We are grateful for funding from the NCI Ovarian Cancer SPORE Program P50CA228991, post-doctoral training program 5T32OD011089, and the Richard W. TeLinde Endowment, Johns Hopkins University. The project was also partly funded by the subsidies for current expenditures to Private Institutions of Higher Education from the Promotion and Mutual Aid Corporation for Private Schools of Japan.

All procedures and experiments involving mice were performed according to protocols approved by Johns Hopkins Animal Care and Use Committee. For all procedures, appropriate PPE was worn, including gloves and disposable isolation gowns. Precautions were taken when handling sharps, which were properly disposed of in red box sharps containers immediately after use. See the Table of Materials for details about all the materials and equipment used in this protocol.
1. Induction of endometrial cancer in iPAD (inducible Pten, Arid1a double deletion) mice with doxycycline
<ol>
	<li>Maintain 10 Pax8-Cre-Arid1a-Pten double deletion (iPAD) transgenic mice (Figure 1) on a mixed genetic background (129S, BALB/C, C57BL/6), as previously described14.</li>
	<li>Collect baseline ultrasound (2D) images of the ovaries, oviduct, and uterus of each mouse before doxycycline treatment.</li>
	<li>Provide exclusively doxycycline-containing mouse chow diets (doxycycline hyclate at 625 mg/kg of feed) to the female iPAD mice for a minimum of 2 weeks starting at 7-8 weeks of age to induce gene deletion.</li>
</ol>
2. Equipment setup
<ol>
	<li>Turn on the heating pad, and cover with a clean absorbent pad (target temperature: 38 &#176;C).</li>
	<li>Confirm that the isoflurane vaporizer and O2 tank are adequately filled. Refill and replace if the contents are low.</li>
	<li>Connect the induction chamber, nose cone, and scavenging system to the vaporizer.</li>
	<li>Set up the ultrasound machine.
	<ol>
		<li>Select a transducer (probe) with a range of 32-56 MHz or up to 70 MHz for imaging of the uterus or ovary, respectively.</li>
		<li>Attach the probe, and power on the machine.</li>
		<li>After system boot-up, use the control panel to log in with the user credentials and access the home screen.</li>
		<li>From the home screen, go to the Applications tab, and select Mouse (small) abdomen mode.</li>
		<li>Click on Scan to return to the home screen, and wait for a live image to be displayed.</li>
		<li>Select B-Mode from the options on the left tool bar.</li>
		<li>Click on More Controls to view additional tools for image refinement, such as image gain and depth, or to adjust the clip acquisition settings, such as the number of frames per second.</li>
		<li>Once the image settings are selected, return to the home screen by clicking on Scan.</li>
	</ol>
	</li>
	<li>Turn on the O2 tank, direct the flow to the induction chamber, and set the flow rate to 1 L/min.</li>
</ol>
3. Preparation of mice for ultrasound screening, including hair removal
<ol>
	<li>Place a mouse in the induction chamber. Set the isoflurane vaporizer to 2%-3% vol/vol for the induction of anesthesia.</li>
	<li>Determine the appropriate depth of anesthesia by a lack of response to toe pinch and a respiratory rate around 1-2 breaths/s.</li>
	<li>Apply sterile ophthalmic lubricant to each eye. Remove the fur from the dorsum and ventrum between the last rib and pelvis with appropriately sized clippers.</li>
	<li>Apply a thin layer of depilatory cream to the ventral and dorsal regions to be imaged (if needed).</li>
	<li>Place the mouse back in the induction chamber for approximately 3-5 min to maintain the appropriate depth of anesthesia while the depilatory cream works to remove the hair. After &#8804;4 minutes, gently wipe away the cream with a clean moist paper towel. 
	&#8203;NOTE: Longer exposure to depilatory cream is irritating and may cause skin lesions.</li>
</ol>
4. Intraperitoneal injection of fluid to increase the contrast between organs
<ol>
	<li>Warm a 3-10 mL syringe filled with sterile isotonic fluid solution (e.g., sterile 0.9% NaCl or Lactated Ringers Solution) to 35-40 &#176;C by placing it between a heating pad and an absorbent pad for several minutes. Place a bottle of ultrasound gel on the heating pad if the machine does not have a warmer.</li>
	<li>For a 20-25 g mouse, inject 1-2 mL of solution into the peritoneal cavity.
	<ol>
		<li>Secure the mouse by the scruff in one hand, exposing the ventrum.</li>
		<li>Hold the mouse at a ~20&#176; angle, with the nose pointed to the floor to direct the organs cranially due to gravity.</li>
		<li>Using a small gauge needle (25 G, 5/8 in length, tuberculin syringe), puncture through the skin and abdominal wall of the caudal right quadrant of the abdomen.</li>
		<li>Before the injection of fluids, to avoid injection into the vasculature or the GI tract, pull back with minimal pressure. If blood or other material enters the syringe, remove the needle. Use a new needle and syringe, and try again at a slightly different position.</li>
