Name: orthotopic implantation and peripheral immune cell monitoring in the ii-45 syngeneic rat mesothelioma model
Uploaded: 2015-10-02T14:00:00.000+00:00
Duration: 9 min 31 s
Description: Watch this Scientific Journal Video about Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model at JoVE.com

The syngeneic rat mesothelioma II-45 cell line was a kind gift from A/Prof. Emanuela Felley-Bosco, Zurich University.

All procedures involving animals were carried out in accordance with the recommendations in the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes. The protocol for this study was approved by the Royal North Shore Hospital Animal Care and Ethics Committee. Female Fischer 344 rats (F344, 150-200 g) were maintained at the Kearns Facility, Kolling Institute under standard conditions (12 hr light/dark cycles and free access to food and water).
Note: A flow chart for all experimental procedures is presented in Figure 1.
1. Preparation of Cells for Implantation
<ol>
	<li>Culture the rat mesothelioma II-45 cell line (also known as IL-45; derived by peritoneal introduction of crocidolite asbestos) in RPMI 1640 (RPMI) media supplemented with 10% fetal bovine serum (FBS) and grow in standard conditions (37 &#176;C humidified incubator with 5% CO2). Maintain by passaging and sub-culturing at approximately 1:50 twice a week in a 75 cm2 flask.</li>
	<li>Prepare reagents for cell culture and warm aliquots at 37 &#176;C. Required reagents include serum free RPMI media (SFM), RPMI with 10% FBS, phosphate buffered saline (PBS) and 0.5% trypsin-EDTA.</li>
	<li>Culture cells for implantation to approximately 70-80% confluence. This ensures they are in the linear growth phase.</li>
	<li>Harvest cells by discarding the media, washing once with 5 ml&#160;of sterile PBS and then adding 3 ml&#160;of 0.5% trypsin-EDTA.</li>
	<li>Return flasks to the incubator for approximately 5 min until all cells become non-adherent.</li>
	<li>Once cells are non-adherent, add 3 ml&#160;of RPMI with 10% FBS to inactivate the trypsin. Collect and centrifuge cells at 300 x g for 3 min.</li>
	<li>Wash the cell pellet in 10 ml&#160;of SFM and centrifuge again at 300 x g for 3 min.</li>
	<li>Wash cell pellet again with 10 ml&#160;of SFM and centrifuge as above.</li>
	<li>Resuspend the cells in 10 ml&#160;of SFM and perform a cell count using a hemocytometer or similar instrumentation.</li>
	<li>Dilute cells so that 100 &#181;l&#160;contains the amount of cells to be implanted. 
	Note: Tumor growth has been demonstrated at a dose as low as 100 cells in 100 &#181;l but a standard dose is 500,000&#160;cells in 100 &#181;l.</li>
	<li>Prepare sufficient cells in media for the number of rats to be implanted (i.e., 100 &#181;l/rat), plus at least 0.5 ml&#160;extra to compensate for losses from priming and the dead volume of the needle.</li>
	<li>Prepare sufficient SFM (without cells) to be implanted into control rats (i.e.,&#160;100 &#181;l/rat), plus at least 0.5 ml&#160;extra. 
	Note: The cells and SFM are now ready for implantation. They should be kept at 37&#160;oC and implanted within 2 hr of harvesting to maintain viability.</li>
</ol>
2. In vivo&#160;Implantation of Cells
<ol>
	<li>Place the F344 rat (&#62; 13 weeks of age) into the induction chamber and anesthetize using 1.4% isoflurane inhalation (or the method in use in the facility). Once the rat appears to be asleep move it from the chamber to a nose cone (with 1.4% isoflurane flowing), place it on its back with chest facing up (ventral view). This allows the internal organs to settle away from the chest cavity. Check reflexes according to institutional protocols to ensure the rat is fully anesthetized.</li>
	<li>Shave the right regio costalis (chest) area to remove the fur.</li>
	<li>Clean the shaven area with 80% v/v ethanol.</li>
	<li>&#160;Identify the injection site: on the right side, find the 2nd gland starting cranial. The injection site is 0.5 cm proximal to this, in between the 3rd and 4th rib from the caudal end of the rib cage.&#160;(Figure 2A).</li>
	<li>Gently mix the II-45 cells to resuspend. Slowly draw the cell suspension (or SFM for control rats) into a 1 ml&#160;syringe without a needle attached. If a needle is attached for the drawing up of cells there is the potential for cells to grow along the needle injection line. Attach a 23G x 1&#188; needle. Prime the needle and remove any air bubbles.</li>
	<li>Once the syringe and needle are primed, place a 20 mm long and 5 mm diameter spacer over the needle shaft. This is used to prevent the needle from penetrating too deeply into the pleural cavity during the injection. Approximately 5 mm-12 mm of exposed needle is sufficient for penetration through the ribs without damaging any organs.</li>
	<li>Slowly insert the needle between the ribs, draw back on the syringe to ensure a blood vessel has not been punctured (no blood should appear in the syringe) then inject 100 &#956;l&#160;cells or SFM.&#160;(Figure 2B).</li>
	<li>Remove the needle and gently roll the rat from side to side to spread cells in the chest cavity.</li>
	<li>Place the rat into a cage and check for recovery. The rat should be awake within 1 min&#160;and starting to move around.</li>
	<li>Repeat for each rat using a new needle. Reusing the same needle will result in cell growth along the injection line of the needle.</li>
	<li>Monitor the well-being of the animals daily.</li>
	<li>Euthanize animals at ethically defined endpoints as governed by the institutional animal ethics committee. The ethical endpoints for the rats in these experiments were weight loss of greater than 10% or labored breathing.</li>
</ol>
3. Tail Vein Blood Collection
<ol>
	<li>If blood is to be collected immediately post-cell implantation, keep the rat anesthetized. If sampling blood at another time point, anesthetize the rat using 1.4% isoflurane inhalation. Check reflexes according to institutional protocols to ensure the rat is fully anaesthetized.</li>
	<li>Place the rat onto its side and locate a lateral tail vein.</li>
	<li>Sterilize the tail with 80% ethanol and label a 0.5 ml&#160;EDTA collection tube.</li>
	<li>To collect blood, always start at the caudal end of the tail (approximately one third of the way along). This allows further attempts closer to the cranial end of the tail in case the first attempt is unsuccessful. Never resample caudally as this can cause a blood clot.</li>
