This work was supported by an unrestricted grant from the Novo Nordisk Center for Basic Metabolic Research and the Lundbeck Foundation.

All methods described were conducted in accordance with the guidelines of Danish legislation governing animal experimentation (1987). Studies were performed with the permission from the Danish Animal Experiments Inspectorate (2013-15-2934-00833) and the local ethical committee.
NOTE: Female C57BL/6J mice (~20&#8722;25 g) were obtained and housed eight per cage in standard 12 h light, 12 h dark cycle with free access to water and standard chow. Animals were left to acclimatize for one week before experiments began.
1. Induction of mucositis using 5-fluorouracil
<ol>
	<li>Obtain 5-fluorouracil (5-FU) in a 50 mg/mL solution.</li>
	<li>Weigh and record the body weight of the mice. From the body weight, calculate the amount of 5-FU for injection (e.g., 400 mg/kg).</li>
	<li>Prepare a 1 mL syringe connected to a 27 G x 30 mm needle and fill the syringe with the calculated amount of 5-FU for injection.</li>
	<li>Restrain the mouse by the scruff method. To do this, grab the base of the tail with one hand and place it on a toe-gripping surface, such as a wire bar lid. While positioning the tail with one hand, hold the scruff of the neck with the other hand.</li>
	<li>Firmly position the body of the mouse across one hand by extending the forefinger and thumb back as far as possible. Place the tail between the fingers of this same hand to secure the mouse.</li>
	<li>While maintaining the mouse in a firm but gentle grip, expose the ventral side of the mouse and insert the needle into the intraperitoneal cavity on the lower right or left quadrant of the abdomen. Aspirate to ensure proper placement and inject the 400 mg/kg 5-FU.</li>
</ol>
2. Tissue collection
<ol>
	<li>Anesthetize the mouse with an intraperitoneal injection of ketamine/xylazine (100/10 mg/kg) diluted in saline solution (0.9% NaCl). Monitor the depth of anaesthesia by observing the pinch withdrawal reflex. 
	NOTE: The anaesthesia is a non-recovery anaesthesia.</li>
	<li>Record the weight of the anesthetized mouse.</li>
	<li>Place the mouse in a supine position and perform a laparotomy to expose the abdominal cavity, followed by an incision of the chest cavity to introduce pneumothorax.</li>
	<li>Using scissors, sacrifice the animal by cutting open the diaphragm.</li>
	<li>Remove the small intestine. Cut superior to the pylorus, carefully retract the small intestine away from the carcass until the cecum is reached and make a cut just before the cecum.</li>
	<li>Using forceps, gently clamp the proximal lumen shut. Using a 1 mL syringe with a 25 G needle attached, flush the small intestine with saline to remove the feces.</li>
	<li>Gently remove the saline from the intestinal lumen with surgical instruments.</li>
	<li>Place the small intestine on clean blotting paper to carefully remove excess saline.</li>
	<li>Record the weight of the flushed tissue.</li>
	<li>Calculate the flushed small intestinal weight as a percentage of body weight.</li>
</ol>
3. Small intestine histology
<ol>
	<li>Tissue preparation
	<ol>
		<li>In a fume hood, use scissors to cut 3 cm sections of the flushed intestinal tissue from each mouse from the duodenum, jejunum and ileum and fixate sections in 10% formalin for 24 h at room temperature. 
		NOTE: The fixative volume should be 5&#8722;10 times of the tissue volume.</li>
		<li>Transfer the fixed tissue to a cutting board.</li>
		<li>Using a scalpel, trim the tissue to approximately 1 cm and transfer into an embedding cassette. 
		NOTE: The cassette should be labelled with the sample identification (ID) in pencil.</li>
		<li>Submerge the cassette in 70% ethanol and store at 4 &#176;C until further processing.</li>
	</ol>
	</li>
	<li>Tissue embedding in paraffin
	<ol>
		<li>Dehydrate the tissue by placing in ascending alcohol solutions. Place the cassette in 70% ethanol for 1 h, followed by 80% ethanol for 1 h, 95% ethanol for 1 h and 100% ethanol for 1.5 h. Transfer the cassette from 100% ethanol to xylene for 1.5 h.</li>
		<li>Immerse the cassette with paraffin wax heated between 58&#8722;60 &#176;C. Use forceps to position the tissue in a transverse orientation and leave the blocks to cool more than 24 h until solid.</li>
	</ol>
	</li>
	<li>Cutting tissue using a microtome
	<ol>
