This research was supported by the Summa Cancer Institute.

Ethical statement: Summa Health System institutional review board approval was obtained for this study.
1. Treatment Consultation
<ol>
	<li>Describe the new lung SBRT treatment to the patient. 
	NOTE: The new SBRT platform delivers coplanar and non-coplanar high radiation dose to cancer targets while lowering radiation dose to non-target organs.</li>
	<li>Discuss the treatment risks. 
	NOTE: SBRT may result in possible short-term skin hyperpigmentation or erythema, tiredness, infrequent cough, nausea, esophagitis, and rare visceral organ injury. Pneumonitis, or inflammation of the lung with low-grade fever and non-productive cough, may occur up to three months after treatment. Acute or late injuries to the heart, other muscles, peripheral nerves or spinal cord, and bone are uncommon. There is a very small hazard for radiation-induced malignancy.</li>
</ol>
2. Fiducial Marker Placement
<ol>
	<li>Perform percutaneous CT-guided or bronchoscopic placement of a single gold-coated marker inserted into the tumor target center-of-mass.
	<ol>
		<li>Ask a radiologist to perform a 3-5 mm thick contiguous axial tomographic imaging of the patient&#8217;s chest7.</li>
		<li>Determine a safe needle approach minimizing aerated lung tissue traversed (avoiding bullae and fissures)7.</li>
		<li>Inject local subcutaneous anesthesia (e.g., 1% lidocaine).</li>
		<li>Introduce 17 or 18 G coaxial needle to place a single short (0.75 x 10 mm) or single long (0.75 x 20 mm) marker10.</li>
	</ol>
	</li>
	<li>Do an electromagnetic navigation bronchoscopy-guided fiducial marker placement by asking a pulmonologist to acquire tomographic imaging of the chest for endobronchial mapping21.
	<ol>
		<li>Wedge the bronchoscope in the suspected bronchial segment.</li>
		<li>Steer the bronchoscope sensor probe to the target lesion.</li>
		<li>Deploy a fiducial marker by transbronchial needle. 
		NOTE: Techniques for fiducial-free radiosurgery in the lung are considered investigational and subject to active research22,23.</li>
	</ol>
	</li>
	<li>As an alternative, order at least three short (1.6 x 3 mm) gold-coated markers to be positioned within a 6 cm &#8216;box&#8217; around the target. If more than one fiducial marker is placed, a physical separation of 2 cm between markers is recommended.</li>
</ol>
3. Treatment Planning
<ol>
	<li>Perform CT-guided simulation (described in steps 3.2 and 3.3) 4-7 days after fiducial marker placement.</li>
	<li>Ask the patient to lie in a supine-head-first on the treatment machine flat tabletop.
	<ol>
		<li>Position the patient&#8217;s arms over their head, supported by upper arm and wrist holders or a vacuum-bag immobilizer. Ensure that the thorax and abdomen are not immobilized.</li>
		<li>Optionally, use a two-pin localized knee sponge for indexing.</li>
		<li>Place at least 4 infrared-tracked body markers on the chest for localization. Infrared-tracked breathing markers overly body regions demonstrating consistent vertical respiratory motion (3 mm or more peak-to-peak motion is recommended).</li>
	</ol>
	</li>
	<li>Conduct a non-contrasted contiguous helical axial CT scan (1 mm slice thickness, voltage 120 kVp, 350 mAs). 
	NOTE: Treating physicians may order a 4D CT thorax scan12 or a 3D CT thorax scan that includes free-breathing, end-inspiration, and end-expiration breath-hold image sets24.</li>
	<li>Obtain 18F-FDG PET/CT scans for enhanced capture of lung tumor motion.
	<ol>
		<li>Ask the patients to lie in a head-first scanning position for contiguous helical CT scan (e.g., voltage 120 kVp, 450 mAs) from the orbitomeatal line to upper thighs during quiet breathing.</li>
		<li>Acquire 18F-FDG PET after intravenous administration of 11 mCi of 18F-FDG on average in the same scanning position from the orbitomeatal line to upper thighs during quiet breathing. If this technique is used, 18F-FDG PET/CT scans are auto-contoured by software set at a 40% SUVmax threshold, and then, co-registered with CT simulation images as described1.</li>
