Name: irradiator commissioning and dosimetry for assessment of lq &alpha; and &beta; parameters, radiation dosing schema, and in vivo dose deposition
Uploaded: 2021-03-11T13:30:00
Duration: 6 min 20 s
Description: Watch this Scientific Journal Video about Irradiator Commissioning and Dosimetry for Assessment of LQ ÃŽÂ± and ÃŽÂ² Parameters, Radiation Dosing Schema, and in vivo Dose Deposition at JoVE.com

The authors would like to thank the Microscope and Animal Models Imaging Facilites at WVU for the use of their equipment supported by grant number P20GM103434. Additionally, this work&#160;was supported by grant&#160;number P20GM121322 from the&#160;National Institue of General&#160;Medical Sciences, by National&#160;Cancer Institute grant number&#160;F99CA25376801,&#160;and the Mylan Chair Endowment Fund.

<ol>
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The authors have no disclosures to make.

The above protocol describes a user-friendly approach for radiation dosimetry, determination of &#945;/&#946; values in cancer cell lines, and a brief example of an approach for irradiation in a preclinical model of breast cancer brain metastasis. These methods can be used to study any model of cancer and are not just limited to brain metastasis of breast cancer. In this section we will discuss the relevant intricacies underlying preclinical radiotherapy experiments.
Dosimetry involves two parts: 1) calibrate the output with a farmer chamber, so that the dose rate of the x-ray unit is established, and 2) prepare a practical dosimetry measurement system using radiochromic film. With regards to output calibration, TG-61 provides a reproducible method in water. The protocol here uses Gammex RMI 457 solid water, as recommended by XStrahl, the manufacturer of the irradiator. Although relative dosimetry (profiles or depth dose curves normalized to maximum dose) analysis with solid water, agrees to better than 1% with that of water, there is a difference of about 3 to 4% in absolute dose due to a higher mass energy absorption coefficient for solid water compared to water. However, as all installations of the XStrahl system use the solid water protocol for output calibration, we did not correct for these differences. Knowing the output allows the calculation of the exposure time required to deliver a desired dose. Placing film in the same setup as the farmer chamber allows us to deliver known doses to the film. Scanning the film then provides optical densities. The dose to the film can then be graphed against the corresponding net optical density (difference in optical density after and before exposure). This produces a film calibration curve. When we change experimental setups, the dose rate in that situation could change, since dose rate depends on field size, depth and the material being irradiated. Exposing film with the experimental setup provides us with a net optical density, and using the film calibration curve, we can then determine the corresponding dose. Dividing this dose by the time the film was irradiated, we get the dose rate. This dose rate can then be used to calculate the exposure time to deliver a desired dose for the given experimental setup. The protocol described above handles several nuances associated with film dosimetry. For example, after exposure, the film requires approximately 24 hours for the chemical reactions in the film&#8217;s active layer to be virtually complete. Not waiting for this amount of time will lead to a lower optical density.
For any study to have reproducible dosimetry it is important to know and understand several of the key elements of a given irradiator. In particular, it is crucial to know and detail to other researchers the make and model of the irradiator used, the source type (x-ray, radioactive, etc.), energy, half-value layer, field size, source to surface and source to isocenter distances, size of material irradiated, attenuation before and backscatter after the irradiated material, experiment-specific dose rate, fractionation schema, exact dosimetry equipment utilized, and the dosimetry protocol used. All of these points of information are what cohesively describe the beam quality of a given irradiator prior to delivering a dose to any animal or cell19. Another pertinent point of information from this protocol and others is that the dose rate achieved in Protocol 1 is simply the output of the irradiator being used. For any given experiment it is important to define the dose rate for that particular setup (Protocol 4) by comparison with a generated radiochromic film calibration curve (Protocol 2).
In vitro experimentation provides important details about the radiobiologic behavior of cancer cell lines. In vitro clonogenic cell survival assays accurately estimate and quantify the inherent radio-sensitivity of a cell line20, aiding in the design of fractionation schedules in subsequent cellular or small animal experiments21. Specifically, these assays approximate values for the parameters &#945; and &#946; that are used in the linear-quadratic model to predict cell death in response to radiotherapy according to the equation:
<img alt="Equation 9" src="/files/ftp_upload/61692/61692equ09.jpg" /> (Equation 9)
where SF is the surviving fraction of clonogenically viable cells, D is radiation dose in Gy, and &#945; and &#946; are fitted parameters22. The ratio &#945;/&#946; provides an inherent measure of cellular radio-sensitivity, with higher values correlating with increased sensitivity of a cell line22. Because this functional relationship is non-linear with respect to dose, the biologic effects of a radiotherapy fractionation scheme are not only related to the total delivered dose but also the number and size of fractions23. The biologic effective dose (BED) is a measure of the true biological dose delivered to a tissue and permits direct comparison of different fractionations schemes24,25. The BED equation only requires an estimate of &#945;/&#946;, and is displayed below:
<img alt="Equation 14" src="/files/ftp_upload/61692/61692equ14.jpg" /> (Equation 14)
where n is the number of fractions of dose D. Clonogenic cell survival assays estimate &#945;/&#946; and facilitate the direct comparison of radiotherapy fractionation schemes via the BED equation. Incorrect conclusions may be drawn regarding a tissue or organ response to radiotherapy (or combinations of radiotherapy with other modalities) if the BED in the treatment groups is not equitable within or between experiments. For example, 2 fractions of 10 Gy compared with 4 fractions of 5 Gy do not yield the same BED, and thus these dosing schemes cannot be directly compared in terms of biologic response. The BED equation, while imperfect due to inherent limitations in the linear-quadratic model, reliably estimates equitable effects for a wide range of experimental treatment conditions24,25.
Clonogenic cell survival assays clearly play an important role in studying radiotherapy effects in cancer models, but in vitro experimentation offers a number of additional options to further explore mechanistic details of cancer cell radiobiology. Simple modifications of the clonogenic cell survival assay were used to determine the modes of action for some radio-sensitizing chemotherapies, such as paclitaxel or etoposide26,27. Further in vitro experimental options include immunocytochemistry studies to examine specific cellular repair pathways, such as &#947;-H2AX foci and/or 53BP1 staining for double-stranded DNA break repair28. These experiments may be of particular interest when comparing radiotherapy as a single modality with combination therapies, especially when probing mechanistic details for a given cell line. Other experimental options include cytokine measurements to examine the innate role of a cell&#8217;s inflammatory response to irradiation or analyses of the mode of cell death (i.e., apoptosis, necrosis, mitotic catastrophe, etc.) under different therapeutic conditions29,30,31. This type of experimentation can complement or replace animal experimentation and provide a more complete understanding of a cancer cell line&#8217;s radiobiology. Regardless of the choice of additional experiments to conduct, a standard clonogenic cell survival assay as described in protocol 3 is an important initial radiobiologic assessment of a cell line.
Clonogenic assays and radiation dosimetry provide the researcher with a means to precisely plan experiments to more directly resemble clinical scenarios. With the addition of preclinical cancer small rodent models, it is possible to study the response to radiation alone or in the context of a treatment plan in vivo. Prior to using animals, it is important to determine the relative dose output of the specific setup if it differs from the setup used for determination of dose output32,33. When it comes to determining a dose rate for field sizes of &#60;10 mm, use of an ionization chamber becomes less accurate due to alignment within a small field and partial volume averaging effects33. The use of radiochromic film to determine output in combination with in vivo immunohistochemical experiments has been used to determine output and dose deposition in the past16,34,35,36,37,38.