	</ol>
	</li>
	<li>If the mouse wakes up during the injections, place it back in the small induction chamber for anesthesia with 2%-3% vol/vol isoflurane.</li>
</ol>
5. Ultrasound imaging from a dorsal approach
<ol>
	<li>Position the mouse in ventral recumbency on the absorbent pad over a heating pad (Figure 2A-C).</li>
	<li>Place a rodent nose cone securely over the mouse&#39;s nose and muzzle. Maintain the anesthetic depth with isoflurane delivered through the nose cone at 1%-2% vol/vol in 100% O2. Apply sterile ophthalmic lubricant, as needed, to each eye.</li>
	<li>Monitor the mouse for a regular respiratory rate (1-2/s) and a lack of toe pinch response to indicate if the anesthesia needs to be adjusted.</li>
	<li>Place a small amount (~0.5-1 mL) of prewarmed ultrasound gel abaxial (lateral) to the spine on either side of the anesthetized mouse, between the last rib and pelvis.</li>
	<li>Put a small amount of gel on the ultrasound probe.</li>
	<li>Place the probe parallel to the vertebrae with the front of the probe on the cranial side. An indicator mark is present on the probe head to indicate the proper probe orientation. Record the day, time, animal ID, probe orientation, and animal side (right, left, dorsal, ventral) for each new set of images being collected.</li>
	<li>With a mouse in ventral recumbency (dorsum skin touching the probe), slowly scan the area for the kidney landmark (Figure 2B and Figure 3). With the kidney in view, pull the probe caudal to find the ovary-a slightly hyperechoic oval to round structure (Figure 4A, B) within a very hyperechoic ovarian fat pad that is bordered cranio-ventrally by the kidney and dorso-laterally by the dorsal abdominal wall. 
	NOTE: Pressure caudal and lateral to the ovary can direct the ovary closer to the abdominal wall and away from the loops of the intestine. The ovary is anatomically positioned up against the dorsal abdominal wall, just ventral and lateral to the epaxial muscles and caudal to the kidney.</li>
	<li>Adjust the signal gain using the slider at the bottom of the control screen to improve the image contrast.</li>
	<li>To improve the imaging of the kidney, apply pressure with a finger to the contralateral abdomen. Vary the pressure and angle from directly parallel to the spine to ~20&#176; ventral.</li>
	<li>Once the organ of interest is in view, collect a video by clicking on Save Clip or Start Recording and then Stop Recording when done to retain images at a preset number of frames.</li>
	<li>Save single frames from either a live image or recording with the Save Frame button.</li>
	<li>To image the uterus, pull the probe caudally until the ovary is in the most cranial aspect of the field of view. Vary the probe pressure and angle until the uterus is in view.</li>
	<li>Repeat video and frame collection for each organ of interest.</li>
	<li>Find the uterus running longitudinal along the dorsal abdominal wall with the lateral leg musculature also in view (Figure 4B). 
	NOTE: The uterus size and lumen diameter may vary with the phase of estrus and disease state.</li>
	<li>Monitor the tissue for peristaltic motion to differentiate the intestinal loops from the uterine stationary horns.</li>
</ol>
6. Collect images from a ventral approach
<ol>
	<li>Place the mouse in dorsal recumbency, and check that the eye lubrication is sufficient and the muzzle is securely in the nose cone (Figure 2A).</li>
	<li>Apply a small amount (~0.5-1 mL) of prewarmed ultrasound gel to the ventral abdomen, and apply the probe at the midline just cranial of the pubis to locate the bladder as a hypoechoic landmark (Figure 5). 
	NOTE: If the bladder is too large and obscures the uterus imaging, gentle pressure can be placed on the lower abdomen to express urine.</li>
	<li>Pull the probe lateral to the bladder to find the uterine horns. Apply light digital pressure from either or both sides of the mouse to bring the horns into the field of view. Hold the probe perpendicular to the mouse, and scan both sides of the abdomen to capture transverse views (cross-sections) of both horns. Rotate the probe to capture sagittal views.</li>
	<li>After the ultrasound, wipe the mouse clean of gel with a paper towel, and return it to its cage to recover. Mice are fully awake in 2-5 min. Once it is fully awake and ambulatory, return the mouse to the animal room. 
	NOTE: A heating pad on a low heat can be placed under the cage to warm the cage for recovery.</li>
	<li>At the experimental or humane endpoint, euthanize the mouse. Ideally, euthanize the mouse in the home cage to reduce stress; alternatively, place the mouse in a clean chamber. Deliver pressurized CO2 at a displacement rate of 10%-30% of the chamber volume per minute. After approximately 5 min of no visible respiration, verify death by cervical dislocation. Proceed with abdominal necropsy for tumor harvest.</li>
</ol>

