The authors would like to thank the National Institutes of Health R01 HL134830 (PKN), K08 HL135343 (KS), and 5F32HL134221 (JWR); the Howard Hughes Medical Institute (ASL); and the Stanford Cardiovascular Institute (ASL) for their support.

This animal experiment was approved and performed under the Institutional Review Board and the Administrative Panel on Laboratory Animal Care at Stanford University.
1. Cell Culture of iPSCs
<ol>
	<li>Grow human iPSCs derived by lentiviral reprogramming on 6-well plates coated with basement membrane matrix (e.g., matrigel, referred to as matrix hereon).</li>
	<li>Daily change the media of the iPSCs with enriched culture medium (see Table of Materials) incubating at 37&#160;&#176;C and 5% CO2.</li>
	<li>Once the cells reach 80&#8211;90% confluence (approximately every 4 days), add 1 mL of recombinant cell-dissociation enzyme (see Table of Materials) per well and incubate at 37&#160;&#176;C for 5 min.</li>
	<li>After 5 min, dissociate the cells from the well by pipetting, transfer them to a 15 mL tube, and centrifuge at 300 x g.</li>
	<li>After the centrifugation, aspirate the supernatant and resuspend the cell pellet into enriched culture medium (see Table of Materials) enriched with Y27632 inhibitor at a 1:1,000 dilution.</li>
	<li>Perform a cell count of the dissociated cells and replate the cells on matrix-coated 6-well plates at a density of 2 x 105 to 4 x 105.</li>
</ol>
2. Transduction of iPSCs with a Double-fusion Reporter Gene
<ol>
	<li>Passage iPSCs in 6-well plates as per routine and add enriched culture medium (see Table of Materials) containing 6 &#181;g/mL hexadimethrine bromide. 
	NOTE: The ideal colony size is 200&#8211;400 cells/colony to yield the highest transduction efficiency.</li>
	<li>Concentrate self-inactivating lentivirus carrying firefly luciferase and green fluorescent protein (FLuc-eGFP) driven by human ubiquitin promotor-C by sediment centrifugation with an SW-29 rotor at 50,000 x g for 2 h at 4&#160;&#176;C.</li>
	<li>Add the viral concentrate to the iPSCs in a 6-well plate at a multiplicity of infection (MOI) of 10 and incubate overnight at 37&#160;&#176;C at 5% CO2. 
	NOTE: The multiplicity of infection was determined by the expression of monomeric fluorescence protein analyzed by a fluorescence-activated cell sorting (FACS) scan.</li>
	<li>The following day, remove the virus by centrifugation of the iPSC 6-well plates at 300 x g for 6 min at room temperature.</li>
	<li>Change the media daily with enriched culture medium (see Table of Materials) and passage as per protocol. Utilize a fluorescence microscope to determine the approximate transduction efficiency for eGFP.</li>
	<li>An efficiency of 30&#8211;40% is sufficient for FACS sorting. Proceed to the FACS of hiPSCs expressing eGFP if at least 30&#8211;40% of the cells express eGFP.</li>
	<li>To confirm FLuc activity ex vivo, plate the cells expressing GFP sorted by FACS at a density of 5,000 cells per well.</li>
	<li>Incubate the transduced cells and non-transduced cells (which will serve as the negative control) with the bioluminescence reporter probe D-luciferin (100 &#956;mol/L) for 6 h. Measure the bioluminescence with a microplate spectrofluorometer.</li>
</ol>
3. Transplantation of PSCs in the Dorsal Flank for Teratoma Formation in Immunodeficient Mice
<ol>
	<li>Add 1 mL of recombinant cell-dissociation enzyme mix per 6-well plate containing human iPSCs transduced with a double-fusion reporter gene (FLuc-GFP) in culture (see section 2) and incubate for 5 min.</li>