		<li>Place block, tissue face down on ice for 5 min. Using a microtome, trim the paraffin blocks at a thickness of 10&#8722;30 &#181;m to expose the tissue surface.</li>
		<li>Cut cross-sections of the tissue block between 3&#8722;5 &#181;m, making transverse sections from the duodenum, the jejunum and the ileum.</li>
		<li>Place paraffin ribbon in a water bath set between 40&#8722;45 &#176;C. Use forceps to separate the sections.</li>
		<li>Use microscope slides to pick up the sections from the water.</li>
		<li>Air dry the slides for 30 min before staining. 
		NOTE: For storage over a longer period of time, store slides in a fridge.</li>
	</ol>
	</li>
	<li>Hematoxylin-eosin staining of tissue
	<ol>
		<li>Place the microscope slides in a histology cradle. Deparaffinize the slides in a heating cabinet set at 60 &#176;C for 60 min. 
		NOTE: Slides directly from the fridge should be left to reach room temperature before placing them in a heating cabinet.</li>
		<li>In the fume hood, rehydrate the tissue in 3 changes of a clearing agent (Table of Materials), make 20 dips in the first clearing agent jar and then let them stand for 7&#8722;8 min in each of the two additional clearing agent jars. Transfer the slides to descending alcohol solutions, start with 3 changes of 99% ethanol, followed by 2 changes of 96% ethanol and 70% ethanol. Make a minimum of 20 dips in each jar. Transfer to running water and let the slides stand for 5 min.</li>
		<li>Immerse the slides in filtered Meyer&#8217;s hematoxylin for 1 min.</li>
		<li>Wash the samples in running tap water for 5 min.</li>
		<li>Immerse sections in the eosin stain for 1&#8722;2 min, then rinse in tap water.</li>
		<li>Dehydrate the tissue by placing in ascending alcohol solutions. Start with 70% ethanol, followed by 96% ethanol, 96% ethanol, and 4 times 99% ethanol. Make a minimum of 20 dips in each jar, and let the cradle stand in 99% ethanol until they are mounted.</li>
		<li>Mount coverslips by applying a small amount of the mounting medium to the surface of the slides. Put the coverslip on top of the mounting medium without creating bubbles under the coverslip. Let it dry for 24 h.</li>
		<li>Examine the tissue using a light microscope connected to a camera to obtain histological photos. Use a 10x objective to take snapshots until a full coverage of the tissue slide is reached.</li>
	</ol>
	</li>
</ol>
4. Measurement of crypt depth and/or villus height
<ol>
	<li>Download and install the analysis software (i.e., Zen Lite, Table of Materials).</li>
	<li>Open the image in the software and connect to the camera. Take snapshots in camera mode with 20x objective.</li>
	<li>In processing mode, open the snapshot.</li>
	<li>To measure the crypt depth: in the 2D view, select the line tool from the graphics tab. Choose a well-orientated crypt and start the line at the bottom of a villus and finish at the bottom of the crypt. Repeat this action for 20 well-orientated crypts (Supplementary Figure 1).</li>
	<li>To measure the villus height: in the 2D view, select the line tool from the graphics tab. Choose a well-orientated villus and start the line at the end of the luminal projection and finish at the start of the crypt. Repeat this action for 20 well-orientated villi (Supplementary Figure 1).</li>
	<li>Switch from 2D view to the Measure view to display the measurements.</li>
	<li>Export the measurements and calculate the average of the crypt depth and villus height.</li>
</ol>
5. BrdU quantification (proliferation) by immunohistochemistry
<ol>
	<li>Injection of the BrdU solution
	<ol>
		<li>Weigh and record the body weight of the mice.</li>
		<li>In a fume hood, prepare a representative amount of 0.5% w/v bromodeoxyuridine (BrdU) solution in phosphate buffered saline (PBS).</li>
		<li>In the fume hood, inject 50 mg/kg of BrdU by intraperitoneal injection. 
		NOTE: To ensure that each mouse is euthanized at exactly 150 min post BrdU injection, inject each individual mouse in succession with 10&#8722;20 min interval in between in order to properly perform the tissue collection.</li>
		<li>Record the time of injection.</li>
		<li>Wait 150 min, then anesthetize mice and collect the tissue as described in steps 2.1&#8722;2.7. Perform BrdU immunohistochemistry as described in section 5.2.</li>
	</ol>
	</li>
	<li>BrdU immunohistochemistry 
	<ol>