Cancers collectively represent the second-leading cause of death in the U.S. and in many countries around the globe1. Radiation therapy is a cornerstone of treatment for many tumor subtypes and is administered to about half of all cancer patients2,3. Patient outcomes for nearly all cancers have improved over time as equipment used to deliver radiation doses has steadily advanced and some effective multimodal therapy approaches were developed4,5,6, but recurrence and mortality rates for patients with certain types of tumors remain high7,8,9. Thus, radiotherapy for cancer continues to be an active area of basic and clinical research. Many pre-clinical radiotherapy studies employ the use of small-scale irradiators to deliver radiation doses to in vitro or animal models of cancers. With a multitude of potential experiments to conduct exploring mechanistic radiobiology details or novel treatments, common pitfalls may be encountered that lead to incorrect conclusions, poor reproducibility, and wasted resources. These pitfalls fall within three important areas: irradiator dosimetry, in vitro characterization of model cell lines, and in vivo irradiation dosing schedule and setup. Accurate and reproducible results from more advanced experiments are difficult to achieve without prior attention to these fundamental aspects of radiotherapy research.
The protocol detailed herein describes a generalized strategy for avoiding or mitigating these issues and draws upon several previously developed methodologies intended for independent use. These distinct methods have been merged so that a researcher interested in beginning or improving preclinical radiotherapy experiments could use this as a robust experimental layout. The suggested framework includes methodology for the commissioning of small-scale animal irradiators, for determining basic radiobiologic properties of model cancer cell lines, and for appropriately designing and administering a dosing and fractionation schedule for in vivo tumor models.