	</ol>
	</li>
	<li>Contour the primary lung gross target volume or volumes (GTVp) by hand drawing on 4D CT datasets, preferably the exhale phase. Expanding the GTVp with a 5 mm margin creates the planning tumor volume (PTV). Radiation dose planning occurs on the end-expiration phase scan for dynamic tracking. 
	NOTE: As an alternative and when using 3D CT datasets, the free-breathing CT simulation scan is the reference scan. Using this technique, the treating radiation oncologist contours the GTV on the free-breathing (GTVfb), inspiration (GTVi), and expiration (GTVe) CT simulation scans. A thresholded 40 percent maximum standard uptake value contour on the 18F-FDG PET images creates an 18F-FDG PET clinical target volume (CTVpet)1. A composite ITV represents the added sum of the GTVfb, GTVi, GTVe, and CTVpet volumes. A 5 mm margin expansion of the composite ITV creates a PTV. Here, radiation dose planning occurs on the free-breathing scan for dynamic tracking.</li>
	<li>Contour nearby normal tissue structures by hand drawing on 4D CT datasets, preferably the exhale phase. This may include normal lung, heart plus pericardium, esophagus, liver, bilateral kidneys, brachial plexus, and spinal cord. A contour of the trachea, right mainstem bronchus, and left mainstem bronchus may be generated, expanded 3 mm, and used as a high-priority planning constraint to avoid late toxicity airway fibrosis.</li>
	<li>Click on the dynamic tracking button in the planning software. This action engages the gimbaled pan-and-tilt tracking on the new SBRT platform.</li>
	<li>Prescribe a radiation dose to the PTV. Consider one of three radiation Monte Carlo dose prescriptions: 3 x 17 Gy = 51 Gy daily for peripheral lung lesions; 4 x 12 Gy = 48 Gy for central lung lesions and peripheral chest wall lesions; or every other day 5 x 10 Gy = 50 Gy. Uncommonly when PTV volume constraints (i.e., 95% coverage) or organ-at-risk constraints are not met, a prescription of 8 x 7.5 Gy = 60 Gy may be used.</li>
</ol>
4. Treatment Delivery and Workflow
<ol>
	<li>Build a quiet breathing correlation model after supine-head-first alignment.
	<ol>
		<li>Place 4 (or up to 6) infrared body markers on the body in the same marked locations identified at CT simulation.</li>
		<li>Verify positional accuracy of the body markers and patient alignment by infrared camera and screens at the treatment console. 
		NOTE: Body marker position serves as a beam-on check for irregular movement, such a cough.</li>
		<li>Acquire cross-plane dual-diagnostic kV x-rays or cone-beam CT images at the treatment console to detect implanted markers for internal positional accuracy.</li>
		<li>Associate and correlate body marker motion (as a surrogate for respiration) and internal implanted marker motion with computer software linked to the new SBRT platform workflow. 
		NOTE: An alternative localization method involves orthogonal alignment of the patient according to anterior and lateral tattooed CT simulation laser triangulation marks and use of a standardized reference array (star) with six impregnated infrared body markers.</li>
	</ol>
	</li>
	<li>Generate a lung tumor motion correlation model.
	<ol>
		<li>Derive a gimbal pan-and-tilt path for the accelerator to track tumor motion using computer software linked to the new SBRT platform workflow.</li>
		<li>Visually assess the lung tumor motion correlation model prior to radiation delivery.</li>
	</ol>
	</li>
	<li>Observe for fiducial marker drift during radiation delivery.</li>
	<li>Evaluate machine-patient collisions due to gantry rotation, O-ring pivot, and gimbal pan-and-tilt actions prior to plan delivery. 
	NOTE: The radiation staff will perform this step. Treatment may entail five to nine static coplanar and noncoplanar treatment beams, manually and visually checked by radiation delivery staff. Treatments may last 15-30 min, with lung tumor motion correlation model verifications done approximately every 7 min.</li>
</ol>