		<li>Prepare the tissue as described in steps 3.1.1&#8722;3.3.4.</li>
		<li>For antigen retrieval, place a slide cradle with the sections in a glass histology jar and fill it with the EDTA buffer (pH 9) in a microwave oven for 1 min at 750 W followed by 9 min at 350 W. Repeat cycle. 
		NOTE: Ensure that the tissue does not dry out, which might require adding more buffer to the jars between heating.</li>
		<li>Wash sections 2&#8722;3 times in tris-buffered saline and polysorbate 20 (TBS-T) buffer for 3 min each.</li>
		<li>Apply 5 drops of peroxide block (Table of Materials) for 10&#8722;15 min at room temperature to block endogenous peroxidase activity. Wash sections 2&#8722;3 times in TBS-T buffer for 3 min each.</li>
		<li>Apply 5 drops of rodent block buffer (Table of Materials) for 30 min at room temperature, to block non-specific binding. Wash sections 2&#8722;3 times in TBS-T buffer for 3 min each.</li>
		<li>Incubate the sections with a monoclonal mouse anti-BrdU antibody diluted 1:500 for 1 h at room temperature. Wash sections 2&#8722;3 times in TBS-T buffer for 3 min each.</li>
		<li>Visualize immunopositivity using 3,3&#8217;-diaminobenzidine (DAB) by applying 5 drops of horseradish peroxidase to each slide and incubate for 15 min at room temperature. Transfer the sections to a fume hood, add 150 &#181;L of DAB and incubate for 5 min. 
		NOTE: Always wear gloves when handling DAB and dispose of waste appropriately.</li>
		<li>Rinse sections in deionized water.</li>
		<li>Dehydrate the tissue and mount a coverslip as described in steps 3.4.6 and 3.4.7.</li>
	</ol>
	</li>
	<li>Visualization of the immunoreactions
	<ol>
		<li>Visualize the tissue sample with a light microscope connected to a camera.</li>
		<li>Use the analysis software to obtain microscope images using a 20x objective of all sections and save files in the .JPG format.</li>
		<li>Take a snapshot of a stage micrometer using a 20x objective, which will be used as a calibration tool in ImageJ software.</li>
	</ol>
	</li>
	<li>Measuring the area of BrdU immunoreactive cells 
	<ol>
		<li>Install ImageJ software (Table of Materials).</li>
		<li>Assigning scale to an image in ImageJ: Open the stage micrometer image using the ImageJ File | Open menu command. Select the straight-line selection tool.</li>
		<li>Select the straight tool and draw a straight line on the micrometer image to define a known distance.</li>
		<li>Select Set Scale under the Analyse menu.</li>
		<li>Enter a value in the Known Distance box and define the unit of length in the Unit of Length box. Select Global so that this calibration applies to all images that are opened in this ImageJ session.</li>
		<li>Open an image of interest by using the ImageJ File | Open menu command to measure the area of the BrdU immunoreactive cells per crypt.</li>
		<li>Set the color graphics to 8-bit under the Image/Type menu.</li>
		<li>Increase the contrast of the image under Process/Enhance contrast and set the saturated pixels to 0.4%.</li>
		<li>Apply a threshold to the image to segment the immunopositivity. Select Image/Adjust under menu, then threshold and select the color Red (dark area shown in red). Choose a threshold of around 10&#8722;20% by moving the threshold bar. 
		NOTE: Choose a threshold that includes all the BrdU positive cells with as little background as possible. The chosen percentage should apply to all sections.</li>
		<li>Choose a well-orientated crypt, i.e., a complete crypt from intact tissue, and select the free hand selection tool to mark the area around it.</li>
		<li>To measure the threshold area, select the Analyse tab, followed by Analyse particles. In the Analyse particle window, set Size to 20-infinity, click the box pixel units, set Circularity to 0.00&#8722;1.00, and set Show to Outlines. Click boxes Display Results, Summarize, and Include holes. 
		NOTE: The values for BrdU positive cells are automatically generated in a result window, showing the individual measurements for each BrdU positive cell. Furthermore, a summary window is automatically generated, showing the number of counts, total area, average size and percentage of area with BrdU positive cells.</li>
		<li>Repeat steps 5.4.10 and 5.4.11 to measure a new crypt. The results of all measured crypts will be displayed in the Summary window.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