<ol>
	<li>Ajayi, A. F., Akhigbe, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Staging+of+the+estrous+cycle+and+induction+of+estrus+in+experimental+rodents:+an+update.">Staging of the estrous cycle and induction of estrus in experimental rodents: an update.</a> Fertility Research and Practice. 6, 5 (2020).</li><li>Kim, S. W., Kim, Y. Y., Kim, H., Ku, S. Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+closer+to+intrauterine+adhesive+pathology.">Animal models closer to intrauterine adhesive pathology.</a> Annals of Translational Medicine. 8 (18), 1125 (2020).</li><li>Shi, D., Vine, D. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Animal+models+of+polycystic+ovary+syndrome:+a+focused+review+of+rodent+models+in+relationship+to+clinical+phenotypes+and+cardiometabolic+risk.">Animal models of polycystic ovary syndrome: a focused review of rodent models in relationship to clinical phenotypes and cardiometabolic risk.</a> Fertility and Sterility. 98 (1), 185-193 (2012).</li><li>Greco, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+biomicroscopy+in+small+animal+research:+applications+in+molecular+and+preclinical+imaging.">Ultrasound biomicroscopy in small animal research: applications in molecular and preclinical imaging.</a> Journal of Biomedicine and Biotechnology. 2012, 519238 (2012).</li><li>Palsdottir, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Interobserver+agreement+of+transvaginal+ultrasound+and+magnetic+resonance+imaging+in+local+staging+of+cervical+cancer.">Interobserver agreement of transvaginal ultrasound and magnetic resonance imaging in local staging of cervical cancer.</a> Ultrasound in Obstetrics and Gynecology. 58 (5), 773-779 (2021).</li><li>Gabrielson, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+imaging+with+confirmation+by+histopathology+for+increased+rigor+and+reproducibility+in+translational+research:+A+review+of+examples,+options,+and+resources.">In vivo imaging with confirmation by histopathology for increased rigor and reproducibility in translational research: A review of examples, options, and resources.</a> ILAR Journal. 59 (1), 80-98 (2018).</li><li>Peterson, R. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Continuing+education+course+#1:+Non-invasive+imaging+as+a+problem-solving+tool+and+translational+biomarker+strategy+in+toxicologic+pathology.">Continuing education course #1: Non-invasive imaging as a problem-solving tool and translational biomarker strategy in toxicologic pathology.</a> Toxicologic Pathology. 39 (1), 267-272 (2011).</li><li>Pfeifer, L. F., Adams, G. P., Pierson, R. A., Singh, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound+biomicroscopy:+A+non-invasive+approach+for+in+vivo+evaluation+of+oocytes+and+small+antral+follicles+in+mammals.">Ultrasound biomicroscopy: A non-invasive approach for in vivo evaluation of oocytes and small antral follicles in mammals.</a> Reproduction, Fertility and Development. 26 (1), 48-54 (2013).</li><li>Cheung, A. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Three-dimensional+ultrasound+biomicroscopy+for+xenograft+growth+analysis.">Three-dimensional ultrasound biomicroscopy for xenograft growth analysis.</a> Ultrasound in Medicine and Biology. 31 (6), 865-870 (2005).</li><li>Snyder, C. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Complementarity+of+ultrasound+and+fluorescence+imaging+in+an+orthotopic+mouse+model+of+pancreatic+cancer.">Complementarity of ultrasound and fluorescence imaging in an orthotopic mouse model of pancreatic cancer.</a> BMC Cancer. 9, 106 (2009).</li><li>Wu, G., Wang, L., Yu, L., Wang, H., Xuan, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+three-dimensional+ultrasound+micro-imaging+to+monitor+prostate+tumor+development+in+a+transgenic+prostate+cancer+mouse+model.">The use of three-dimensional ultrasound micro-imaging to monitor prostate tumor development in a transgenic prostate cancer mouse model.</a> The Tohoku Journal of Experimental Medicine. 207 (3), 181-189 (2005).</li><li>Rinaldi, S. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound-guided+intrauterine+injection+of+lipopolysaccharide+as+a+novel+model+of+preterm+birth+in+the+mouse.">Ultrasound-guided intrauterine injection of lipopolysaccharide as a novel model of preterm birth in the mouse.</a> The American Journal of Pathology. 185 (5), 1201-1206 (2015).</li><li>Wang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasonography+in+experimental+reproductive+investigations+on+rats.">Ultrasonography in experimental reproductive investigations on rats.</a> Journal of Visualized Experiments. 130, e56038 (2017).</li><li>Suryo Rahmanto, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inactivation+of+Arid1a+in+the+endometrium+is+associated+with+endometrioid+tumorigenesis+through+transcriptional+reprogramming.">Inactivation of Arid1a in the endometrium is associated with endometrioid tumorigenesis through transcriptional reprogramming.</a> Nature Communications. 11, 2717 (2020).</li><li>Pani, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pre-existing+thyroiditis+ameliorates+papillary+thyroid+cancer:+Insights+from+a+new+mouse+model.">Pre-existing thyroiditis ameliorates papillary thyroid cancer: Insights from a new mouse model.</a> Endocrinology. 162 (10), bqab144 (2021).</li></ol>