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The authors have nothing to disclose.

Promising early stereotactic radiosurgery clinical experiences have driven clinical trial investigation of ablative radiation for treatment of lung cancers25,26. Experience has led investigators to use ablative radiation against a variety of tumor types metastasizing to the lung27,28. The new SBRT platform introduces a radiation delivery system particularly attuned to the treatment of moving tumors.
The new SBRT platform delivers an unseen x-ray treatment that is generated by a linear accelerator mounted within a pivoting O-ring gantry. A gimbal mechanism enables pan-and-tilt motion of the linear accelerator, providing in-time dynamic tumor motion tracking. Dual cross-plane kV x-rays are obtained before and during treatment to verify 6 degree-of-freedom patient positioning. Coplanar and non-coplanar unique degrees of freedom enhance delivery of high radiation dose to cancer targets while simultaneously minimizing radiation dose to critical visceral organs. It is anticipated that treatment sterilizes cancer cell targets without irreparable damage to normal cells&#8212;lowering radiation-related toxicity. Future study of the new SBRT platform will document any gains in target control and any reduction in side effects.
Initial experience with the new SBRT platform shows promise10. Nuances of dynamic tracking of lung tumors continue to be explored; however, some generalizations are apparent. Lung tumors demonstrating motion less than seven millimeters may be best treated by a composite ITV plus 5 mm expansion approach. For lung tumors showing 7 mm or more vertical translation, a dynamic tracking approach using a GTVp plus 5 mm expansion may be best for treatment. Further research defining these limits is needed. Also, 18F-FDG PET images superimposed upon 3D CT image datasets usually increase composite ITV volumes. This approach assumes volume expansion due to 18F-FDG signal smear occurring during the PET scanner&#8217;s 3-5 min bin time. A 40% thresholded 18F-FDG clinical target volume has been studied and has been used in one of our programs1. Further research characterizing whether 18F-FDG PET images adequately replicates tumor hysteresis is needed. Lastly, up to 3 lesions in a single lung may be considered for treatment at one time. Otherwise, a sequential approach is done.
Dynamic tracking on the new SBRT platform uses a lung tumor motion correlation model to predict lung tumor motion up to 40 msec into the future. Position and velocity of the infrared body and respiratory markers are included in the model. A marker detection rate of 70% in acquired kV x-rays is a requisite for dynamic tracking. Fiducials are tracked in three dimensions (i.e., x, y, z). Images generated by the kV x-ray units are automatically registered and compared real-time. Observed latency in dynamic tracking is due to limitations in pan-and-tilt gimbal hardware, software processing, and positional control performance of the kV x-ray units. Research investigators are engaged in improving tracking latency.
During radiation delivery using dynamic tracking on the new SBRT platform, it is critical to watch for fiducial marker drift. Trends in fiducial marker drift beyond predefined 3 mm tolerances in any direction results in operator-initiated treatment pause or in automatic beam hold. If a treatment pause occurs, it is recommended that operators allow for resumption of quiet breathing motion over the next several patient breaths and then treatment resumption prior to correlation model rebuild. If pauses are unsuccessful, patient repositioning, infrared breathing marker motion detection, kV marker detection, correlation modeling rebuild are done to resume treatment. In our experience, breathing correlation models are accurate for up to 7 min, often limited by patient tension or relaxation while resting on the treatment tabletop.
Unanswered questions remain. What are the radiobiological consequences and mode of cell death in normal cells and cancer cells occurring after ablative radiation dose? Why has it been so difficult to merge high-precision ablative radiation with radiosensitizing chemotherapies? While it is essential to investigate other modalities of delivering ablative radiation in the chest, it remains unclear as to whether ablative radiation can provide equivalent therapeutic effectiveness as thoracic surgery. Indeed, thoracic surgery is the more commonly used and validated technique to achieve tumor eradication in the lung when conventional therapies have already been applied. Here, the new SBRT platform provides an innovative non-invasive means of therapy for women and men with lung tumors showing motion.

Lung cancers account for the greatest number of cancer-related deaths in women and men worldwide2. Up to 63% of persistent or recurrent lung cancers involve lung tissue that is already taxed by chemotherapy or previously irradiated.3,4. Further irradiation at sites of persistent or recurrent lung tumors may lead to intolerable lung morbidity5,6, especially when conventional surgery, chemotherapy, and radiation therapy already have been tried. Thus, women and men in such clinical circumstances have need of new cancer therapy strategies similar to treatments presented before in this journal7. Stereotactic ablative body radiation therapy (SBRT) may satisfy this therapeutic need by sterilizing lung tumors through precisely targeted, high-dose radiation8,9.
There is a novel SBRT platform capable of this therapeutic task10-12. It separates itself from other SBRT platforms by integrating dual-diagnostic Exactrac kV x-ray units (capable of cone-beam computed tomography target localization) and an infrared camera unit (capable of body surface marker tracking as a surrogate for internal motion) that both allow stereoscopic open-loop feedback of cancer target intrafraction motion. It also has a unique &#177;4 cm gimbaled pan-and-tilt radiation accelerator that has its radiation beam shaped by 60 tungsten alloy leaves (0.25 cm physical width, 11 cm physical height). It uses a full over-center-travel multi-leaf collimator for a maximum 15 x 15 cm field size. It incorporates a robot-driven &#177;60&#176; pivoting O-ring and &#177;185&#176; rotational gantry allowing for coplanar and non-coplanar accelerator beam set-up angles and unique treatment degrees of freedom. Lastly, it has submillimeter accuracy (0.4 mm at isocenter)13. In contrast, other SBRT radiotherapy platforms mount a clinical radiation accelerator either to an industrial robotic arm14, or to a helical slice-by-slice gantry15, or within a conventional machine linked to image-guided intensity modulated radiation therapy or dynamic arc delivery software16. Each platform engages a variety of machine components to track motion resulting from respiration motion, heartbeat, or digestion. Lung radiosurgery has had clinical success17,18, rendering the modality a novel treatment option in radiation oncology19,20. This how-to article provides one new radiation therapy protocol that describes dynamic lung tumor tracking for therapeutic treatment intent.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Vero SBRT Linac System 1.0</td>
			<td>Brainlab, Inc. (Munich, Germany)</td>
			<td>46300</td>
			<td>High accuracy first-of-its-kind gimbaled irradiation head with tilt function and gantry rotation</td>
		</tr>
		<tr>
			<td></td>
			<td>Mitsubishi Heavy Industries, Ltd. (Tokyo, Japan)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Visicoil fiducial marker</td>
			<td>IBA Dosimetry America (Bartlett, TN, USA)</td>
			<td>67245</td>
			<td>0.75 mm x 10 mm marker -or- 0.75 mm x 20 mm marker</td>
		</tr>
		<tr>
			<td>Gold fiducial marker</td>
			<td>Civco Medical Solutions (Orange City, IA, USA)</td>
			<td>MTNW887860</td>
			<td>Sterile placement needle (14GA ETW x 20cm) with one 1.6mm &#215; 3mm marker</td>
		</tr>
	</tbody>
</table>