Here, we demonstrate a widely accessible method to study SI injury and regeneration in a mouse model. A wide variety of preclinical animal models of intestinal injury exist, but it is vital we understand that each model is unique and that the endpoints must be appropriate to answer the research question. This model is excellent to study adaptive response to injury, but the endpoints should be modified when using the model as a pre-clinical model of mucositis. However, translation from animal models to patients is challen...

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to disease or surgery1,2. After trauma, the gut undergoes a morphometric and functional adaptive response, characterized by crypt cell proliferation and increased nutrient absorption3. This step is critical in recovery, yet poorly understood. Experimental studies of the intestinal adaptive response have focused on the changes occurring after small bowel resection in mice, rats, and pigs, but understanding the molecular mechanism behind the adaptive response in other kinds of injuries (e.g., chemical or bacterial) is crucial to facilitate the identification of nutrients or drugs to enhance adaptation. Experimentally, different approaches have been used to describe the complex molecular and cellular index of small intestinal pathology, including histopathological scoring and measuring the outcome of injury. Despite this, what is absent from the literature is a detailed description of how to perform the procedures that are needed to obtain reproducible data. When identifying factors involved in adaptation, such as gut hormones, an easy, low cost, and reproducible animal model is warranted and here we suggest using a model of chemotherapy-induced intestinal mucositis (CIM).
One of the simplest and very informative endpoints of both injury and adaptation is to measure the mass of the small intestine (SI). We know that a hallmark of mucositis is apoptosis of enterocytes, time-dependent villus atrophy and reduced mitosis. Therefore, examining intestinal morphology is highly relevant in preclinical models4,5. In humans, a decline in plasma citrulline, a marker of functioning enterocytes, correlates with toxicity scores and inflammatory markers6 in addition to the absorptive capacity7, suggesting this amino acid is an excellent biomarker of mucositis. Citrulline can be measured in both mice and rats, and has shown excellent correlations with villus length8, crypt survival9, and radiation-induced mucositis10.
A major advantage of measuring plasma citrulline is the ability to collect repeated measurements from one animal. However, multiple blood sampling in mice is restricted to a total blood volume of 6 &#181;L/g/week and requires general anaesthesia. This unfortunately also limits the use of citrulline measurements in mice. Furthermore, the measurement of citrulline requires high-performance liquid chromatography11,12, which is costly and time-consuming. Recently, we showed that citrulline levels in mice correlate significantly with SI weight (p &#60; 0.001) (unpublished data), making citrulline a direct measurement reflecting enterocyte mass. A limitation to the measurement of SI weight is the necessity for the mice to be sacrificed and thus no repeated measurements within the same mouse are possible. Still the method provides the possibility to perform a variety of other tissue analyses directed to the research question, and these facts can conceivably make up for the additional use of animals. We, therefore, suggest using SI weight as an easy, low-cost, and fast biomarker of injury and adaptation in mice. To ensure reproducibility and acceptable analytic variation, the intestines should be carefully removed from the animal, flushed with saline, emptied and dried before weighing. In this article, we show exactly how this procedure is performed.
Another hallmark of mucositis is the loss of the proliferating cells in the crypts and a compensatory hyperproliferation during the regenerative period3. The cellular marker Ki67 has been frequently used to determine fast proliferative cells by means of immunohistochemistry13. Even though Ki67 is a simple marker of proliferation, it has a tendency for imprecision as Ki67 is present during all active phases of the cell cycle (G1, S, G2, and M)14. Specific labelling is essential to detect replicating cells, which is why we suggest in situ incorporation of 5-bromo-2&#39;-deoxyuridine (BrdU), a synthetic analogue of thymidine, as it is largely restricted to replicating cells in the S-phase15. BrdU is injected in the animals 150 minutes before sacrificing and cells can be subsequently detected with immunohistochemistry using BrdU specific antibodies. In this method article, we show exactly how to measure the area of BrdU immunopositive cells within a crypt using a free image software.
Morphologic and functional changes are often studied in 5-FU induced mucositis models, where the intestinal adaptation is assessed by villus height and crypt depth. During this study, we found that during the acute phase of mucositis, which is equal to the injury phase, proliferation measured by BrdU incorporation is not correlated with crypt depth. In contrast to this, crypt depth is significantly correlated with proliferation seen in the repair phase of mucositis, 3 to 5 days after induction. This suggests that the acute phase of mucositis is not measurable by crypt depth alone. We suggest that when using proliferation as an endpoint in the acute phase of mucositis mice, BrdU incorporation should preferably be used but when quantitating hyperproliferation in the later stage during the regenerative phase, crypt depth is a reasonable alternative to BrdU incorporation. The goal of this study was to describe this model in a way that it can be used by all researchers, both in the field of oncology but especially researchers not familiar with intestinal injury models.
The described model can be used to phenotype transgenic models according to the adaptive response using body weight, SI weight and crypt depth as endpoints. As an example, we show here how we used the model of 5-fluorouracil (5-FU) induced mucositis in a cellular knock out model with insufficient L-cell secretion16. Glucagon-like peptide-1 (GLP-1) and glucagon-like peptide-2 (GLP-2) are intestinal hormones co-secreted from the enteroendocrine L-cells in response to food intake17,18. GLP-2 is recognized as an important factor for intestinal healing, the regulation of mucosal apoptosis and the improvement of the barrier function of the SI19,20,21,22. Based on the literature, we hypothesized that endogenous hormones are essential for compensatory hyperproliferation occurring in the adaptive response after injury.