	<li>Position a 23G x 1&#188; needle parallel to the lateral vein and slide it into the vein at a shallow angle so it penetrates approximately 10 mm (Figure 3A).</li>
	<li>Note: If the vein has been successfully punctured blood will be visible in the attachment end of the needle (Figure 3B).</li>
	<li>A drop of blood will form on the tail at the site of the needle puncture. Collect this blood using a pipette and transfer into the labelled 0.5 ml (or smaller) EDTA collection tube. For the immune cell assay 25 &#956;l is sufficient. Apply gauze with pressure to puncture site until bleeding stops.</li>
	<li>Flick the blood tube to mix the blood and EDTA to prevent clotting. Keep the time between blood collection and mixing with the EDTA as short as possible to prevent clotting.</li>
	<li>When collecting blood from multiple rats store EDTA-blood samples in a rack at RT until analysis. Process blood within 2 hr&#160;of collection.</li>
</ol>
4. Sample Preparation for Immune Cell Profiling Using the Bead-based Method
Note: This single platform method relies on using commercially available absolute counting tubes that have a known number of beads for each sample. These tubes contain lyophilized pellets that dissolve during sample preparation, releasing the beads. The beads are fluorescently labelled and by gating on the bead population, absolute counts can be calculated.
<ol>
	<li>Ensure the EDTA whole blood sample is well mixed by placing it on a slow rotary mixer for several min. Label one absolute counting tube for each sample. A pellet containing the beads should be visible underneath the metal bead holder at the bottom of the tube.</li>
	<li>Transfer 25 &#181;l&#160;of EDTA whole blood into a labelled absolute counting tube. The bead pellet will dissolve upon addition of the blood.</li>
	<li>To each tube add 20 &#181;l&#160;of anti-rat T/B/Natural Killer (NK) cell cocktail, 10 &#181;l&#160;of anti-rat CD8a PE, 10 &#181;l&#160;of anti-rat CD4 (domain 1) FITC and 10 &#181;l of anti-rat CD45 PE/Cy7 (Figure 4A). Fluorophores are defined in Table 1.</li>
	<li>Centrifuge the tube briefly (300 x g) to ensure the antibodies and cells are in the bottom of the tube and not stuck to the side of the tube. Vortex to mix and incubate for 15 min&#160;at RT.</li>
	<li>To lyse red blood cells add 400 &#181;l&#160;of 10 mM Tris, 0.15 M ammonium chloride buffer (pH 7.5) and vortex to mix. Lysis is complete when the sample appears translucent and not cloudy (Figures 4B and C). Failure to lyse the sample completely will lead to increased background and falsely elevated counts when analyzing by flow cytometry.</li>
</ol>
5. Flow Cytometric Processing of Samples
Note: Perform on a 4 color flow cytometer.
<ol>
	<li>Open the software in acquisition mode and a new template with 8 plots as depicted in Figure 5.</li>
	<li>Adjust instrument settings to those listed in Table 1 and set up gate R1 (FITC [FL-1] versus APC [FL-4]), Figure 5Ai) to count the fluorescent beads. The other gates are not as important at this acquisition stage but will be required for analysis. The absolute counting beads used in this protocol contain fluorescent dyes and can be detected in any channel although are weakest in the blue channel.</li>
	<li>Using a prepared control blood sample, vortex and then load onto the cytometer and run at a low speed (12 &#181;l/min) on setup mode so data acquisition gates can be adjusted.</li>
	<li>Set the acquisition to collect 10,000 events in the R1 bead gate.</li>
	<li>Set up a folder to record data and set file number and label sample file in acquisition menu.</li>
	<li>Load the sample to be analyzed onto the cytometer and set the flow rate to medium (35 &#181;l/min). Run each sample at the same flow rate. The flow rate may need to be varied to low (12 &#181;l/min) or high (60 &#181;l/min), but medium is generally appropriate. At this rate it takes approximately 90 to 120 sec&#160;to acquire 10,000 bead events for each sample.</li>
	<li>Once the sample is loaded watch the scatter plots to make sure events are appearing in the R1 bead gate. Initially there can be some instability in the sample pressure causing drift in the scatter plots. Wait for this to stabilize.</li>
	<li>Once stabilized, click on acquire and allow sample to run. Once the cytometer has finished acquiring 10,000 bead events in R1 the cytometer will stop acquiring and save all data.</li>
	<li>Remove the sample and discard flow tube. The cytometer is now ready for the next sample. Run all samples and then proceed to analysis mode.</li>
</ol>
6. Immune Cell Analysis
Note: Gating strategies and Boolean algebra are used to define each cell population. Boolean algebra is a logic based analysis method that allows for multiple operations in a single definition. The analysis software of the flow cytometer (e.g., BD CELLQuest) allows for the use of Boolean algebra. The equations are used to actively account for the significant negative reactivity that assists in defining the cell to more specifically identify each cell population. &#8216;Regions&#8217; are used to define a &#8216;gate&#8217;. Regions define a 2 dimensional space whereas gates can be composed of numerous regions connected by algebraic operators (+, *,-, defined in Table 2).
<ol>
	<li>Switch the software to analysis mode. An analysis template should be generated to match Figure 5 with the plots and gates shown.</li>
	<li>Analyze each individual file (i.e., each individual sample) separately. Set up gates R1 through to R9 and then set up the algorithms for each cell type as defined in Table 2 (also shown in Figure 5) .</li>
	<li>Use the cell statistics counter to calculate individual cell populations defined by gates and algorithms (Table 2 and Figure 5). The algorithms will adjust cell numbers automatically in the cell statistics counter.</li>
	<li>Calculate cell subsets using the following equation: 
	<img alt="Equation 1" src="/files/ftp_upload/53019/53019eq1.jpg" /> 
	Note: Number of cell events counted (e.g., CD4 T cell events) is enumerated using the above equation to give cell number per &#181;l of blood. Examples are shown in Figure 5.</li>
</ol>