dynamic lung tumor tracking for stereotactic ablative body radiation therapy

Physicians considering stereotactic ablative body radiation therapy (SBRT) for the treatment of extracranial cancer targets must be aware of the sizeable risks for normal tissue injury and the hazards of physical tumor miss. A first-of-its-kind SBRT platform achieves high-precision ablative radiation treatment through a combination of versatile real-time imaging solutions and sophisticated tumor tracking capabilities. It uses dual-diagnostic kV x-ray units for stereoscopic open-loop feedback of cancer target intrafraction movement occurring as a consequence of respiratory motions and heartbeat. Image-guided feedback drives a gimbaled radiation accelerator (maximum 15 x 15 cm field size) capable of real-time &#177;4 cm pan-and-tilt action. Robot-driven &#177;60&#176; pivots of an integrated &#177;185&#176; rotational gantry allow for coplanar and non-coplanar accelerator beam set-up angles, ultimately permitting unique treatment degrees of freedom. State-of-the-art software aids real-time six dimensional positioning, ensuring irradiation of cancer targets with sub-millimeter accuracy (0.4 mm at isocenter). Use of these features enables treating physicians to steer radiation dose to cancer tumor targets while simultaneously reducing radiation dose to normal tissues. By adding respiration correlated computed tomography (CT) and 2-[18F] fluoro-2-deoxy-&#7429;-glucose (18F-FDG) positron emission tomography (PET) images into the planning system for enhanced tumor target contouring, the likelihood of physical tumor miss becomes substantially less1. In this article, we describe new radiation plans for the treatment of moving lung tumors.

SBRT on the new platform currently involves multiple static radiation beams converging on single or multiple closely-associated clinical radiation targets, as depicted for example in Figure 1. A representative good planning outcome delivers ablative radiation with 95% coverage of cancer target volume and cancer target dose conformity. Figure 1 shows 5 coplanar and 4 non-coplanar beams (i.e., ring rotation +20&#176; for beams 2, 4, 6, and 8) used to treat a single PTV representing squamous cell carcinoma in the right lung. Beam margins for the PTV were one-millimeter. Radiation dose, prescribed to the 95% isodose line, rendered 95% PTV coverage with a conformity index of 1.48. The prescription was 50 Gy in five every other day 10 Gy fractions. Structures depicted here include the planning target volume (red), internal target volume (white), spinal cord (green), and esophagus (light blue). Isodose lines are as indicated.
<img alt="Figure 1" src="/files/ftp_upload/52875/52875fig1highres.jpg" width="700" /> 
Figure 1: Dynamic tumor tracking of a right-sided lung tumor. Pictured is an example of ablative radiation dose (50 Gy in five 10 Gy every other day fractions) delivered to a single right-sided lung tumor using nine static beams (blue/green, 34&#176; apart). The 4 planning software windows depict: (A) a beam and critical structure collision map, (B) beam&#8217;s-eye-view (here, beam 1), (C) three-dimensional CT and beam reconstruction map, and (D) axial CT with dose distribution.
<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>Structure</td>
			<td>Metric</td>
			<td>Volume&#160;</td>
			<td>Acceptable variation</td>
		</tr>
		<tr>
			<td>PTV</td>
			<td>V50Gy</td>
			<td>&#8805;95%</td>
			<td>&#8805;90%</td>
		</tr>
		<tr>
			<td>Minimum dose</td>
			<td>0.03 cm3</td>
			<td>&#8805;46 Gy (92%)</td>
			<td>&#8805;45 Gy (90%)</td>
		</tr>
		<tr>
			<td>Maximum dose</td>
			<td>0.03 cm3</td>
			<td>&#8804;60 Gy (120%)</td>
			<td>&#8804;62.5 Gy (125%)</td>
		</tr>
		<tr>
			<td>Spinal cord</td>
			<td>0.03 cm3</td>
			<td>&#8804;15 Gy</td>
			<td>&#8804;22 Gy</td>
		</tr>
		<tr>
			<td>Lung (minus GTV)</td>
			<td>V20Gy</td>
			<td>&#8804;10%</td>
			<td>&#8804;15%</td>
		</tr>
		<tr>
			<td></td>
			<td>Mean dose</td>
			<td>&#8804;8 Gy</td>
			<td>&#8804;10 Gy</td>
		</tr>
		<tr>
			<td>Heart / Pericardium</td>
			<td>15 cm3</td>
			<td>&#8804;32 Gy</td>
			<td>&#8804;36 Gy</td>
		</tr>
		<tr>
			<td>Esophagus</td>
			<td>Mean dose</td>
			<td>&#8804;18 Gy</td>
			<td>&#8804;20 Gy</td>
		</tr>
		<tr>
			<td></td>
			<td>0.03 cm3</td>
			<td>&#8804;27 Gy</td>
			<td>&#8804;30 Gy</td>
		</tr>
		<tr>
			<td>Brachial plexus</td>
			<td>0.03 cm3</td>
			<td>&#8804;24 Gy</td>
			<td>&#8804;30 Gy</td>
		</tr>
	</tbody>
</table>
Table 1: Structure treatment planning constraints.