<table>
	<tbody>
		<tr>
			<td>Acetic acid, glacial</td>
			<td>Sigma-Aldrich</td>
			<td>A6283</td>
			<td>This or comparable glacial acetic acid products are acceptable.</td>
		</tr>
		<tr>
			<td>Crystal Violet</td>
			<td>Sigma-Aldrich</td>
			<td>C6158</td>
			<td>This or comparable crystal violet products are acceptable.</td>
		</tr>
		<tr>
			<td>Digital Baraometer</td>
			<td>Fisher Scientific</td>
			<td>14-650-118</td>
			<td>For pressure and temperature measurements.</td>
		</tr>
		<tr>
			<td>Electrometer</td>
			<td>Standard Imaging</td>
			<td>CDX 2000B</td>
			<td>Calibrated by an ADCL; Need correction factor, Pelec</td>
		</tr>
		<tr>
			<td>Film</td>
			<td>Gafchromic</td>
			<td>EBT3 Film</td>
			<td>Comes in sheets of 25; calibration films and experimental films must come from same set</td>
		</tr>
		<tr>
			<td>Ionization Chamber</td>
			<td>Farmer</td>
			<td>PTW TN30013</td>
			<td>Calibrated by an ADCL @ two calibration points</td>
		</tr>
		<tr>
			<td>Methanol</td>
			<td>Sigma-Aldrich</td>
			<td>34860</td>
			<td>This or comparable methanol products are acceptable.</td>
		</tr>
		<tr>
			<td>Photo Scanner</td>
			<td>Epson</td>
			<td>Perfection V700</td>
			<td>Equivalent scanners are V800, V10000, V11000, V12000</td>
		</tr>
		<tr>
			<td>XenX</td>
			<td>Xstrahl</td>
			<td>NA</td>
			<td>Irradiator used.</td>
		</tr>
	</tbody>
</table>

irradiator commissioning and dosimetry for assessment of lq &alpha; and &beta; parameters, radiation dosing schema, and in vivo dose deposition

Radiation dosimetry is critical in the accurate delivery and reproducibility of radiation schemes in preclinical models for high translational relevance. Prior to performing any in vitro or in vivo experiments, the specific dose output for the irradiator and individual experimental designs must be assessed. Using an ionization chamber, electrometer, and solid water setup, the dose output of wide fields at isocenter can be determined. Using a similar setup with radiochromic films in the place of the ionization chamber, dose rates for smaller fields at different depths can also be determined. In vitro clonogenic survival assays of cancer cells in response to radiation treatment are inexpensive experiments that provide a measure of inherent radio-sensitivity of cell lines by fitting these data with the traditional linear-quadratic model. Model parameters estimated from these assays, combined with the principles of biologic effective doses, allows one to develop varying fractionation schedules for radiation treatment that provide equivalent effective doses in tumor-bearing animal experiments. This is an important factor to consider and correct for in comparing in vivo radiation therapy schedules to eliminate potential confounding of results due to variance in the delivered effective doses. Taken together, this article provides a general method for dose output verification preclinical animal and cabinet irradiators, in vitro assessment of radio-sensitivity, and verification of radiation delivery in small living organisms.