The authors have no conflicts of interest to disclose.

This protocol examines the utility of ultrasound for assessing uterine morphological changes in the progression of adenocarcinoma in the uterus in mice. In this study, by following the induction of endometrial cancer in mice longitudinally, the anatomical details detected by ultrasound were found to be indicators of gross and histological pathology. This opens the door for the use of longitudinal studies with smaller numbers of mice monitored by ultrasound at multiple time points to follow the progression of uterine canc...

Mice remain one of the most important animal models for reproductive disorders1,2,3. There are several genetically modified or induced rodent models of ovarian and uterine cancers. These studies typically rely on multiple cohorts euthanized at different time points to capture longitudinal trends in morphologic and pathologic changes. This prevents the ability to acquire continuous data on cancer development in an individual mouse. Additionally, without knowing the individual mouse disease progression state, intervention studies are based on predetermined time points and averaged findings of previous cohorts rather than individual thresholds for the detection of progression in a specific animal4,5. Therefore, imaging approaches that allow for longitudinal assessment in live animals are needed to facilitate preclinical models for testing new drugs or compounds and accelerate the understanding of pathobiology while also increasing the rigor and reproducibility6.
Ultrasound imaging (US) is an appealing method for the longitudinal monitoring of mouse uterine cancer progression because it is relatively facile and inexpensive compared to other imaging methods, is easy to perform, and can have remarkable resolution6,7. This non-invasive modality can capture features to the micron scale in awake mice or with mice under brief sedation using a 5-10 min exam. Ultrasound microscopy has been validated as a method to measure mouse ovarian follicle development 8 and the growth of implanted or induced neoplasia9,10,11. High-frequency US has also been used for percutaneous intrauterine injections12 and observing rat uterine change over the estrus cycle13. High-frequency US can be used with mice held on specialized stationary platforms using a rail system to hold the transducer/probe to capture high-resolution images with standardized position and pressure; however, this equipment is not available at all institutions. Hand-held transducer scanning methods can be adopted with less dedicated equipment and used for both clinical diagnostics and research applications in mice.
The question remains as to whether US imaging with hand-held, high-frequency probes could be used to monitor uterine cancer development over multiple weeks. Similar to the intestines, the rodent uterus is a thin-walled, slender structure that is very mobile within the abdomen and is contiguous through multiple tissue depths, making imaging more challenging than with relatively immobile organs such as the kidneys. This study sought to establish the correlation between tissues observed by ultrasound and histopathology, define landmarks for locating the mouse uterus, and determine the feasibility of the longitudinal assessment of endometrial cancer. This study presents data showing a qualitative correspondence between the appearance of uteri imaged by US and histopathology, as well as serial imaging of mice over several weeks. These results indicate that hand-held US can be used to monitor endometrial cancer development in mice, thus creating an opportunity for collecting individual mouse longitudinal data to study uterine cancer without the need for dedicated equipment.