<table>
	<tbody>
		<tr>
			<td>5-Fluorouracil</td>
			<td>Hospira Nordic AB, Sweden</td>
			<td>137853</td>
			<td></td>
		</tr>
		<tr>
			<td>Ketaminol&#174;Vet</td>
			<td>Merck, New Jersey, USA</td>
			<td>511485</td>
			<td></td>
		</tr>
		<tr>
			<td>Rompun&#174;Vet Xylazine</td>
			<td>Rompunvet, Bayer, Leverkusen, Germany.</td>
			<td>148999</td>
			<td></td>
		</tr>
		<tr>
			<td>10% nautral formalin buffer</td>
			<td>Cell Path Ltd, Powys, United Kingdom</td>
			<td>BAF-5000-08A</td>
			<td></td>
		</tr>
		<tr>
			<td>HistoClear</td>
			<td>National Diagnostics, United Kingdom</td>
			<td>HS-200</td>
			<td></td>
		</tr>
		<tr>
			<td>Pertex</td>
			<td>HistoLab&#174;, Sweden</td>
			<td>840</td>
			<td></td>
		</tr>
		<tr>
			<td>BrdU</td>
			<td>Sigma-Aldrich, Germany.</td>
			<td>B5002</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris/EDTA pH 9 buffer</td>
			<td>Thermofisher scientific, Denmark</td>
			<td>TA-125-PM4X</td>
			<td></td>
		</tr>
		<tr>
			<td>Peroxide Block</td>
			<td>Ultravision Quanto Mouse on Mouse kit, Thermofisher Scientific, Denmark</td>
			<td>TL-060-QHDM</td>
			<td></td>
		</tr>
		<tr>
			<td>Rodent Block buffer</td>
			<td>Ultravision Quanto Mouse on Mouse kit, Thermofisher Scientific, Denmark</td>
			<td>TL-060-QHDM</td>
			<td></td>
		</tr>
		<tr>
			<td>Monoclonal mouse anti-BrdU antibody</td>
			<td>Thermofisher Scientific, Denmark.</td>
			<td>MA1-81890</td>
			<td></td>
		</tr>
		<tr>
			<td>Lab Vision Antibody Diluent OP Quanto</td>
			<td>Thermofisher Scientific, Denmark.</td>
			<td>TA-125-ADQ</td>
			<td></td>
		</tr>
		<tr>
			<td>Horseradish peroxidase</td>
			<td>Ultravision Quanto Mouse on Mouse kit, Thermofisher Scientific, Denmark</td>
			<td>TL-060-QHDM</td>
			<td></td>
		</tr>
		<tr>
			<td>DAB Quanto Substrate</td>
			<td>DAB Substrate Kit, Thermofisher Scientific, Denmark</td>
			<td>TA-125-QHDX</td>
			<td></td>
		</tr>
		<tr>
			<td>DAB Quanto Chromogen</td>
			<td>DAB Substrate Kit, Thermofisher Scientific, Denmark</td>
			<td>TA-125-QHDX</td>
			<td></td>
		</tr>
		<tr>
			<td>Zen Lite Software (Blue edition)</td>
			<td>Carl Zeiss A/S</td>
			<td></td>
			<td>https://www.zeiss.com/microscopy/int/products/microscope-software/zen-lite.html</td>
		</tr>
		<tr>
			<td>ImageJ Software</td>
			<td>LOCI, University of Wisconsin</td>
			<td></td>
			<td>https://imagej.nih.gov/ij/</td>
		</tr>
	</tbody>
</table>