	<li>After the incubation period, disperse the cells by pipette aspiration and expression. Add an equal volume of culture medium and then centrifuge at 250 x g for 4 min.</li>
	<li>After centrifugation is complete, aspirate the supernatant solution, resuspend the cell pellet in 30 &#181;L of matrix, and place it on ice to preserve its viability prior to injection. Confirm a harvest of 1 x 106 cells using a hemocytometer.</li>
	<li>If utilizing double-fusion reporter-gene-transfected cells, suspend the double-positive FACS cells (from section 2) in 30 &#181;L of matrix.</li>
	<li>Induce anesthesia in 8-10 week athymic nude mice using 2% isoflurance with 1L/min oxygen and then use maintenance 2% isoflurane with 1L/min of oxygen.</li>
	<li>Using a 28.5 G syringe, inject cell/matrix mixture (see Table of Materials) suspension into the subcutaneous space at the&#160;flank, aiming for an injection of in total 5 x 103 to 5 x 106 cells (approximately 100 ul per injection).</li>
	<li>Consider a more caudal injection site if planning to maintain mice for prolonged period and anticipating large tumor growth.&#160;</li>
	<li>Anesthetized animals are allowed to recover on a heated pad until ambulatory (typically &#60;1h) with monitoring of respiratory rate, skin color of toes until ambulatory.&#160;</li>
</ol>
4. Bioluminescence Imaging (BLI) of Transplanted Cells to Assess Cell Survival and Teratoma Growth
<ol>
	<li>At the desired timepoints after inoculation, perform an intraperitoneal (IP) injection of 375 mg/kg of the reporter probe D-Luciferin into the mice.</li>
	<li>10 min after an IP injection, image the bioluminescence signal in the anesthetized animals (performed as described in step 3.5) for 30 min using 1 min acquisition windows at 5-min intervals. 
	NOTE: Weekly image acquisitions are recommended. Anesthesia is maintained during imaging by delivering inhaled isoflurane via a nose cone.</li>
	<li>For data analysis, draw a region of interest (ROI) over the BLI signal and, then, normalize for the acquisition time to quantify emissions in units of maximum photons per second per square centimeter per steradian (photons/s/cm2/sr).</li>
</ol>
5. Teratoma Irradiation Using a Preclinical Image-guided Irradiator (Figure 1)
<ol>
	<li>Anesthetize a mouse in a knockdown box using 2% isoflurane in 100% oxygen at a flow rate of 1 L/min. After the mouse is fully anesthetized, transfer it to the bed of an image-guided pre-clinical irradiator (see Table of Materials). Maintain anesthesia by 2% isoflurane continuously via a nose cone.</li>
	<li>Acquire micro-CT images as a set of 400 projection images over 360&#176; using a 40 kVp, 2 mA X-ray beam, and reconstruct those into volumetric images with an isotropic pixel size of 0.2 mm.</li>
	<li>Plan a radiation treatment using the micro-CT images using the RT_Image software package (http://rtimage.sourceforge.net/) and perform the treatment. 
	NOTE: The treatment plan used consists of two 225 kVp X-ray beams, oriented to pass through the superficial target teratoma while skirting the surface of the rest of the mouse and sparing the underlying viscera. The exposure times for the beams are adjusted based on quarterly system calibration data so that the dose at the center of the target tumor was 6 Gy.</li>
	<li>Repeat the treatment process on three consecutive days to deliver a total of 18 Gy to the target tumor.</li>
	<li>Maintain standard post-treatment care of the animals.</li>
</ol>

