group with drug dosing but no radiation treatment will also be required.
4. Proceed with this step when seeding cells before radiation treatment (Figure 3B).
   1. Extract cells using preferred trypsinization method and create a single cell suspension.
   2. Place cells at desired densities in multiple replicates.
   3. Allow cells to adhere to plate overnight.
   4. Perform desired radiation doses. Additional treatments, such as drug dosing, may be performed at any point before or after this step, so long as cells have attached to their treatment plates. For the representative results in Figure 3C, 1250 brain-tropic MDA-MB-231 cells were plated prior to treatment (step 3.4). Then, cells were treated with 15 nM doxorubicin 3 hours prior to irradiation with 3 Gy of X-rays.
   5. Replace the media after the initial 24 h.
   6. Replace media every 2-3 days.
   7. Culture the treated cells until control group colonies exceed 50 cells, ~9-14 days. Control colonies are those treatment groups that receive no radiation doses. For experiments using drug treatments as well, another control group with drug dosing but no radiation treatment will also be required.
5. Remove culture media from wells or dishes, and wash with PBS.
6. Fix cells for 15 minutes in a 1:7 (v:v) solution of glacial acetic acid and methanol.
7. Remove the fixation solution.
8. After fixation, stain cells for 30 minutes, or 2 h if time is available, at room temperature with a 2.5-5.0 mg/mL solution of crystal violet in a 4:1 (v:v) solution of distilled water and methanol.
9. Remove staining solution and wash cells in a large, room-temperature water bath.
   NOTE: Do not wash under running water.
10. Count the resultant number of colonies in each treatment group and calculate the survival fraction of each plate.
11. Plot the survival fraction against the corresponding dose delivered, and fit the curve with an exponential fit.
12. To estimate the α/β value, use an exponential fit of the above plot to estimate the values for each of the adjustable parameters in the linear-quadratic equation found below:
    (Equation 9)
    NOTE: Irradiation of cells can typically be done at isocenter without any collimation provided the field size is large enough to accommodate well-plates or petri dishes. Potential pitfalls in this protocol may include yields such as no colony formation, significant cell migration with clear cell growth but no true colonies, or contamination due to treatment in a non-sterile irradiator chamber.

4. Determination of the specific dose output for variable experimental designs

1. Decide upon the desired field size and distance from source.
   NOTE: Collimation will alter the dose rate no matter the size or distance of the collimator from the x-ray source.
2. Using solid water phantoms to provide buildup and backscatter, position a piece of film in the correct orientation that best portrays the experimental design.
   NOTE: For any experimental setup solid water may not provide the most accurate representation of a given design. Instead we recommend using the actual experiment’s vessels (i.e., Petri dish, well-plates, small animal phantoms, etc.).
3. Irradiate films for 1 (N=3) and 2 (N=3) minutes.
4. Allow films to rest for 24 hours protected from light.
5. Determine the net optical density of each the films following the procedures from Section 2. Use the film calibration curve to determine dose from the net optical density.
6. Determine the dose at 1 minute, D₁, as the output dose rate, Ḋ, for this experimental setup defined by Equation 10 as follows:
   (Equation 10)
7. Similarly, caluclate the dose at 2 minutes by Equation 11 as follows:
   (Equation 11)
8. Due to the end effect, the dose rate for the above calculations may be slightly different. For this reason to calculate D_exp for the desired experimental design, use an average of the individual D_exp as indicated in Equation 12:
   (Equation 12)
9. Using this average, define the time for treating to any desired dose for this particular setup in Equation 13:
   (Equation 13)

5. Treating mice bearing tumors in anatomical location of interest

1. Anesthetize mouse with safe and humane anesthesia techniques approved by the institution’s IACUC.
2. Place anesthetized animal in restraint as indicated in the desired experimental design.
3. This step is optional, if not available proceed to step 5.6. Obtain a radiogram, using an onboard portal camera, of the mouse without collimation using an aluminum filter.
4. Obtain a second radiogram with collimation in place.
5. Overlay radiograms in ImageJ to demonstrate beam positioning.
6. Using the predetermined α/β value, determine the dose scheme that provides the most reasonable approach to answer a research question (i.e., if wanting to model the effects of a dose of 30Gy delivered in 10 fractions of 3 Gy, but only wish to give four fractions). Using equation 14, with an assumed α/β value of 10 (this value can be determined for individual cancer cell lines in protocol 3) and a BED similar to that of 30 Gy/10 F, treat with 24 Gy in 4 fractions of 6 Gy.
7. Treat animal for the prescribed time given for the desired dose.

6. Histological confirmation of dose deposition in vivo

1. Following protocol 5, collect tissue of interest within 1 hour of treatment^15,16. Following tissue harvest, proceed with preferred immunohistochemistry protocol. An example is given below.
2. Perfuse animal with ice cold 4% paraformaldehyde (PFA).
3. Post-fix in PFA at 4 °C.
4. Following fixation, fix tissue sequentially in 10%, 20%, and 30% sucrose for 24 hours each at room temperature.
5. Embed tissue in gelatin and sequentially fix in 4% PFA and again in 10-30% sucrose for 24 hours each at room temperature.
6. Trim block and place at -80 °C for 30 minutes.
7. Slice tissue into 20-30 µm sections.
8. Immunostain slides as free floating sections in a six well-plate^17,18.
   1. Wash three times and permeabilize for 30 minutes on a shaker with 1.83% lysine in 1% Triton, and 4% heat-inactivated goat serum.
   2. Incubate sections with anti-γH2AX antibody for 24 hours, followed by a 2 hour incubation with desired secondary antibody.
   3. Coverslip slides with glass coverslips using preferred mounting media.
9. Image on a fluorescent microscope.