<table><tbody><tr><td>&lt;strong&gt;Reagents and Equipment Used for Animal Care&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Rodent Diet (2018, 625 Doxycycline)</td><td>Envigio</td><td>TD.01306</td><td>Mouse Feed</td></tr><tr><td>&lt;strong&gt;Reagents and Equipment Used for Ultrasound Imaging&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10 mL injectable 0.9% NaCl&amp;nbsp;</td><td>Hospira, Inc</td><td>RL-7302</td><td>Isotonic Fluid</td></tr><tr><td>Absorbent Pad with Plastic Backing</td><td>Daigger</td><td>EF8313</td><td>Absorbant Pads</td></tr><tr><td>Anesthesia Induction Chambers</td><td>Harvard Apparatus</td><td>75-2029</td><td>Induction Chamber</td></tr><tr><td>Anesthetic absorber kit with absorber canister, holder, tubing, &amp;amp; adapters</td><td>CWE, Inc</td><td>13-20000</td><td>Nose Cone and Tubing</td></tr><tr><td>Aquasonic Clear Ultrasound Gel (0.25 Liter)</td><td>Parker Laboratoies</td><td>08-03</td><td>Ultrasound Gel</td></tr><tr><td>BD Plastipak 3 mL Syringe</td><td>BD Biosciences</td><td>309657</td><td>Syringe</td></tr><tr><td>F/Air Scavenger Charcoal Canister</td><td>OMNICON</td><td>80120</td><td>Scavenging System for Anesthesia</td></tr><tr><td>Isoflurane, USP</td><td>Vet One</td><td>502017</td><td>Anesthesia Agent</td></tr><tr><td>M1050 Non-Rebreathing Mobile Anesthesia Machine</td><td>Scivena Scientific</td><td>M1050</td><td>Anestheic Vaporizer</td></tr><tr><td>MX550S, 25-55 MHz Transducer, 15mm, Linear</td><td>VisualSonics</td><td>MX550S</td><td>Ultrasound Transducer (Probe)</td></tr><tr><td>Nair Hair Aloe &amp;amp; Lanolin Hair Removal Lotion - 9.0 oz</td><td>Nair</td><td></td><td>Depilliating Cream</td></tr><tr><td>Philips Norelco Multigroomer All-in-One Trimmer Series 7000</td><td>Philips North America</td><td>MG7750</td><td>Clippers</td></tr><tr><td>PrecisionGlide 25 G 1&quot; Needle</td><td>BD Biosciences</td><td>305125</td><td>Needle</td></tr><tr><td>Puralube Ophthalmic Ointment</td><td>Dechra</td><td>17033-211-38</td><td>Lubricating Eye Drops</td></tr><tr><td>Vevo 3100 Imaging System</td><td>VisualSonics</td><td>Vevo 3100</td><td>Ultrasound Machine</td></tr><tr><td>Vevo LAB 5.6.1</td><td>VisualSonics</td><td>Vevo LAB 5.6.1</td><td>Ultrasound Analysis Software</td></tr><tr><td>Vinyl Heating Pad with cover, 12 x 15&quot;</td><td>Sunbeam</td><td>731-500-000R</td><td>Heating Pad</td></tr><tr><td>Wd Elements 2TB Basic Storage</td><td>Western Digital Elements</td><td>WDBU6Y0020BBK-WESN</td><td>Data Storage</td></tr><tr><td>&lt;strong&gt;Reagents and Equipment Used for Immunohistochemistry&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>10% w/v Formalin</td><td>Fischer Scientific</td><td>SF98-4</td><td>Tissue Fixation Buffer</td></tr><tr><td>Animal-Free Blocker and Diluent, R.T.U.</td><td>Vector Laboratories Inc.</td><td>&amp;nbsp;SP5035</td><td>Antibody Blocker</td></tr><tr><td>Charged Super Frost Plus Glass Slides</td><td>VWR</td><td>4831-703</td><td>Tissue Mounting Slides</td></tr><tr><td>Citrate Buffer</td><td>MilliporeSigma</td><td>&amp;nbsp;C9999-1000ML</td><td>Epitope Retrival Buffer (pTEN)</td></tr><tr><td>Cytoseal &amp;ndash; 60</td><td>Thermo Scientific</td><td>8310-4</td><td>Resin for Slide Sealing</td></tr><tr><td>Gold Seal Cover Glass</td><td>Thermo Scientific</td><td>3322</td><td>Coverslide</td></tr><tr><td>Harris Modified Hematoxylin</td><td>MilliporeSigma</td><td>HHS32-1L</td><td>Counterstain Buffer</td></tr><tr><td>Hybridization Incubator (Dual Chamber)</td><td>Fischer Scientific</td><td>13-247-30Q</td><td>Oven to Melt Parraffin</td></tr><tr><td>ImmPACT DAB Substrate, Peroxidase (HRP)</td><td>Vector Laboratories Inc.</td><td>SK-4105</td><td>Signal Development Substrate</td></tr><tr><td>ImmPRESS HRP Goat Anti-Rabbit IgG Polymer Detection Kit, Peroxidase</td><td>Vector Laboratories Inc.</td><td>MP-7451</td><td>Secondary IHC Antibody</td></tr><tr><td>Oster 5712 Digital Food Steamer</td><td>Oster</td><td>5712</td><td>Vegetable Steamer for Epitope Retrival</td></tr><tr><td>rabbit mAB anti-ARID1a</td><td>abcam</td><td>ab182560</td><td>Primary IHC Antibody (1:1,000)</td></tr><tr><td>rabbit mAB anti-PTEN</td><td>Cell Signaling</td><td>9559</td><td>Primary IHC Antibody (1:100)</td></tr><tr><td>Scotts Tap Water Substitute</td><td>MilliporeSigma</td><td>S5134-100ML</td><td>&quot;Blueing&quot; Buffer</td></tr><tr><td>Tissue Path IV Cassette</td><td>Fischer Scientific</td><td>22272416</td><td>Tissue Fixation Cassette</td></tr><tr><td>Trilogy Buffer</td><td>Cell Marque</td><td>&amp;nbsp;920P-10</td><td>Epitope Retrival Buffer (ARID1a)</td></tr></tbody></table>