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The authors have nothing to disclose.

This paper details a method for the generation of a rat syngeneic orthotopic model of pleural mesothelioma and a simple method for monitoring disease progression through longitudinal blood sampling.
The II-45 model was developed by exposing Fischer 344 rats to asbestos fibers13. Although this exposure represents the true dynamics of host-asbestos-immune system interactions for mesothelioma pathogenesis, it has a long lag time (taking years to generate) and can be dangerous for the r...

Malignant mesothelioma (MM) is an aggressive malignancy which arises from transformed cells in the membrane (mesothelium) that lines the lung and abdominal cavities, heart and internal reproductive organs, and is the most common primary tumor of the lung cavity or pleura1,2. Exposure to asbestos fibres accounts for 80% of all MM, and while bans on asbestos use were introduced decades ago in most western countries, its widespread use in the community has left a deadly legacy. The World Health Organization has estimated that 107,000 people worldwide die each year from asbestos related diseases, with mortality rates continuing to increase. A new non-occupational incidence wave is also emerging and there is little understanding of when, and at what level this will peak3.
The majority of people with MM are diagnosed late when systemic chemotherapy represents one of the only viable options4. The most effective chemotherapy and current &#8216;standard of care&#8217; (pemetrexed together with cisplatin5) was identified over 10 years ago. However failure of this treatment is inevitable and there are no proven second line options, leaving patients with a grim prognosis and median survival of only 12 months2. Therefore, there is an urgent unmet need for more effective treatments. Despite the examination of a number of novel therapies in clinical trials none has resulted in changes in practice. This is due in part to the low (5%) transference of pre-clinical results, generally performed in xenograft mouse models, to the clinical setting6-8. Such models do not faithfully recapitulate the complex aspects of the tumor microenvironment occurring in non-physiological locations, frequently in the absence of a functioning immune system9.
Syngeneic orthotopic models create a significantly more realistic tumor environment than the commonly used subcutaneous xenograft models as the tumors occur in the correct physiological location with an intact immune system10,11. The larger size of the rat enhances its use as a rodent disease model, especially in drug studies where serial blood draws are required to assess treatment response and toxicity12. Furthermore, in models in which monitoring disease progression is difficult due to the location of the tumors (such as in the pleural cavity), the ability to monitor disease progression using factors found in the circulation is extremely attractive. The generation of a syngeneic orthotopic model of pleural mesothelioma using immune-competent rats is described. In addition, an easy and relatively non-invasive method for monitoring pleural disease progression by measuring circulating immune cells is also described.