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The authors have nothing to disclose.

Preclinical data and anecdotal cases from victims of &#34;stem cell tourism&#34; confirm that the risk of developing teratomas is a serious drawback associated with PSC treatments23. Development of careful approaches to prevent and treat the neoplastic risk associated with stem cell therapies is, therefore, an important step in facilitating the clinical translation of regenerative stem cell therapies. In this article, we described a method of therapeutic targeting of PSC-associated teratomas using...

The development of stem cell therapies for tissue regeneration has encountered a number of barriers in the past several decades, hampering efforts for efficient clinical deployment. These hurdles include poor cell retention at sites of delivery, stem cell immunogenicity, and the neoplastic potential to form teratomas1. Tumorigenicity is of particular clinical concern as it can potentially harm stem cell transplant recipients2. Accounts of tumor formation due to unregulated stem cell injections have already been reported in multiple clinical settings3,4,5. The potential for teratoma formation is the most frequently cited clinical concern in pluripotent stem cell (PSC) development and has resulted in delays and cancellations of multiple high-profile embryonic stem cell (ESC) and induced pluripotent stem cell (iPSC) trials6,7,8,9. Thus, there is a pressing need for a translational investigation dedicated toward providing appropriate treatment, should these iatrogenic tumors arise.
To date, most strategies to control stem cell misbehavior have focused on reducing the number of PSCs with tumorigenic potential2,10. Unfortunately, only a small number of residual cells (e.g., 1 x 104 to 1 x 105 cells11) is required for teratoma formation, which is far below the detection limit quoted by currently available assays12,13. Other limitations of using these preseparation methods include low efficiency and high expense, reliance on single-cell suspensions that may not be appropriate for newer tissue-engineering approaches, and the potential impairment of cell survival and engraftment.
Few studies have addressed treatment options following teratoma formation. Perhaps the most well-studied strategy is the incorporation of &#34;suicide&#34; genes into stem cells14,15. This method involves genetically manipulating the stem cells to incorporate an inducible apoptosis-activating gene that can be activated by pharmacological stimulation postinjection, thereby providing a rescue approach if injected cells produce teratomas. This approach, however, suffers from significant drawbacks, including off-target effects of genetic modifications of PSCs and the potential for a gradual development of drug resistance16. A similar approach utilizes small molecules to induce selective cell death of PSCs via the inhibition of anti-apoptotic pathways17. Other groups have targeted cell death of PSCs using antibodies against pluripotency surface markers, such as podocalyxin-like protein-1 (PODXL)18. The timing of small-molecule or antibody delivery stands to have a significant impact on the therapeutic potential of PSCs if delivered too early and may lack therapeutic efficacy if delivered too late. In addition, the systemic effects of small molecules and antibodies used in this fashion have not been studied.
An alternative approach to treating these tumors relies on using external beam radiation therapy (EBRT). EBRT is one of the primary modalities currently employed in the treatment of solid tumors19. Innovations in EBRT, including the development of the proton beam and stereotactic radiosurgery, have enabled the enhanced targeting of pathological structures while avoiding damage to normal tissue, making conformal EBRT ideal for addressing teratoma formation in anatomically sensitive structures20. Additionally, this method avoids the genetic manipulation or viral transduction of stem cells, which are both fraught with additional clinical safety and efficacy concerns15. Finally, advances in micro-irradiators have enabled the application of EBRT in rodents21.
In this article, we demonstrate how to create a small-animal model of teratoma formation by injecting human iPSCs in mice. We then show how to apply EBRT to selectively eradicate these tumors in vivo with minimal damage to surrounding tissue. This approach provides a targeted therapy for PSC-derived teratomas while avoiding the off-target effects of the systemic delivery of biological molecules and peptides and the genetic manipulation of the PSCs. For investigational purposes, we offer an optional step to transduce stem cells with reporter genes to track tumor response to radiation therapy via bioluminescence imaging (BLI).

<table><tbody><tr><td>Induced Pluripotent Stem Cell Control Line</td><td>Stanford University</td><td>Nguyen Lab</td><td>Cell culture of iPSC</td></tr><tr><td>Corning matrigel basement membrane matrix 354234</td><td>Fisher Scientific</td><td>CB-40234</td><td>Cell culture of iPSC</td></tr><tr><td>Essential 8 culture medium</td><td>ATCC-The global bioresource center</td><td>30-2203</td><td>Cell culture of iPSC</td></tr><tr><td>Tryple E</td><td>Gibco</td><td>12605-036</td><td>Cell culture of iPSC</td></tr><tr><td>Y27632 inhibitor 2 HCL (ROCK Inhibitor)</td><td>Fisher Scientific</td><td>S104950MG</td><td>Cell culture of iPSC</td></tr><tr><td>Lentivirus</td><td>Cyagen</td><td>P170721-1001cjn</td><td>Transduction of iPSC with double fusion reporter gene</td></tr><tr><td>Polyrbrene Infection/Transfection Reagent</td><td>Millipore Sigma</td><td>TR-1003-G</td><td>Transduction of iPSC with double fusion reporter gene</td></tr><tr><td>Fluc-eGFP reporter gene driven by ubiquitin promoter</td><td>Stanford University</td><td>Sam Gambhir lab</td><td>Transduction of iPSC with double fusion reporter gene</td></tr><tr><td>D-luciferin</td><td>Perkin Elmer</td><td>122799</td><td>Transduction of iPSC with double fusion reporter gene and BLI</td></tr><tr><td>Flow cytometer (BD FACSARIA III)</td><td>BD Biosciences&amp;nbsp;</td><td>FACSAria</td><td>Transduction of iPSC with double fusion reporter gene</td></tr><tr><td>microplate spectrofluorometer (Glomax Navigator System)</td><td>Promega Bio Systems, Sunnyvale, CA</td><td>GM2000</td><td>Transduction of iPSC with double fusion reporter gene</td></tr><tr><td>Xenogen IVIS 200&amp;nbsp;</td><td>Perkin Elmer</td><td>124262</td><td>BLI</td></tr><tr><td>Isoflurane</td><td>Sigma-Aldrich</td><td>CDS019936</td><td>irradiation</td></tr><tr><td>X-Rad SmART image-guided irradiator&amp;nbsp;</td><td>Precision X-ray Inc., North Branford, CT</td><td>X-Rad SmART</td><td>irradiation</td></tr><tr><td>RT_Image software package</td><td>Stanford University (http://rtimage.sourceforge.net/)</td><td>RT_Image v0.2&amp;beta;</td><td>Irradiation</td></tr></tbody></table>