Following protocol 1 will provide a dose rate in Gy/min, which is specific to the irradiator being used. However, regardless of the type of irradiator, with a known dose rate a calibration curve can be generated using protocol 2 yielding similar films and a similar calibration curve to that in Figure 2A-B. A successful assay from protocol 3 will yield distinct, well-demarcated colonies of cells that stain homogenously violet. The estimate of &#945;/&#946; can be compared to literature values or other treatment groups to interpret the radio-sensitivity of the given cell line. Utilizing the calibration curve developed following protocol 2 and displayed in Figure 2B, protocol 4 will yield two film samples resembling Figure 2A that can be used to estimate required experimental irradiation times. If an on-board portal imaging camera is available for the irradiator being used, radiograms of small animals can be obtained with and without collimation. Overlaying these images will demonstrate the exact positioning of the collimated radiation beam relative to the small animal being treated as depicted in Figure 4A. Successful dose-deposition in protocol 5 can be confirmed following protocol 6. One indication that radiation is being deposited in an in vivo or in vitro systems is through the detection of double stranded DNA breaks. Illustrated in Figure 4B, the same mouse treated solely through the right hemisphere in Figure 4A, demonstrates positive &#947;H2AX staining only in the treated hemisphere. In this figure, the nuclei are stained with DAPI to show two things; 1) the whole are of the brain which the anti &#947;H2AX antibody was applied to during histological analysis, and 2) the untreated hemisphere of the brain remains unstained.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/61692/61692fig01.jpg" /> 
Figure 1: Rough set up of ionization chamber and water phantom set up for determination of dose output.&#160;The pictogram illustrates a basic setup utilizing the various components required for dosimetry using an ionization chamber and solid water phantoms within the cabinet of the irradiator. <a href="https://www.jove.com/files/ftp_upload/61692/61692fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/61692/61692fig02.jpg" /> 
Figure 2: Generation of a calibration curve using radiochromic film.&#160;(A) Representative color change of radiochromic film with increasing dose. Top left (0 cGy); bottom right (2000 cGy). (B) Potential radiochromic film calibration curve comparing net optical density and dose. <a href="https://www.jove.com/files/ftp_upload/61692/61692fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/61692/61692fig03.jpg" /> 
Figure 3: Clonogenic Assay of Cancer Cells.&#160;Radiation treatment of cells can be done prior to plating in six well plates/petri dishes (A), or after (B). In panel (C), a representative image is displayed of a successful clonogenic assay with MDA-MB-231 breast cancer cells after following Protocol Section 3. <a href="https://www.jove.com/files/ftp_upload/61692/61692fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" class="xfigimg" src="/files/ftp_upload/61692/61692fig04.jpg" /> 
Figure 4: Use of dual overlayed radiograms for positioning (if available) and positive &#947;H2AX immunohistochemical staining for confirmation of dose deposition.&#160;(A) Representative overlayed radiograms depicting placement of radiation beam. (B) Representative results indicating dose deposition to the right hemisphere as demonstrated by increased &#947;H2AX intensity. <a href="https://www.jove.com/files/ftp_upload/61692/61692fig04large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Correction Factor</td>
			<td colspan="2">Explanation</td>
		</tr>
		<tr>
			<td>N&#954;</td>
			<td colspan="2">Air kerma calibration factor</td>
		</tr>
		<tr>
			<td>[(&#181;en/&#961;)Wair]water</td>
			<td colspan="2">Ration of mass energy absorption coefficients of water to air; approximately 1.05</td>
		</tr>
		<tr>
			<td>Pq,Cham</td>
			<td colspan="2">Correction accounting for chamber stem affecting photon fluence perterbation by chamber; approximately 1.022</td>
		</tr>
		<tr>
			<td>Psheath</td>
			<td colspan="2">Correction accounting for sheath protecting ionization chamber; value of 1, as chamber is waterproof</td>
		</tr>
		<tr>
			<td>Ppol</td>
			<td colspan="2">Correction factor accounting for polarity; determined in Protocol 1</td>
		</tr>
		<tr>
			<td>Pion</td>
			<td colspan="2">Correction factor accounting for ion recombination; determined in Protocol 1</td>
		</tr>
		<tr>
			<td>PTp</td>
			<td colspan="2">Correction factor acocunting for temerpature and pressure on day of experiment; determined in Protocol 1</td>
		</tr>
	</tbody>
</table>
Table 1: Correction factors needed for determination of dose rate in Protocol 1.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Dose</td>
			<td>N</td>
		</tr>
		<tr>
			<td>0.5</td>
			<td>3</td>
		</tr>
		<tr>
			<td>1</td>
			<td>3</td>
		</tr>
		<tr>
			<td>2</td>
			<td>3</td>
		</tr>
		<tr>
			<td>3</td>
			<td>3</td>
		</tr>
		<tr>
			<td>4</td>
			<td>3</td>
		</tr>
		<tr>
			<td>6</td>
			<td>3</td>
		</tr>
		<tr>
			<td>8</td>
			<td>3</td>
		</tr>
		<tr>
			<td>10</td>
			<td>3</td>
		</tr>
		<tr>
			<td>12*</td>
			<td>3</td>
		</tr>
		<tr>
			<td>15*</td>
			<td>3</td>
		</tr>
		<tr>
			<td>20*</td>
			<td>3</td>
		</tr>
		<tr>
			<td colspan="2">*Only necessary for doses exceeding 10 for individual experiments.</td>
		</tr>
	</tbody>
</table>
Table 2: Doses to be used in generation of radiochromic film calibration curve.

Watch this Scientific Journal Video about Irradiator Commissioning and Dosimetry for Assessment of LQ ÃŽÂ± and ÃŽÂ² Parameters, Radiation Dosing Schema, and in vivo Dose Deposition at JoVE.com

Irradiator Commissioning and Dosimetry for Assessment of LQ ÃƒÅ½Ã‚Â± and ÃƒÅ½Ã‚Â² Parameters, Radiation Dosing Schema, and in vivo Dose Deposition

Radiation dosimetry provides a technique for enhancing the accuracy of preclinical experiments and ensuring that the radiation doses delivered are closely related to clinical parameters. This protocol describes steps to be taken at each phase during preclinical radiation experiments to ensure proper experimental design.

Irradiator Commissioning and Dosimetry for Assessment of LQ &alpha; and &beta; Parameters, Radiation Dosing Schema, and in vivo Dose Deposition

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