<table><tbody><tr><td>EDTA Collection tube (0.5 ml)</td><td>Greiner Bio One GmbH</td><td>450480</td><td></td></tr><tr><td>Rat T/B/NK Cell cocktail</td><td>BD Pharmingen</td><td>558509</td><td>anti-Rat CD3&amp;nbsp; APC (IgM clone 1F4), anti-Rat CD45RA Fitc (IgG1 clone OX-33), anti-Rat CD161a PE (IgG1 Clone 10/78)</td></tr><tr><td>anti-RAT CD8a PE&amp;nbsp;</td><td>Biolegend</td><td>200608</td><td>(IgG1vClone G28)</td></tr><tr><td>anti-Rat CD4 FITC (Domain 1)&amp;nbsp;</td><td>Biolegend</td><td>203406</td><td>(IgG&lt;sub&gt;1&lt;/sub&gt; Clone OX-38)</td></tr><tr><td>anti-Rat CD45 PE/Cy7&amp;nbsp;</td><td>Biolegend</td><td>202214</td><td>(IgG&lt;sub&gt;1 &lt;/sub&gt;Clone OX-1)</td></tr><tr><td>TruCount Tubes</td><td>Becton Dickinson</td><td>340334</td><td>Box of 50 absolute counting tubes</td></tr><tr><td>RPMI 1640 media</td><td>Life Technologies</td><td>11875-119</td><td></td></tr><tr><td>foetal bovine serum (FBS)</td><td>Scientifix</td><td>FBS500-S (lot# 010101-1)</td><td></td></tr><tr><td>trypsin-EDTA</td><td>Life Technologies</td><td>15400-054</td><td></td></tr><tr><td>PBS tablets</td><td>Medicago AB</td><td>09-9400-100</td><td></td></tr><tr><td>23Gx1&amp;frac14; Needle</td><td>Becton Dickinson</td><td>302008</td><td></td></tr><tr><td>1 ml Syringe</td><td>Becton Dickinson</td><td>302 100</td><td></td></tr><tr><td>Fischer 344 Rat</td><td>Animal Resources Centre, Perth Australia</td><td>F344</td><td></td></tr><tr><td>I.S.O (Isoflurane USP)</td><td>Veterinary Companys Australia (VCA)&amp;nbsp;</td><td>B7058</td><td></td></tr><tr><td>II-45 Rat Mesothelioma line</td><td>Zurich University</td><td></td><td>Note: The cell line was given as a gift and is not commercially available at the ATCC</td></tr><tr><td>FACSCalibur 4 color</td><td>Becton Dickinson</td><td>342975</td><td></td></tr><tr><td>TRIS-HCL</td><td>SIGMA</td><td>T3253</td><td></td></tr><tr><td>Ammonium Chloride</td><td>SIGMA</td><td>9718</td><td></td></tr><tr><td>Anaesthetic Machine (The stinger)</td><td>Advanced Anaesthesia specialists</td><td>#00449</td><td></td></tr></tbody></table>

orthotopic implantation and peripheral immune cell monitoring in the ii-45 syngeneic rat mesothelioma model

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of the role of the tumor microenvironment in treatment response, collectively point to the need for immune-competent orthotopic models for pre-clinical testing of these new therapies. This paper demonstrates how to establish an orthotopic immune-competent rat model of pleural malignant mesothelioma. Monitoring disease progression in orthotopic models is confounded by the internal location of the tumors. To longitudinally monitor disease progression and its effect on circulating immune cells in this and other rat models of cancer, a single tube flow cytometry assay requiring only 25 &#181;l whole blood is described. This provides accurate quantification of seven immune parameters: total lymphocytes, monocytes and neutrophils, as well as the T-cell subsets CD4 and CD8, B-cells and Natural Killer cells. Different subsets of these parameters are useful in different circumstances and models, with the neutrophil to lymphocyte ratio having the greatest utility for monitoring disease progression in the mesothelioma model. Analyzing circulating immune cell levels using this single tube method may also assist in monitoring the response to immune-based treatments and understanding the underlying mechanisms leading to success or failure of treatment.