important endpoints and proliferative markers to assess small intestinal injury and adaptation using a mouse model of chemotherapy-induced mucositis

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell proliferation and increased nutrient absorption, are critical in recovery, yet poorly understood. Understanding the molecular mechanism behind the adaptive responses is crucial to facilitate the identification of nutrients or drugs to enhance adaptation. Different approaches and models have been described throughout the literature, but a detailed descriptive way to essentially perform the procedures is needed to obtain reproducible data. Here, we describe a method to estimate important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis in mice. We demonstrate the detection of proliferating cells using a cell cycle specific marker, as well as using small intestinal weight, crypt depth, and villus height as endpoints. Some of the critical steps within the described method are the removal and weighing of the small intestine and the rather complex software system suggested for the measurement of this technique. These methods have the advantages that they are not time-consuming, and that they are cost-effective and easy to carry out and measure.

In the first experiment, we induced mucositis in mice at day 0 and sacrificed a group of mice each day for 5 consecutive days. When measuring the SI weight, we found that this parameter decreased from day 2 until day 4 suggesting a loss in the enterocyte mass. We also found that at day 5, the SI weight was not significantly different from day 0 (untreated mice) (Figure 1). The proliferation measured by the incorporation of BrdU was almost abolished at day 1 a...

Watch this Scientific Journal Video about Important Endpoints and Proliferative Markers to Assess Small Intestinal Injury and Adaptation using a Mouse Model of Chemotherapy-Induced Mucositis at JoVE.c

Important Endpoints and Proliferative Markers to Assess Small Intestinal Injury and Adaptation using a Mouse Model of Chemotherapy-Induced Mucositis

Here, we present a protocol to establish important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis. We demonstrate the detection of proliferating cells using a cell cycle specific marker and using small intestinal weight, crypt depth, and villus height as endpoints.

Intestinal adaptation is the natural compensatory mechanism that occurs when the bowel is lost due to trauma. The adaptive responses, such as crypt cell ...

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Research

JoVE Journal

Cancer Research

5.7K Views.  University of Copenhagen. Here, we present a protocol to establish important endpoints and proliferative markers of small intestinal injury and compensatory hyperproliferation using a model of chemotherapy-induced mucositis. We demonstrate the detection of proliferating cells using a cell cycle specific marker and using small intestinal weight, crypt depth, and villus height as endpoints.

Video: Important Endpoints and Proliferative Markers to Assess Small Intestinal Injury and Adaptation using a Mouse Model of Chemotherapy-Induced Mucositis

A Mouse Model of Fatigue Induced by Peripheral Irradiation

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. Here we describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. With appropriate shielding, the irradiation targets the lower abdominal/pelvic region of the mouse, sparing the brain, in an effort to model radiation treatment received by individuals with pelvic cancers. We deliver an irradiation dose that is sufficient to induce fatigue-like behavior in mice, measured by voluntary wheel-running activity (VWRA), without causing obvious morbidity. Since wheel running is a normal, voluntary behavior in mice, its use should have little confounding effect on other behavioral tests or biological measures. Hence, wheel running can be used as a feasible outcome measure in understanding the behavioral and biological correlates of fatigue. CRF is a complex condition with frequent comorbidities, and likely has causes related both to cancer and its various treatments. The methods described in this paper are useful for investigating radiation-induced changes that contribute to the development of CRF and, more generally, to explore the biological networks that can explain the development and persistence of a peripherally-triggered but centrally-driven behavior like fatigue.