Watch this Scientific Journal Video about Dynamic Lung Tumor Tracking for Stereotactic Ablative Body Radiation Therapy at JoVE.com

Dynamic Lung Tumor Tracking for Stereotactic Ablative Body Radiation Therapy

Stereotactic ablative body radiation therapy (SBRT) involves the precise delivery of high-dose radiation to cancer tumor targets. A novel SBRT platform offers a first-of-its-kind gimbaled radiation accelerator mounted within a pivoting O-ring gantry capable of dynamic image-guided tumor tracking. Here, we describe dynamic tumor tracking for lung targets.

Physicians considering stereotactic ablative body radiation therapy (SBRT) for the treatment of extracranial cancer targets must be aware of the sizeable ...

dynamic-lung-tumor-tracking-for-stereotactic-ablative-body-radiation

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JoVE Journal

Medicine

15.2K ビュー.  Summa Cancer Institute. Stereotactic ablative body radiation therapy (SBRT) involves the precise delivery of high-dose radiation to cancer tumor targets. A novel SBRT platform offers a first-of-its-kind gimbaled radiation accelerator mounted within a pivoting O-ring gantry capable of dynamic image-guided tumor tracking. Here, we describe dynamic tumor tracking for lung targets.

ビデオ: Dynamic Lung Tumor Tracking for Stereotactic Ablative Body Radiation Therapy

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Combination Radiotherapy in an Orthotopic Mouse Brain Tumor Model

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the understanding of the mechanics of tumor invasion, and direct intracerebral inoculation of tumor provides the opportunity of observing the invasive process in a physiologically appropriate environment2. As far as human brain tumors are concerned, the orthotopic models currently available are established either by stereotaxic injection of cell suspensions or implantation of a solid piece of tumor through a complicated craniotomy procedure3. In our technique we harvest cells from tissue culture to create a cell suspension used to implant directly into the brain. The duration of the surgery is approximately 30 minutes, and as the mouse needs to be in a constant surgical plane, an injectable anesthetic is used. The mouse is placed in a stereotaxic jig made by Stoetling (figure 1). After the surgical area is cleaned and prepared, an incision is made; and the bregma is located to determine the location of the craniotomy. The location of the craniotomy is 2 mm to the right and 1 mm rostral to the bregma. The depth is 3 mm from the surface of the skull, and cells are injected at a rate of 2 &mu;l every 2 minutes. The skin is sutured with 5-0 PDS, and the mouse is allowed to wake up on a heating pad. From our experience, depending on the cell line, treatment can take place from 7-10 days after surgery. Drug delivery is dependent on the drug composition. For radiation treatment the mice are anesthetized, and put into a custom made jig. Lead covers the mouse's body and exposes only the brain of the mouse. The study of tumorigenesis and the evaluation of new therapies for GBM require accurate and reproducible brain tumor animal models. Thus we use this orthotopic brain model to study the interaction of the microenvironment of the brain and the tumor, to test the effectiveness of different therapeutic agents with and without radiation.

The purpose of this article is to describe the use of an orthotopic glioblastoma model for chemoradiation studies. This article will go though cell processing, implanting, and radiotherapy of the mouse using an intracranial model.

Glioblastoma multiforme (GBM) are the most common and aggressive adult primary brain tumors1. In recent years there has been substantial progress in the ...