The method used in this paper for the generation of an orthotopic model of pleural mesothelioma using II-45 cells resulted in animals succumbing to mesothelioma in a reproducible and rapid timeframe, with no rats dying due to the implantation method. Titration of the number of cells implanted determined that 1x 103 cells was the minimum number required for a fully penetrant model (100% engraftment). The different number of cells implanted in the rats changed the time course of the disease without appearing to ...

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Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model

The generation of an orthotopic rat model of pleural malignant mesothelioma by implantation of II-45 mesothelioma cells into the pleural cavity of immune competent rats is presented. A flow cytometric method to analyze seven immune cell subsets in these animals from a 25 &#181;l&#160;blood sample is also described.

The enormous upsurge of interest in immune-based treatments for cancer such as vaccines and immune checkpoint inhibitors, and increased understanding of ...

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9.1K Views.  Royal North Shore Hospital, University of Sydney. The generation of an orthotopic rat model of pleural malignant mesothelioma by implantation of II-45 mesothelioma cells into the pleural cavity of immune competent rats is presented. A flow cytometric method to analyze seven immune cell subsets in these animals from a 25 µl blood sample is also described.

Video: Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or bioartificial scaffold materials, entrapment of cardiac myocytes in hydrogels such as fibrin or collagen and stacking of myocyte monolayers 1. These concepts aim at restoration of compromised cardiac function (e.g. after myocardial infarction) or as experimental models (e.g. predictive toxicology and substance screening or disease modelling). Precise monitoring of cell survival after implantation of engineered heart tissue (EHT) has now become possible using in-vivo bioluminescence imaging (BLI) techniques 2. Here we describe the generation of fibrin-based EHT from a transgenic rat strain with ubiquitous expression of firefly luciferase (ROSA/luciferase-LEW Tg; 3). Implantation is performed into the greater omentum of different rat strains to assess immune responses of the recipient organism following EHT implantation. Comparison of results generated by BLI and the Enzyme Linked Immuno Spot Technique (ELISPOT) confirm the usability of BLI for the assessment of immune responses.

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue EHT

This video demonstrates the use of in vivo bioluminescence imaging to study immune responses after implantation of Engineered Heart Tissue (EHT) in rats.

Various techniques of cardiac tissue engineering have been pursued in the past decades including scaffolding strategies using either native or ...

Bioengineering

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all gynecologic malignancies among US women 1,2,3. Serous tumors are the most widespread forms of ovarian cancer and 4,5 the Tg-MISIIR-TAg transgenic represents the only mouse model that spontaneously develops this type of tumors. Tg-MISIIR-TAg mice express SV40 transforming region under control of the Mullerian Inhibitory Substance type II Receptor (MISIIR) gene promoter 6. Additional transgenic lines have been identified that express the SV40 TAg transgene, but do not develop ovarian tumors. Non-tumor prone mice exhibit typical lifespan for C57Bl/6 mice and are fertile. These mice can be used as syngeneic allograft recipients for tumor cells isolated from Tg-MISIIR-TAg-DR26 mice.
Objective: Although tumor imaging is possible 7, early detection of deep tumors is challenging in small living animals. To enable preclinical studies in an immunologically intact animal model for serous ovarian cancer, we describe a syngeneic mouse model for this type of ovarian cancer that permits in vivo imaging, studies of the tumor microenvironment and tumor immune responses.
Methods: We first derived a TAg+ mouse cancer cell line (MOV1) from a spontaneous ovarian tumor harvested in a 26 week-old DR26 Tg-MISIIR-TAg female. Then, we stably transduced MOV1 cells with TurboFP635 Lentivirus mammalian vector that encodes Katushka, a far-red mutant of the red fluorescent protein from sea anemone Entacmaea quadricolor with excitation/emission maxima at 588/635 nm 8,9,10. We orthotopically implanted MOV1Kat in the ovary 11,12,13,14 of non-tumor prone Tg-MISIIR-TAg female mice. Tumor progression was followed by in vivo optical imaging and tumor microenvironment was analyzed by immunohistochemistry.
Results: Orthotopically implanted MOV1Kat cells developed serous ovarian tumors. MOV1Kat tumors could be visualized by in vivo imaging up to three weeks after implantation (fig. 1) and were infiltrated with leukocytes, as observed in human ovarian cancers 15 (fig. 2).
Conclusions: We describe an orthotopic model of ovarian cancer suitable for in vivo imaging of early tumors due to the high pH-stability and photostability of Katushka in deep tissues. We propose the use of this novel syngeneic model of serous ovarian cancer for in vivo imaging studies and monitoring of tumor immune responses and immunotherapies.