We describe a method using targeted peripheral irradiation to induce fatigue-like behavior in mice. The selected non-lethal irradiation dose leads to a week-long reduction in voluntary wheel-running activity.

Cancer-related fatigue (CRF) is a distressing and costly condition that often affects patients receiving cancer treatments, including radiation therapy. ...

A Genetically Engineered Mouse Model of Sporadic Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Genetically engineered mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in large organs such as the colon in which only a single tumor is desired.
As a result, an immunocompetent, genetically engineered mouse model of colorectal cancer was developed which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor development is initiated by surgical, segmental infection of the distal colon with adeno-cre virus in compound conditionally mutant mice. The tumors can be easily detected and monitored via colonoscopy. We here describe the surgical technique of segmental adeno-cre infection of the colon, the surveillance of the tumor via high-resolution colonoscopy and present the resulting colorectal tumors.

A protocol for the establishment of a genetically engineered mouse model of colorectal cancer by segmental adeno-cre infection and its surveillance via high-resolution colonoscopy is presented.

Despite the advantages of easy applicability and cost-effectiveness, colorectal cancer mouse models based on tumor cell injection have severe limitations ...

Tumor Engraftment in a Xenograft Mouse Model of Human Mantle Cell Lymphoma

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when stimulated by T lymphocytes, oncogenic B cells survive and expand autonomously in undefined organ niches. Mantle cell lymphoma (MCL) is one such B cell disorder, where the median survival rate of patients is 4 - 5 years. This calls for the need of effective mechanisms by which the homing and engraftment of these cells are blocked in order to increase the survival and longevity of patients. Therefore, the effort to develop a xenograft mouse model to study the efficacy of MCL therapeutics by blocking the homing mechanism in vivo is of utmost importance. Development of animal recipients for human cell xenotransplantation to test early stage drugs have long been pursued, as relevant preclinical mouse models are crucial to screen new therapeutic agents. This animal model is developed to avoid human graft rejection and to establish a model for human diseases, and it may be an extremely useful tool to study disease progression of different lymphoma types and to perform preclinical testing of candidate drugs for hematologic malignancies, like MCL. We established a xenograft mouse model that will serve as an excellent resource to study and develop novel therapeutic approaches for MCL.

Mantle cell lymphoma (MCL) is a difficult to treat B cell disorder and it is equally difficult to establish a xenograft mouse model of primary MCL to study and develop therapeutics. Here, we describe the successful establishment of MCL xenografts in mice to help understand its underlying biology.

B lymphocytes are key players in immune cell circulation and they mainly home to and reside in lymphoid organs. While normal B cells only proliferate when ...

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 ...

A Preclinical Mouse Model of Osteosarcoma to Define the Extracellular Vesicle-mediated Communication Between Tumor and Mesenchymal Stem Cells

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under the influence of specific environmental signals, these adult stem cells can release paracrine mediators leading to accelerated tumor growth and metastasis. Defining the crosstalk between tumor and MSCs is of primary importance to understand the mechanisms underlying cancer progression and identify novel targets for therapeutic intervention. 
Cancer cells produce high amounts of extracellular vesicles (EVs), which can profoundly affect the behavior of target cells in the tumor microenvironment or at distant sites. Tumor EVs enclose functional biomolecules, including inflammatory RNAs and (onco)proteins, that can educate stromal cells to enhance the metastatic behavior of cancer cells or to participate in the pre-metastatic niche formation. In this article, we describe the development of a preclinical cancer mouse model that enables specific evaluation of the EV-mediated crosstalk between tumor and mesenchymal stem cells. First, we describe the purification and characterization of tumor-secreted EVs and the assessment of the EV internalization by MSCs. We then make use of a multiplex bead-based immunoassay to evaluate the alteration of the MSC cytokine expression profile induced by cancer EVs. Finally, we illustrate the generation of a bioluminescent orthotopic xenograft mouse model of osteosarcoma that recapitulates the tumor-MSC interaction, and show the contribution of EV-educated MSCs to tumor growth and metastasis formation. 
Our model provides the opportunity to define how cancer EVs shape a tumor-supporting environment, and to evaluate whether blockade of the EV-mediated communication between tumor and MSCs prevents cancer progression.

Direct injection of cancer-derived extracellular vesicles (EVs) leads to reprogramming of bone marrow supporting tumor progression; however, which cells mediate this effect is unclear. Herein, we describe a step-by-step protocol to investigate EV-mediated tumor-mesenchymal stem cell (MSC) interactions in vivo, revealing a crucial role for EV-educated MSCs in metastasis.