targeted and selective treatment of pluripotent stem cell-derived teratomas using external beam radiation in a small-animal model

The growing number of victims of &#34;stem cell tourism,&#34; the unregulated transplantation of stem cells worldwide, has raised concerns about the safety of stem cell transplantation. Although the transplantation of differentiated rather than undifferentiated cells is common practice, teratomas can still arise from the presence of residual undifferentiated stem cells at the time of transplant or from spontaneous mutations in differentiated cells. Because stem cell therapies are often delivered into anatomically sensitive sites, even small tumors can be clinically devastating, resulting in blindness, paralysis, cognitive abnormalities, and cardiovascular dysfunction. Surgical access to these sites may also be limited, leaving patients with few therapeutic options. Controlling stem cell misbehavior is, therefore, critical for the clinical translation of stem cell therapy.
External beam radiation offers an effective means of delivering targeted therapy to decrease the teratoma burden while minimizing injury to surrounding organs. Additionally, this method avoids genetic manipulation or viral transduction of stem cells-which are associated with additional clinical safety and efficacy concerns. Here, we describe a protocol to create pluripotent stem cell-derived teratomas in mice and to apply external beam radiation therapy to selectively ablate these tumors in vivo.

Injected mice typically will demonstrate teratoma growth formation after 4&#8211;8 weeks as confirmed by BLI imaging (Figure 2). Tumors will shrink dramatically when irradiated with a cumulative dose of 18 Gy given one month after cell delivery, resulting in a significant decrease in luciferase signal (Figure 2). Importantly, normal tissues taken 5 mm from the irradiated site do not appear to have any significant damage (<strong ...

Watch this Scientific Journal Video about Targeted and Selective Treatment of Pluripotent Stem Cell-derived Teratomas Using External Beam Radiation in a Small-animal Model at JoVE.com

Targeted and Selective Treatment of Pluripotent Stem Cell-derived Teratomas Using External Beam Radiation in a Small-animal Model

Research on treatment strategies for pluripotent stem cell-derived teratomas is important for the clinical translation of stem cell therapy. Here, we describe a protocol to, first, generate stem cell-derived teratomas in mice and, then, to selectively target and treat these tumors in vivo using a small-animal irradiator.

The growing number of victims of &#34;stem cell tourism,&#34; the unregulated transplantation of stem cells worldwide, has raised concerns about the ...

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JoVE Journal

Medicine

6.1K Views.  Stanford University School of Medicine. Het algemene doel van deze procedure is om selectief stamcel-afgeleide teratomen in muizen te richten voor evaluatie van de in vivo effectiviteit van kleine dierlijke externe straalstralingtherapie. Het belangrijkste voordeel van externe stralingsstraaltherapie is dat het een gerichte therapie voor stamcel geassocieerde teramtomas die de off-targets nadelige effecten van systemisch geleverde therapieën vermijdt. Vooruitgang in externe straalstraling hebben de specifieke targeting van kleine ...