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

To study in vivo tumor growth and tumor microenvironment, we used a syngeneic and orthotopic mouse model of ovarian cancer in immunocompetent animals. We transduced a mouse tumor cell line (MOV1) with Katushka fluorescent protein (MOV1KAT) and here we show its orthotopic implantation in ovary and in vivo imaging.

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

Immunology and Infection

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

Orthotopic Transplantation of Syngeneic Lung Adenocarcinoma Cells to Study PD-L1 Expression

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, including genetically engineered models as well as transplantation models. However, genetically engineered mouse models are time-consuming and expensive, whereas some orthotopic transplantation models are difficult to reproduce. Here, a non-invasive intratracheal delivery method of lung tumor cells as an alternative orthotopic transplantation model is described. The use of mouse lung adenocarcinoma cells and syngeneic graft recipients allows studying tumorigenesis under the presence of a fully active immune system. Furthermore, genetic manipulations of tumor cells before transplantation makes this model an attractive time-saving approach to study the impact of genetic factors on tumor growth and tumor cell gene expression profiles under physiological conditions. Using this model, we show that lung adenocarcinoma cells express increased levels of the T-cell suppressor programmed death-ligand 1 (PD-L1) when grown in their natural environment as compared to cultivation in vitro.

Here we describe a minimally invasive syngeneic orthotopic transplantation model of mouse lung adenocarcinoma cells as a time- and cost-reducing model to study non-small cell lung cancer.

The use of mouse models is indispensable for studying the pathophysiology of various diseases. With respect to lung cancer, several models are available, ...

Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop better therapeutic strategies, understanding tumor microenvironmental factors that contribute to the treatment resistance is important. A major impediment to understanding disease mechanisms and improving therapy has been a lack of murine cell lines that resemble the aggressive and metastatic nature of human HNSCCs. Furthermore, a majority of murine models employ subcutaneous implantations of tumors which lack important physiological features of the head and neck region, including high vascular density, extensive lymphatic vasculature, and resident mucosal flora. The purpose of this study is to develop and characterize an orthotopic model of HNSCC. We employ two genetically distinct murine cell lines and established tumors in the buccal mucosa of mice. We optimize collagenase-based tumor digestion methods for the optimal recovery of single cells from established tumors. The data presented here show that mice develop highly vascularized tumors that metastasize to regional lymph nodes. Single-cell multiparametric mass cytometry analysis shows the presence of diverse immune populations with myeloid cells representing the majority of all immune cells. The model proposed in this study has applications in cancer biology, tumor immunology, and preclinical development of novel therapeutics. The resemblance of the orthotopic model to clinical features of human disease will provide a tool for enhanced translation and improved patient outcomes.

Here we present a reproducible method for developing an orthotopic murine model of head and neck squamous cell carcinoma. Tumors demonstrate clinically relevant histopathological features of the disease, including necrosis, poor differentiation, nodal metastases, and immune infiltration. Tumor-bearing mice develop clinically relevant symptoms including dysphagia, jaw displacement, and weight loss.

Head and neck squamous cell carcinoma (HNSCC) is a debilitating and deadly disease with a high prevalence of recurrence and treatment failure. To develop ...

A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate

The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival compared to surgery alone. To address this problem, the aim of the study is to refine a lesser-known model of OS in rats with a comprehensive histologic, imaging, biologic, implantation, and amputation surgical approach that prolongs survival. We used an immunocompetent, outbred Sprague-Dawley (SD), syngeneic rat model with implanted UMR106 OS cell line (originating from a SD rat) with orthotopic tibial tumor implants into 3-week-old male and female rats to model pediatric OS. We found that rats develop reproducible primary and metastatic pulmonary tumors, and that limb amputations at 3 weeks post implantation significantly reduce the incidence of pulmonary metastasis and prevent unexpected deaths. Histologically, the primary and metastatic OSs in rats were very similar to human OS. Using immunohistochemistry methods, the study shows that rat OS are infiltrated with macrophages and T cells. A protein expression survey of OS cells reveals that these tumors express ErbB family kinases. Since these kinases are also highly expressed in most human OSs, this rat model could be used to test ErbB pathway inhibitors for therapy.

Here, a syngeneic orthotopic implantation followed by an amputation procedure of the osteosarcoma with spontaneous pulmonary metastasis that can be used for preclinical investigation of metastasis biology and development of novel therapeutics is described.

The most recent advance in the treatment of osteosarcoma (OS) occurred in the 1980s when multi-agent chemotherapy was shown to improve overall survival ...