Within the tumor microenvironment, resident or recruited mesenchymal stem cells (MSCs) contribute to malignant progression in multiple cancer types. Under ...

Long-term Live-cell Imaging to Assess Cell Fate in Response to Paclitaxel

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over the past decade, new and innovative technologies have greatly enhanced the practicality of live-cell imaging. Cells can now be kept in focus and continuously imaged over several days while maintained under 37 &#176;C and 5% CO2 cell culture conditions. Moreover, multiple fields of view representing different experimental conditions can be acquired simultaneously, thus providing high-throughput experimental data. Live-cell imaging provides a significant advantage over fixed-cell imaging by allowing for the direct visualization and temporal quantitation of dynamic cellular events. Live-cell imaging can also identify variation in the behavior of single cells that would otherwise have been missed using population-based assays. Here, we describe live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel. We demonstrate methods to visualize whether mitotically arrested cells die directly from mitosis or slip back into interphase. We also describe how the fluorescent ubiquitination-based cell cycle indicator (FUCCI) system can be used to assess the fraction of interphase cells born from mitotic slippage that are capable of re-entering the cell cycle. Finally, we describe a live-cell imaging method to identify nuclear envelope rupture events.

Live-cell imaging provides a wealth of information on single cells or whole populations that is unattainable by fixed cell imaging alone. Here, live-cell imaging protocols to assess cell fate decisions following treatment with the anti-mitotic drug paclitaxel are described.

Live-cell imaging is a powerful technique that can be used to directly visualize biological phenomena in single cells over extended periods of time. Over ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

Using Computer-based Image Analysis to Improve Quantification of Lung Metastasis in the 4T1 Breast Cancer Model

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. The leading cause of breast cancer related deaths is the metastatic burden. Therefore, preclinical models for breast cancer need to analyze metastatic burden to be clinically relevant. The 4T1 breast cancer model provides a spontaneously-metastasizing, quantifiable mouse model for stage IV human breast cancer. However, most 4T1 protocols quantify the metastatic burden by manually counting stained colonies on tissue culture plates. While this is sufficient for tissues with lower metastatic burden, human error in manual counting causes inconsistent and variable results when plates are confluent and difficult to count. This method offers a computer-based solution to human counting error. Here, we evaluate the protocol using the lung, a highly metastatic tissue in the 4T1 model. Images of methylene blue-stained plates are acquired and uploaded for analysis in Fiji-ImageJ. Fiji-ImageJ then determines the percentage of the selected area of the image that is blue, representing the percentage of the plate with metastatic burden. This computer-based approach offers more consistent and expeditious results than manual counting or histopathological evaluation for highly metastatic tissues. The consistency of Fiji-ImageJ results depends on the quality of the image. Slight variations in results between images can occur, thus it is recommended that multiple images are taken and results averaged. Despite its minimal limitations, this method is an improvement to quantifying metastatic burden in the lung by offering consistent and rapid results.

We describe a more consistent and expeditious method to quantify lung metastasis in the 4T1 breast cancer model by using Fiji-ImageJ.

Breast cancer is a devastating malignancy, accounting for 40,000 female deaths and 30% of new female cancer diagnoses in the United States in 2019 alone. ...

A Mouse Model to Investigate the Role of Cancer-Associated Fibroblasts in Tumor Growth

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role of CAFs in the tumor microenvironment can be helpful for understanding the functional importance of fibroblasts, fibroblasts from different tissues, and specific genetic factors in fibroblasts. Mouse models are essential for understanding the contributors to tumor growth and progression in an in vivo context. Here, a protocol in which cancer cells are mixed with fibroblasts and introduced into mice to develop tumors is provided. Tumor sizes over time and final tumor weights are determined and compared among groups. The protocol described can provide more insight into the functional role of CAFs in tumor growth and progression.

A protocol to co-inject cancer cells and fibroblasts and monitor tumor growth over time is provided. This protocol can be used to understand the molecular basis for the role of fibroblasts as regulators of tumor growth.

Cancer-associated fibroblasts (CAFs) can play an important role in tumor growth by creating a tumor-promoting microenvironment. Models to study the role ...

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 ...