Video: Targeted and Selective Treatment of Pluripotent Stem Cell-derived Teratomas Using External Beam Radiation in a Small-animal Model

An Orthotopic Model of Murine Bladder Cancer

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and subsequent cell adhesion. After their bladders are transurethrally catheterized and drained, animals are again catheterized to permit insertion of a platinum wire into bladders without damaging the urethra or bladder. The catheters are made of Teflon to serve as an insulator for the wire, which will conduct electrical current into the bladder to create a burn injury. An electrocautery unit is used to deliver 2.5W to the exposed end of the wire, burning away extracellular layers and providing attachment sites for carcinoma cells that are delivered in suspension to the bladder through a subsequent catheterization. Cells remain in the bladder for 90 minutes, after which the catheters are removed and the bladders allowed to drain naturally. The development of tumor is monitored via ultrasound. Specific attention is paid to the catheterization technique in the accompanying video.

Bladder tumors are established in female mice in a minimally invasive fashion through catheterization, local cauterization, and subsequent adhesion of carcinoma cells to the burn sites.

In this straightforward procedure, bladder tumors are established in female C57 mice through the use of catheterization, local cauterization, and ...

PET and MRI Guided Irradiation of a Glioblastoma Rat Model Using a Micro-irradiator

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without the ability to target a specific or well-delineated tumor volume. The delivery of radiation was achieved using fixed radiation sources or linear accelerators producing megavoltage (MV) X-rays. These devices are unable to achieve sub-millimeter precision required for small animals. Furthermore, the high doses delivered to healthy surrounding tissue hamper response assessment. To increase the translation between small animal studies and humans, our goal was to mimic the treatment of human glioblastoma in a rat model. To enable a more accurate irradiation in a preclinical setting, recently, precision image-guided small animal radiation research platforms were developed. Similar to human planning systems, treatment planning on these micro-irradiators is based on computed tomography (CT). However, low soft-tissue contrast on CT makes it very challenging to localize targets in certain tissues, such as the brain. Therefore, incorporating magnetic resonance imaging (MRI), which has excellent soft-tissue contrast compared to CT, would enable a more precise delineation of the target for irradiation. In the last decade also biological imaging techniques, such as positron emission tomography (PET) gained interest for radiation therapy treatment guidance. PET enables the visualization of e.g., glucose consumption, amino-acid transport, or hypoxia, present in the tumor. Targeting those highly proliferative or radio-resistant parts of the tumor with a higher dose could give a survival benefit. This hypothesis led to the introduction of the biological tumor volume (BTV), besides the conventional gross target volume (GTV), clinical target volume (CTV), and planned target volume (PTV).
At the preclinical imaging lab of Ghent University, a micro-irradiator, a small animal PET, and a 7 T small animal MRI are available. The goal was to incorporate MRI-guided irradiation and PET-guided sub-volume boosting in a glioblastoma rat model.

In the past, small animal irradiation was usually performed without the ability to target a well-delineated tumor volume. The goal was to mimic the treatment of human glioblastoma in rats. Using a small animal irradiation platform, we performed MRI-guided 3D conformal irradiation with PET-based sub-volume boosting in a preclinical setting.

For decades, small animal radiation research was mostly performed using fairly crude experimental setups applying simple single-beam techniques without ...

Cancer Research

Teratoma Generation in the Testis Capsule

Pluripotent stem cells (PSCs) have the unique characteristic that they can differentiate into cells from all three germ layers. This makes them a potentially valuable tool for the treatment of many different diseases. With the advent of induced pluripotent stem cells (iPSCs) and continuing research with human embryonic stem cells (hESCs) there is a need for assays that can demonstrate that a particular cell line is pluripotent. Germline transmission has been the gold standard for demonstrating the pluripotence of mouse embryonic stem cell (mESC) lines1,2,3. Using this assay, researchers can show that a mESC line can make all cell types in the embryo including germ cells4. With the generation of human ESC lines5,6, the appropriate assay to prove pluripotence of these cells was unclear since human ESCs cannot be tested for germline transmission. As a surrogate, the teratoma assay is currently used to demonstrate the pluripotency of human pluripotent stem cells (hPSCs)7,8,9. Though this assay has recently come under scrutiny and new technologies are being actively explored, the teratoma assay is the current gold standard7. In this assay, the cells in question are injected into an immune compromised mouse. If the cells are pluripotent, a teratoma will eventually develop and sections of the tumor will show tissues from all 3 germ layers10. In the teratoma assay, hPSCs can be injected into different areas of the mouse. The most common injection sites include the testis capsule, the kidney capsule, the liver; or into the leg either subcutaneously or intramuscularly11. Here we describe a robust protocol for the generation of teratomas from hPSCs using the testis capsule as the site for tumor growth.