Stereotactic Radiosurgery for Gynecologic Cancer

Stereotactic body radiotherapy (SBRT) distinguishes itself by necessitating more rigid patient immobilization, accounting for respiratory motion, intricate treatment planning, on-board imaging, and reduced number of ablative radiation doses to cancer targets usually refractory to chemotherapy and conventional radiation. Steep SBRT radiation dose drop-off permits narrow 'pencil beam' treatment fields to be used for ablative radiation treatment condensed into 1 to 3 treatments.
Treating physicians must appreciate that SBRT comes at a bigger danger of normal tissue injury and chance of geographic tumor miss. Both must be tackled by immobilization of cancer targets and by high-precision treatment delivery. Cancer target immobilization has been achieved through use of indexed customized Styrofoam casts, evacuated bean bags, or body-fix molds with patient-independent abdominal compression.1-3 Intrafraction motion of cancer targets due to breathing now can be reduced by patient-responsive breath hold techniques,4 patient mouthpiece active breathing coordination,5 respiration-correlated computed tomography,6 or image-guided tracking of fiducials implanted within and around a moving tumor.7-9 The Cyberknife system (Accuray [Sunnyvale, CA]) utilizes a radiation linear accelerator mounted on a industrial robotic arm that accurately follows patient respiratory motion by a camera-tracked set of light-emitting diodes (LED) impregnated on a vest fitted to a patient.10 Substantial reductions in radiation therapy margins can be achieved by motion tracking, ultimately rendering a smaller planning target volumes that are irradiated with submillimeter accuracy.11-13
Cancer targets treated by SBRT are irradiated by converging, tightly collimated beams. Resultant radiation dose to cancer target volume histograms have a more pronounced radiation "shoulder" indicating high percentage target coverage and a small high-dose radiation "tail." Thus, increased target conformality comes at the expense of decreased dose uniformity in the SBRT cancer target. This may have implications for both subsequent tumor control in the SBRT target and normal tissue tolerance of organs at-risk. Due to the sharp dose falloff in SBRT, the possibility of occult disease escaping ablative radiation dose occurs when cancer targets are not fully recognized and inadequate SBRT dose margins are applied. Clinical target volume (CTV) expansion by 0.5 cm, resulting in a larger planning target volume (PTV), is associated with increased target control without undue normal tissue injury.7,8 Further reduction in the probability of geographic miss may be achieved by incorporation of 2-[18F]fluoro-2-deoxy-D-glucose (18F-FDG) positron emission tomography (PET).8 Use of 18F-FDG PET/CT in SBRT treatment planning is only the beginning of attempts to discover new imaging target molecular signatures for gynecologic cancers.

Stereotactic body radiotherapy (SBRT) involves image-guided, ablative radiation delivered to cancer targets refractory to chemotherapy or to conventional radiation treatment. The robotic-armed Cyberknife SBRT system, using sophisticated target localization, delivers hypofractionated radiation doses capable of sterilizing cancer targets. This article will consider new therapeutic roles of SBRT for gynecological cancers.

Stereotactic body radiotherapy (SBRT) distinguishes itself by necessitating more rigid patient immobilization, accounting for respiratory motion, ...

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality treatment consisting of surgical resection, post-operative radiation therapy and temozolomide chemotherapy 1. New therapeutic approaches are clearly needed to improve patient survival and quality of life. The development of more effective treatment strategies would be aided by animal models of GBM that recapitulate human disease yet allow serial imaging to monitor tumor growth and treatment response. In this paper, we describe our technique for the precise stereotactic implantation of bio-imageable GBM cancer cells into the brains of nude mice resulting in tumor xenografts that recapitulate key clinical features of GBM 2. This method yields tumors that are reproducible and are located in precise anatomic locations while allowing in vivo bioluminescent imaging to serially monitor intracranial xenograft growth and response to treatments 3-5. This method is also well-tolerated by the animals with low perioperative morbidity and mortality.

Stereotactic Intracranial Implantation and In vivo Bioluminescent Imaging of Tumor Xenografts in a Mouse Model System of Glioblastoma Multiforme

We describe an integrated method for the precise, stereotactic implantation of human glioblastoma multiforme cells into the brains of nude mice and subsequent serial in vivo imaging to monitor growth and response to treatment of the resultant xenografts.

Glioblastoma multiforme (GBM) is a high-grade primary brain cancer with a median survival of only 14.6 months in humans despite standard tri-modality ...