An Immunocompetent Murine Model for Laser Interstitial Thermal Therapy of Glioblastoma

Glioblastoma (GB), the most aggressive form of primary brain cancer, accounts for approximately half of all high-grade primary brain tumors in adults and has no cure. Laser interstitial thermal therapy (LITT) is a Food and Drug Administration (FDA)-approved treatment for GB and is used in patients who may not be candidates for conventional surgical resection. While the clinical efficacy of LITT has been established, research beyond clinical case studies and case series is limited and hindered by the lack of an established animal model. This protocol uses C57BL/6 mice and syngeneic CT2A glioma cancer cell line to closely recapitulate human GB&#160;while also using a 1064 nm Neodymium-doped Yttrium Aluminum Garnet (Nd:YAG) laser, such as is used in one of the two FDA-approved LITT systems, providing excellent pre-clinical relevance. The successful establishment of this LITT murine model will provide a valuable platform for investigating the unique features of LITT ablation and its effects on the tumor microenvironment, potentially leading to improved therapeutic strategies.

Glioblastoma is a devastating form of primary brain cancer, and laser interstitial thermal therapy is emerging as a promising alternative to conventional surgical resection for inoperable glioblastoma. This protocol describes an optimized pre-clinical mouse model that can be used to study treatment effects or adjuvant and combinatorial treatments.

Glioblastoma (GB), the most aggressive form of primary brain cancer, accounts for approximately half of all high-grade primary brain tumors in adults and ...

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms requires reliable models. Here, we describe models of tolerance to cardiac allograft in rat, induced by blockade of costimulation signals or by upregulation of immunoregulatory molecules through gene transfer. Each of these models allowed in vivo generation of regulatory cells such as regulatory T cells (Tregs), regulatory B cells (Bregs) or regulatory myeloid cells (RegMCs). In this manuscript, we describe two complementary protocols that have been used to identify and define in vitro and in vivo regulatory cell activity to determine their responsibility in tolerance induction and maintenance. First, an in vitro suppressive assay allowed rapid identification of cells with suppressive capacity on effector immune responses in a dose dependent manner, and can be used for further analysis such as cytokine measurement or cytotoxicity. Second, the adoptive transfer of cells from a tolerant treated recipient to a newly irradiated grafted recipient, highlighted the tolerogenic properties of these cells in controlling graft directed immune responses and/or converting new regulatory cells (termed infectious tolerance). These methods are not restricted to cells with known phenotypic markers and can be extended to any cell population. Furthermore, donor directed allospecificity of regulatory cells (an important goal in the field) can be assessed by using third party donor cells or graft either in vitro or in vivo. Finally, to determine the specific tolerogenic capacity of these regulatory cells, we provide protocols to assess the humoral anti-donor antibody responses and the capacity of the recipient to develop humoral responses against new or former known antigens. The models of tolerance described can be used to further characterize regulatory cells, to identify new biomarkers, and immunoregulatory molecules, and are adaptable to other transplantation models or autoimmune diseases in rodent or human.

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Here, we present a protocol to induce tolerance in transplantation, and assess in vitro and in vivo the suppressive capacity of distinct cell subsets from the recipient and the immune status of the recipient toward donor or exogenous antigens.

The main concern in transplantation is to achieve specific tolerance through induction of regulatory cells. The understanding of tolerance mechanisms ...

Rat Mesentery Angiogenesis Assay

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in vivo [see review papers], which is the dominating form of angiogenesis in human tumors and non-tumor 
tissues, as discussed in invited review papers1,2. Angiogenesis induced in the membranous mesenteric parts by intraperitoneal (i.p.) injection of a pro-angiogenic factor can be modulated
by subcutaneous (s.c.), intravenous (i.v.) or oral (p.o.) treatment with modifying agents of choice. Each membranous part of the mesentery is 
translucent and framed by fatty tissue, giving it a window-like appearance.
The assay has the following advantageous features: (i) the test tissue is natively vascularized, albeit 
sparsely, and since it is extremely thin, the microvessel network is virtually two-dimensional, which allows the entire network 
to be assessed microscopically in situ; (ii) in adult rats the test tissue lacks significant physiologic angiogenesis, which characterizes
 most normal adult mammalian tissues; the degree of native vascularization is, however, correlated with age, as discussed in1; 
(iii) the negligible level of trauma-induced angiogenesis ensures high sensitivity; (iv) the 
assay replicates the clinical situation, as the angiogenesis-modulating test drugs are administered systemically and the responses 
observed reflect the net effect of all the metabolic, cellular, and molecular alterations induced by the treatment; (v) the assay 
allows assessments of objective, quantitative, unbiased variables of microvascular spatial extension, density, and network pattern 
formation, as well as of capillary sprouting, thereby enabling robust statistical analyses of the dose-effect and molecular 
structure-activity relationships; and (vi) the assay reveals with high sensitivity the toxic or harmful effects of treatments in 
terms of decreased rate of physiologic body-weight gain, as adult rats grow robustly.
Mast-cell-mediated angiogenesis was first demonstrated using this assay3,4. The model demonstrates a high level of 
discrimination regarding dosage-effect relationships and the measured effects of systemically administered chemically or functionally closely related drugs and proteins, 
including: (i) low-dosage, metronomically administered standard chemotherapeutics that yield diverse, drug-specific effects (i.e., 
angiogenesis-suppressive, neutral or angiogenesis-stimulating activities5); (ii) natural iron-unsaturated human lactoferrin, which stimulates 
VEGF-A-mediated angiogenesis6, and natural iron-unsaturated bovine lactoferrin, which inhibits VEGF-A-mediated angiogenesis7; and (iii) 
low-molecular-weight heparin fractions produced by various means8,9. Moreover, the assay is highly suited to studies of the combined 
effects on angiogenesis of agents that are administered systemically in a concurrent or sequential fashion.
The idea of making this video originated from the late Dr. Judah Folkman when he visited our laboratory and witnessed the methodology being demonstrated.
Review papers (invited) discussing and appraising the assay
Norrby, K. In vivo models of angiogenesis. J. Cell. Mol. Med. 10, 588-612 (2006).
Norrby, K. Drug testing with angiogenesis models. Expert Opin. Drug. Discov. 3, 533-549 (2008).