non-invasive ultrasound assessment of endometrial cancer progression in pax8-directed deletion of the tumor suppressors arid1a and pten in mice

Uterine cancers can be studied in mice due to the ease of handling and genetic manipulation in these models. However, these studies are often limited to assessing pathology post-mortem in animals euthanized at multiple time points in different cohorts, which increases the number of mice needed for a study. Imaging mice in longitudinal studies can track the progression of disease in individual animals, reducing the number of mice needed. Advances in ultrasound technology have allowed for the detection of micrometer-level changes in tissues. Ultrasound has been used to study follicle maturation in ovaries and xenograft growth but has not been applied to morphological changes in the mouse uterus. This protocol examines the juxtaposition of pathology with in vivo imaging comparisons in an induced endometrial cancer mouse model. The features observed by ultrasound were consistent with the degree of change seen by gross pathology and histology. Ultrasound was found to be highly predictive of the observed pathology, supporting the incorporation of ultrasonography into longitudinal studies of uterine diseases such as cancer in mice.

Pax8-Cre-Arid1a-Pten double deletion (iPAD) transgenic mice were maintained on a mixed genetic background (129S, BALB/C, C57BL/6), as previously described14. The mice were all fed a doxycycline feed for 2 weeks to induce Cre recombinase. In previous work by our group, doxycycline was dosed by gavage14; however, in this current study, the doxycycline feed induction method worked efficiently and reduced the stress of gavage for the mice. It is important to c...

Watch this Scientific Journal Video about Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice at JoVE.com

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

This protocol describes a method for monitoring the progression of morphological changes over time in the uterus in an inducible mouse model of endometrial cancer using ultrasound imaging with correlation to gross and histological changes.

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

Uterine cancers can be studied in mice due to the ease of handling and genetic manipulation in these models. However, these studies are often limited to ...

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Cancer Research

1.4K Views.  Johns Hopkins University School of Medicine. Deze studie toont aan dat handmatig geleide echografie zeer geschikt is om de ontwikkeling van baarmoederpathologie te volgen in een geïnduceerd muismodel van endometriumkanker. Echografie is niet-invasief en maakt dus longitudinale studies van individuele muizen gedurende dagen en weken mogelijk. Hierdoor kan individuele variatie worden vastgelegd en kunnen uiteindelijk minder dieren worden gebruikt voor onderzoek.Deze methode kan gemakkelijk worden toegepast om pathologische verande...