Human Neuroendocrine Tumor Cell Lines as a Three-Dimensional Model for the Study of Human Neuroendocrine Tumor Therapy

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human malignancies.1 Other than surgery for the minority of patients who present with localized disease, there is little or no survival benefit of systemic therapy. Therefore, there is a great need to better understand the biology of NETs, and in particular define new therapeutic targets for patients with nonresectable or metastatic neuroendocrine tumors. 3D cell culture is becoming a popular method for drug screening due to its relevance in modeling the in vivo tumor tissue organization and microenvironment.2,3 The 3D multicellular spheroids could provide valuable information in a more timely and less expensive manner than directly proceeding from 2D cell culture experiments to animal (murine) models.
To facilitate the discovery of new therapeutics for NET patients, we have developed an in vitro 3D multicellular spheroids model using the human NET cell lines. The NET cells are plated in a non-adhesive agarose-coated 24-well plate and incubated under physiological conditions (5% CO2, 37 &deg;C) with a very slow agitation for 16-24 hr after plating. The cells form multicellular spheroids starting on the 3rd or 4th day. The spheroids become more spherical by the 6th day, at which point the drug treatments are initiated. The efficacy of the drug treatments on the NET spheroids is monitored based on the morphology, shape and size of the spheroids with a phase-contrast light microscope. The size of the spheroids is estimated automatically using a custom-developed MATLAB program based on an active contour algorithm. Further, we demonstrate a simple method to process the HistoGel embedding on these 3D spheroids, allowing the use of standard histological and immunohistochemical techniques. 
This is the first report on generating 3D spheroids using NET cell lines to examine the effect of therapeutic drugs. We have also performed histology on these 3D spheroids, and displayed an example of a single drug's effect on growth and proliferation of the NET spheroids. Our results support that the NET spheroids are valuable for further studies of NET biology and drug development.

We present a simple agarose overlay platform to grow 3D multicellular spheroids using neuroendocrine cancer cell lines. This method provides a very convenient way to examine the effect of therapeutic drugs on the neuroendocrine tumor cells. It could also help us establish human neuroendocrine tumor spheroids for cancer therapy.

Neuroendocrine tumors (NETs) are rare tumors, with an incidence of two per 100,&#x200A;000 individuals per year, and they account for 0.5% of all human ...

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a novel platform that allows for direct long-term visualization of tissue structure changes intracranially. Imaging at a single cell resolution in a real-time fashion provides supplementary dynamic information beyond that provided by standard end-point histological analysis, which looks solely at 'snap-shot' cross sections of tissue.
Establishing this intravital imaging technique in fluorescent chimeric mice, we are able to image four fluorescent channels simultaneously. By incorporating fluorescently labeled cells, such as GFP+ bone marrow, it is possible to track the fate of these cells studying their long-term migration, integration and differentiation within tissue. Further integration of a secondary reporter cell, such as an mCherry glioma tumor line, allows for characterization of cell:cell interactions. Structural changes in the tissue microenvironment can be highlighted through the addition of intra-vital dyes and antibodies, for example CD31 tagged antibodies and Dextran molecules.
Moreover, we describe the combination of our ICW imaging model with a small animal micro-irradiator that provides stereotactic irradiation, creating a platform through which the dynamic tissue changes that occur following the administration of ionizing irradiation can be assessed.
Current limitations of our model include penetrance of the microscope, which is limited to a depth of up to 900 &mu;m from the sub cortical surface, limiting imaging to the dorsal axis of the brain. The presence of the skull bone makes the ICW a more challenging technical procedure, compared to the more established and utilized chamber models currently used to study mammary tissue and fat pads 5-7. In addition, the ICW provides many challenges when optimizing the imaging.

A Novel High-resolution In vivo Imaging Technique to Study the Dynamic Response of Intracranial Structures to Tumor Growth and Therapeutics

We describe a novel in vivo imaging technique that couples fluorescent chimeric mice with intracranial windows and high-resolution 2-photon microscopy. This imaging platform aids studies of dynamic changes in brain tissue and microvasculature, at a single-cell level, following pathological insults and is adaptable to assess intracranial drug delivery and distribution.

We have successfully integrated previously established Intracranial window (ICW) technology 1-4 with intravital 2-photon confocal microscopy to develop a ...

In vivo 19F MRI for Cell Tracking

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. Here, we describe a general protocol for cell labeling, imaging, and image processing. The technique is applicable to various cell types and animal models, although here we focus on a typical mouse model for tracking murine immune cells. The most important issues for cell labeling are described, as these are relevant to all models. Similarly, key imaging parameters are listed, although the details will vary depending on the MRI system and the individual setup. Finally, we include an image processing protocol for quantification. Variations for this, and other parts of the protocol, are assessed in the Discussion section. Based on the detailed procedure described here, the user will need to adapt the protocol for each specific cell type, cell label, animal model, and imaging setup. Note that the protocol can also be adapted for human use, as long as clinical restrictions are met.

In vivo 19F MRI for Cell Tracking

We describe a general protocol for in vivo cell tracking using MRI in a mouse model with ex vivo labeled cells. A typical protocol for cell labeling, image acquisition processing and quantification is included.