Normal adult vascularized mammalian tissue that lacks physiologic angiogenesis and that has not been exposed to surgical intervention is used to study: (i) the initiation and development of angiogenesis following intraperitoneal administration of test agents; and (ii) modification of angiogenesis following systemic administration of selected test agents.

The adult rat mesentery window angiogenesis assay is biologically appropriate and is exceptionally well suited to the 
study of sprouting angiogenesis in ...

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. MPM development is mainly associated with asbestos. Treatments provide only modest survival since the median survival average is 9&#8211;18 months from the time of diagnosis. Therefore, more effective treatments must be identified. Most data describing new therapeutic targets were obtained from in vitro experiments and need to be validated in reliable in vivo preclinical models. This article describes one such reliable MPM orthotopic model obtained after injection of a human MPM cell line H2052/484 into the pleural cavity of immunodeficient athymic mice. Transplantation in the orthotopic site allows studying the progression of tumor in the natural in vivo environment. Positron emission tomography/computed tomography (PET/CT) molecular imaging using the clinical [18F]-2-fluoro-2-deoxy-D-glucose ([18F]FDG) radiotracer is the diagnosis method of choice for examining patients with MPM. Accordingly, [18F]FDG-PET/CT was used to longitudinally monitor the disease progression of the H2052/484 orthotopic model. This technique has a high 3R potential (Reduce the number of animals, Refine to lessen pain and discomfort, and Replace animal experimentation with alternatives) since the tumor development can be monitored non-invasively and the number of animals required could be significantly reduced.
This model displays a high development rate, a rapid tumor growth, is cost-efficient and allows for rapid clinical translation. By using this orthotopic xenograft MPM model, researchers can assess biological responses of a reliable MPM model following therapeutic interventions.

This article describes the generation of an orthotopic mouse model of human pleural mesothelioma by implantation of H2052/484 mesothelioma cells into the pleural cavity of immunocompromised athymic mice. The longitudinal monitoring of the development of intrapleural tumors was assessed by non-invasive multimodal [18F]-2-fluoro-2-deoxy-D-glucose positron emission tomography and computed tomography imaging.

Malignant pleural mesothelioma (MPM) is a rare and aggressive tumor arising in the mesothelium that covers the lungs, the heart, and the thoracic cavity. ...

Orthotopic Implantation and Peripheral Immune Cell Monitoring in the II-45 Syngeneic Rat Mesothelioma Model

Summary

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Chapters in this video

Bioluminescence Imaging for Assessment of Immune Responses Following Implantation of Engineered Heart Tissue (EHT)

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

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

Orthotopic Transplantation of Syngeneic Lung Adenocarcinoma Cells to Study PD-L1 Expression

Intramucosal Inoculation of Squamous Cell Carcinoma Cells in Mice for Tumor Immune Profiling and Treatment Response Assessment

A Syngeneic Orthotopic Osteosarcoma Sprague Dawley Rat Model with Amputation to Control Metastasis Rate

An Immunocompetent Murine Model for Laser Interstitial Thermal Therapy of Glioblastoma

In Vitro and In Vivo Assessment of T, B and Myeloid Cells Suppressive Activity and Humoral Responses from Transplant Recipients

Rat Mesentery Angiogenesis Assay

Implantation and Monitoring by PET/CT of an Orthotopic Model of Human Pleural Mesothelioma in Athymic Mice