Video: Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

A Murine Orthotopic Bladder Tumor Model and Tumor Detection System

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified to secrete human Prostate Specific Antigen (PSA), and the procedure for the confirmation of tumor implantation. In brief, mice are anesthetized using injectable drugs and are made to lay in the dorsal position. Urine is vacated from the bladder and 50 &#181;L of poly-L-lysine (PLL) is slowly instilled at a rate of 10 &#181;L/20 s using a 24 G IV catheter. It is left in the bladder for 20 min by stoppering the catheter. The catheter is removed and PLL is vacated by gentle pressure on the bladder. This is followed by instillation of the murine bladder cancer cell line (1 x 105 cells/50 &#181;L) at a rate of 10 &#181;L/20 s. The catheter is stoppered to prevent premature evacuation. After 1 h, the mice are revived with a reversal drug, and the bladder is vacated. The slow instillation rate is important, as it reduces vesico-ureteral reflux, which can cause tumors to occur in the upper urinary tract and in the kidneys. The cell line should be well re-suspended to reduce clumping of cells, as this can lead to uneven tumor sizes after implantation.
This technique induces tumors with high efficiency. Tumor growth is monitored by urinary PSA secretion. PSA marker monitoring is more reliable than ultrasound or fluorescence imaging for the detection of the presence of tumors in the bladder. Tumors in mice generally reach a maximum size that negatively impacts health by about 3 - 4 weeks if left untreated. By monitoring tumor growth, it is possible to differentiate mice that were cured from those that were not successfully implanted with tumors. With only end-point analysis, the latter may be mistakenly assumed to have been cured by therapy.

This protocol describes the generation of murine orthotopic bladder tumors in female C57BL/6J mice and the monitoring of tumor growth.

This protocol describes the generation of bladder tumors in female C57BL/6J mice using the murine bladder cancer cell line MB49, which has been modified ...

Utilizing High Resolution Ultrasound to Monitor Tumor Onset and Growth in Genetically Engineered Pancreatic Cancer Models

The LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mouse model represents an established and frequently used transgenic model to evaluate novel therapies in pancreatic cancer. Tumor onset is variable in the KPC model between 8 weeks and several months. Therefore, non-invasive imaging tools are required to screen for tumor onset and monitor for response to treatment. To address this issue, different approaches have emerged over the last years. High resolution ultrasound has major advantages such as non-invasiveness, fast session times and a high image resolution without radiation exposure. However, ultrasound in mice is not trivial and sufficient anatomical knowledge and practical skills are required to successfully perform high resolution ultrasound in preclinical pancreatic cancer models. With the following article, a detailed hands-on guide for abdominal ultrasound in murine models with a particular focus on endogenous pancreatic cancer models is presented. Furthermore, a summary of common mistakes and how to avoid them is provided.

This article describes the utilization of high-resolution ultrasound in genetically engineered pancreatic cancer mice. The primary aim is to provide a detailed instruction for detection and evaluation of endogenous pancreatic tumors.

The LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) mouse model represents an established and frequently used transgenic model to evaluate novel ...

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic alterations in oncogenes such as the KRAS oncogene, which constitutes ~25% of lung cancer cases. The difficulty in therapeutically targeting KRAS-driven lung cancer partly stems from having poor models that can mimic the progression of the disease in the lab. We describe a method that permits the relative quantification of primary KRAS lung tumors in a Cre-inducible LSL-KRAS G12D mouse model via ultrasound imaging. This method relies on brightness (B)-mode acquisition of the lung parenchyma. Tumors that are initially formed in this model are visualized as B-lines and can be quantified by counting the number of B-lines present in the acquired images. These would represent the relative tumor number formed on the surface of the mouse lung. As the formed tumors develop with time, they are perceived as deep clefts within the lung parenchyma. Since the circumference of the formed tumor is well-defined, calculating the relative tumor volume is achieved by measuring the length and width of the tumor and applying them in the formula used for tumor caliper measurements. Ultrasound imaging is a non-invasive, fast and user-friendly technique that is often used for tumor quantifications in mice. Although artifacts may appear when obtaining ultrasound images, it has been shown that this imaging technique is more advantageous for tumor quantifications in mice compared to other imaging techniques such as computed tomography (CT) imaging and bioluminescence imaging (BLI). Researchers can investigate novel therapeutic targets using this technique by comparing lung tumor initiation and progression between different groups of mice.

This protocol describes the steps taken to induce KRAS lung tumors in mice as well as the quantification of formed tumors by ultrasound imaging. Small tumors are visualized in early timepoints as B-lines. At later timepoints, relative tumor volume measurements are achieved by the measurement tool in the ultrasound software.

With ~1.6 million victims per year, lung cancer contributes tremendously to the worldwide burden of cancer. Lung cancer is partly driven by genetic ...