In vivo 19F MRI allows quantitative cell tracking without the use of ionizing radiation. It is a noninvasive technique that can be applied to humans. ...

Tumor Treating Field Therapy in Combination with Bevacizumab for the Treatment of Recurrent Glioblastoma

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA approved in April 2011 for the treatment of patients 22 years or older with rGBM. The device delivers alternating electric fields and is programmed to ensure maximal tumor cell kill1.
Glioblastoma is the most common type of glioma and has an estimated incidence of approximately 10,000 new cases per year in the United States alone2. This tumor is particularly resistant to treatment and is uniformly fatal especially in the recurrent setting3-5. Prior to the approval of the TTF System, the only FDA approved treatment for rGBM was bevacizumab6. Bevacizumab is a humanized monoclonal antibody targeted against the vascular endothelial growth factor (VEGF) protein that drives tumor angiogenesis7. By blocking the VEGF pathway, bevacizumab can result in a significant radiographic response (pseudoresponse), improve progression free survival and reduce corticosteroid requirements in rGBM patients8,9. Bevacizumab however failed to prolong overall survival in a recent phase III trial26. A pivotal phase III trial (EF-11) demonstrated comparable overall survival between physicians&#8217; choice chemotherapy and TTF Therapy but better quality of life were observed in the TTF arm10.
There is currently an unmet need to develop novel approaches designed to prolong overall survival and/or improve quality of life in this unfortunate patient population. One appealing approach would be to combine the two currently approved treatment modalities namely bevacizumab and TTF Therapy. These two treatments are currently approved as monotherapy11,12, but their combination has never been evaluated in a clinical trial. We have developed an approach for combining those two treatment modalities and treated 2 rGBM patients. Here we describe a detailed methodology outlining this novel treatment protocol and present representative data from one of the treated patients.

A novel methodology that is employed for the treatment of recurrent glioblastomas is described. This treatment approach employs the application of alternating electric tumor treating fields (TTFields), known as TTF Therapy in combination with bevacizumab, a targeted agent that is currently FDA approved as monotherapy.

A novel device that employs TTF therapy has recently been developed and is currently in use for the treatment of recurrent glioblastoma (rGBM). It was FDA ...

Primary Tumor and MEF Cell Isolation to Study Lung Metastasis

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered genotypes now possess the ability to invade and survive in other tissues. In this protocol, mouse mammary tumors are removed and primary cells are prepared from tumors. The cells isolated from this procedure are then available for gene profiling experiments. For successful metastasis, these cells must be able to intravasate, survive in circulation, extravasate to distant organs, and survive in that new organ system. The lungs are the typical target of breast cancer metastasis. A set of genes have been discovered that mediates the selectivity of metastasis to the lung. Here we describe a method of studying lung metastasis from a genetically engineered mouse model.. Furthermore, another protocol for analyzing mouse embryonic fibroblasts (MEFs) from the mouse embryo is included. MEF cells from the same animal type provide a clue of non-cancer cell gene expression. Together, these techniques are useful in studying mouse mammary tumorigenesis, its associated signaling mechanisms and pathways of the abnormalities in embryos.

The goal of this protocol is to study breast tumorigenesis. With this technique, mouse mammary tumors are removed and primary cells are prepared from tumors. A lung extraction protocol is included for studying lung metastasis. Furthermore, another protocol for analyzing mouse embryonic fibroblasts from the mouse embryo is included.

In breast tumorigenesis, the metastatic stage of the disease poses the greatest threat to the affected individual. Normal breast cells with altered ...

Dynamic Contrast Enhanced Magnetic Resonance Imaging of an Orthotopic Pancreatic Cancer Mouse Model

Dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) has been limitedly used for orthotopic pancreatic tumor xenografts due to severe respiratory motion artifact in the abdominal area. Orthotopic tumor models offer advantages over subcutaneous ones, because those can reflect the primary tumor microenvironment affecting blood supply, neovascularization, and tumor cell invasion. We have recently established a protocol of DCE-MRI of orthotopic pancreatic tumor xenografts in mouse models by securing tumors with an orthogonally bent plastic board to prevent motion transfer from the chest region during imaging. The pressure by this board was localized on the abdominal area, and has not resulted in respiratory difficulty of the animals. This article demonstrates the detailed procedure of orthotopic pancreatic tumor modeling using small animals and DCE-MRI of the tumor xenografts. Quantification method of pharmacokinetic parameters in DCE-MRI is also introduced. The procedure described in this article will assist investigators to apply DCE-MRI for orthotopic gastrointestinal cancer mouse models.

The goal of this protocol is to apply dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) for orthotopic pancreatic tumor xenografts in mice. DCE-MRI is a non-invasive method to analyze microvasculature in a target tissue, and useful to assess vascular response in a tumor following a novel therapy.