Human pluripotent stem cells (hPSCs) have the potential to treat a myriad of different diseases. The utility of these cells lies in the fact that they can differentiate into any cell type in the body. Here we describe the teratoma assay, which is used to demonstrate the pluripotence of hPSCs.

Pluripotent stem cells (PSCs) have the unique characteristic that they can differentiate into cells from all three germ layers.  This makes them a ...

Utilization of Ultrasound Guided Tissue-directed Cellular Implantation for the Establishment of Biologically Relevant Metastatic Tumor Xenografts

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard subcutaneous flank models include procedural ease and the ability to monitor tumor progression and response without invasive imaging. Such models are often inconsistent in translational clinical trials and have limited biologically relevant characteristics with low proclivity to produce metastasis, as there is a lack of a native microenvironment. In comparison, orthotopic xenograft models at native tumor sites have been shown to mimic the tumor microenvironment and replicate important disease characteristics such as distant metastatic spread. These models often require tedious surgical procedures with prolonged anesthetic time and recovery periods. To address this, cancer researchers have recently utilized ultrasound-guided injection techniques to establish cancer xenograft models for preclinical experiments, which allows for rapid and reliable establishment of tissue-directed murine models. Ultrasound visualization also provides a noninvasive method for longitudinal assessment of tumor engraftment and growth. Here, we describe the method for ultrasound-guided injection of cancer cells, utilizing the adrenal gland for NB and renal sub capsule for ES. This minimally invasive approach overcomes tedious open surgery implantation of cancer cells in tissue-specific locations for growth and metastasis, and abates morbid recovery periods. We describe the utilization of both established cell lines and patient derived cell lines for orthotopic injection. Pre-made commercial kits are available for tumor dissociation and luciferase tagging of cells. Injection of cell suspension using image-guidance provides a minimally invasive and reproducible platform for the creation of preclinical models. This method is utilized to create reliable preclinical models for other cancers such as bladder, liver and pancreas exemplifying its untapped potential for numerous cancer models.

Here, we present a protocol to utilize ultrasound-guided injection of neuroblastoma (NB) and Ewing's sarcoma (ES) cells (established cell lines and patient-derived tumor cells) at biologically relevant sites to create reliable preclinical models for cancer research.

Preclinical testing of anticancer therapies relies on relevant xenograft models that mimic the innate tendencies of cancer. Advantages of standard ...

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 ...

One Minute, Sub-One-Watt Photothermal Tumor Ablation Using Porphysomes, Intrinsic Multifunctional Nanovesicles

We recently developed porphysomes as intrinsically multifunctional nanovesicles. A photosensitizer, pyropheophorbide &alpha;, was conjugated to a phospholipid and then self-assembled to liposome-like spherical vesicles. Due to the extremely high density of porphyrin in the porphyrin-lipid bilayer, porphysomes generated large extinction coefficients, structure-dependent fluorescence self-quenching, and excellent photothermal efficacy. In our formulation, porphysomes were synthesized using high pressure extrusion, and displayed a mean particle size around 120 nm. Twenty-four hr post-intravenous injection of porphysomes, the local temperature of the tumor increased from 30 &deg;C to 62 &deg;C rapidly upon one minute exposure of 750 mW (1.18 W/cm2), 671 nm laser irradiation. Following the complete thermal ablation of the tumor, eschars formed and healed within 2 weeks, while in the control groups the tumors continued to grow and all reached the defined end point within 3 weeks. These data show how porphysomes can be used as potent photothermal therapy (PTT) agents.

We developed novel intrinsic multifunctional nanovesicles called porphysomes, which have structure-dependent fluorescence self-quenching and unique photothermal properties, thus functioning as potent photothermal therapy agents. We formulated porphysomes using high pressure extrusion and investigated their photothermal therapy efficacy in a xenograft tumor model.

We recently developed porphysomes as intrinsically multifunctional nanovesicles. A photosensitizer, pyropheophorbide &alpha;, was conjugated to a ...

Bioengineering

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 ...

Testing Targeted Therapies in Cancer using Structural DNA Alteration Analysis and Patient-Derived Xenografts

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic target analyses and drug response monitoring using patient derived xenografts (PDX). This strategy was validated using ovarian tumors as an example. The mate-pair next generation sequencing (MPseq) protocol was used to identify structural alterations and followed by analysis of potentially targetable alterations. Human tumors grown in immunocompromised mice were treated with drugs selected based on the genomic analyses. Results demonstrated a good correlation between the predicted and the observed responses in the PDX model. The presented approach can be used to test the efficacy of combination treatments and aid personalized treatment for patients with recurrent cancer, specifically in cases when standard therapy fails and there is a need to use drugs off label.

Here we present a protocol to test the efficacy of targeted therapies selected based on the genomic makeup of a tumor. The protocol describes identification and validation of structural DNA rearrangements, engraftment of patients&#8217; tumors into mice and testing responses to corresponding drugs.

We present here an integrative approach for testing efficacy of targeted therapies that combines the next generation sequencing technolo-gies, therapeutic ...

Positron Emission Tomography-based Dose Painting Radiation Therapy in a Glioblastoma Rat Model using the Small Animal Radiation Research Platform

A rat glioblastoma model to mimic chemo-radiation treatment of human glioblastoma in the clinic was previously established. Similar to the clinical treatment, computed tomography (CT) and magnetic resonance imaging (MRI) were combined during the treatment-planning process. Positron emission tomography (PET) imaging was subsequently added to implement sub-volume boosting using a micro-irradiation system. However, combining three imaging modalities (CT, MRI, and PET) using a micro-irradiation system proved to be labor-intensive because multimodal imaging, treatment planning, and dose delivery have to be completed sequentially in the preclinical setting. This also results in a workflow that is more prone to human error. Therefore, a user-friendly algorithm to further optimize preclinical multimodal imaging-based radiation treatment planning was implemented. This software tool was used to evaluate the accuracy and efficiency of dose painting radiation therapy with micro-irradiation by using an in silico study design. The new methodology for dose painting radiation therapy is superior to the previously described method in terms of accuracy, time efficiency, and intra- and inter-user variability. It is also an important step towards the implementation of inverse treatment planning on micro-irradiators, where forward planning is still commonly used, in contrast to clinical systems.

Here we present a protocol to perform preclinical positron emission tomography-based radiotherapy in a rat glioblastoma model using algorithms developed in-house to optimize the accuracy and efficiency.

A rat glioblastoma model to mimic chemo-radiation treatment of human glioblastoma in the clinic was previously established. Similar to the clinical ...

Minimally Invasive Establishment of Murine Orthotopic Bladder Xenografts

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable cell lines are inoculated either by intravesical instillation (model of nonmuscle invasive growth) or intramural injection into the bladder wall (model of invasive growth). Both procedures are complex and highly time-consuming. Additionally, the superficial model has its shortcomings due to the lack of cell lines that are tumorigenic following instillation. Intramural injection, on the other hand, is marred by the invasiveness of the procedure and the associated morbidity for the host mouse.
With these shortcomings in mind, we modified previous methods to develop a minimally invasive approach for creating orthotopic bladder cancer xenografts. Using ultrasound guidance we have successfully performed percutaneous inoculation of the bladder cancer cell lines UM-UC1, UM-UC3 and UM-UC13 into 50 athymic nude. We have been able to demonstrate that this approach is time efficient, precise and safe. With this technique, initially a space is created under the bladder mucosa with PBS, and tumor cells are then injected into this space in a second step. Tumor growth is monitored at regular intervals with bioluminescence imaging and ultrasound. The average tumor volumes increased steadily in in all but one of our 50 mice over the study period.
In our institution, this novel approach, which allows bladder cancer xenograft inoculation in a minimally-invasive, rapid and highly precise way, has replaced the traditional model.

The established technique to inoculate primary invasive orthotopic bladder cancer xenografts requires laparotomy and mobilization of the bladder. This procedure inflicts significant morbidity on the mice, is technically challenging and time-consuming. We therefore developed a high-precision, percutaneous approach utilizing ultrasound guidance.

Orthotopic bladder cancer xenografts are the gold standard to study molecular cellular manipulations and new therapeutic agents&#160;in vivo. Suitable ...

Targeted and Selective Treatment of Pluripotent Stem Cell-derived Teratomas Using External Beam Radiation in a Small-animal Model

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