A Novel Use of Three-dimensional High-frequency Ultrasonography for Early Pregnancy Characterization in the Mouse

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is routinely used as an in vivo model to study embryo implantation and pregnancy progression. Unfortunately, such murine studies require pregnancy interruption to enable follow-up phenotypic analysis. To address this issue, we used three-dimensional (3-D) reconstruction of HFUS imaging data for early detection and characterization of murine embryo implantation sites and their individual developmental progression in utero. Combining HFUS imaging with 3-D reconstruction and modeling, we were able to accurately quantify embryo implantation site number as well as monitor developmental progression in pregnant C57BL6J/129S mice from 5.5 days post coitus (d.p.c.) through to 9.5 d.p.c. with the use of a transducer. Measurements included: number, location, and volume of implantation sites as well as inter-implantation site spacing; embryo viability was assessed by cardiac activity monitoring. In the immediate post-implantation period (5.5 to 8.5 d.p.c.), 3-D reconstruction of the gravid uterus in both mesh and solid overlay format enabled visual representation of the developing pregnancies within each uterine horn. As genetically engineered mice continue to be used to characterize female reproductive phenotypes derived from uterine dysfunction, this method offers a new approach to detect, quantify, and characterize early implantation events in vivo. This novel use of 3-D HFUS imaging demonstrates the ability to successfully detect, visualize, and characterize embryo-implantation sites during early murine pregnancy in a non-invasive manner. The technology offers a significant improvement over current methods, which rely on the interruption of pregnancies for gross tissue and histopathologic characterization. Here we use a video and text format to describe how to successfully perform ultrasounds of early murine pregnancy to generate reliable and reproducible data with reconstruction of the uterine form in mesh and solid 3-D images.

Mice are widely used to study gestational biology. However, pregnancy termination is required for such studies which precludes longitudinal investigations and necessitates the use of large numbers of animals. Therefore, we describe a non-invasive technique of high-frequency ultrasonography for early detection and monitoring of post-implantation events in the pregnant mouse.

High-frequency ultrasonography (HFUS) is a common method to non-invasively monitor the real-time development of the human fetus in utero. The mouse is ...

Developmental Biology

Analysis of Lymph Node Volume by Ultra-High-Frequency Ultrasound Imaging in the Braf/Pten Genetically Engineered Mouse Model of Melanoma

Tyr::CreER+,BrafCA/+,Ptenlox/lox&#160;genetically engineered mice (Braf/Pten mice) are widely used as an in vivo model of metastatic melanoma. Once a primary tumor has been induced by tamoxifen treatment, an increase in metastatic burden is observed within 4-6 weeks after induction. This paper shows how Ultra-High-Frequency UltraSound (UHFUS) imaging can be exploited to monitor the increase in metastatic involvement of the inguinal lymph nodes by measuring the increase in their volume.
The UHFUS system is used to scan anesthetized mice with a UHFUS linear probe (22-55 MHz, axial resolution 40 &#181;m). B-mode images from the inguinal lymph nodes (both left and right sides) are acquired in a short-axis view, positioning the animals in dorsal recumbency. Ultrasound records are acquired using a 44 &#181;m step size on a motorized mechanical arm. Afterward, two-dimensional (2D) B-mode acquisitions are imported into the software platform for ultrasound image post-processing, and inguinal lymph nodes are identified and segmented semi-automatically in the acquired cross-sectional 2D images. Finally, a total reconstruction of the three-dimensional (3D) volume is automatically obtained along with the rendering of the lymph node volume, which is also expressed as an absolute measurement.
This non-invasive in vivo technique is very well tolerated and allows the scheduling of multiple imaging sessions on the same experimental animal over 2 weeks. It is, therefore, ideal to assess the impact of pharmacological treatment on metastatic disease.

Melanoma is a very aggressive disease that quickly spreads to other organs. This protocol describes the application of ultra-high-frequency ultrasound imaging, coupled with 3D rendering, to monitor the volume of the inguinal lymph nodes in the Braf/Pten mouse model of metastatic melanoma.

Tyr::CreER+,BrafCA/+,Ptenlox/lox&#160;genetically engineered mice (Braf/Pten mice) are widely used as an in vivo model of metastatic melanoma. Once a ...

High-Resolution Ultrasonography for the Analysis of Orthotopic ATC Tumors in a Genetically Engineered Mouse Model

Anaplastic thyroid carcinoma (ATC) is associated with a poor prognosis and short median survival time, but no effective treatment improves the outcomes significantly. Genetically engineered murine models that mimic ATC&#39;s progression may help researchers to study treatments for this disease. Crossing three different genotypes of mice, a TPO-cre/ERT2; BrafCA/wt; Trp53&#916;ex2-10/&#916;ex2-10&#160;transgenic ATC model was developed. The ATC murine model was induced by an intraperitoneal injection of tamoxifen with overexpression of BrafV600E and deletion of Trp53, and the tumors were generated within about 1 month. High-resolution ultrasound was applied to investigate the tumor initiation and progression, and the dynamic growth curve was obtained by measuring the tumor sizes. Compared to magnetic resonance imaging (MRI) and computed tomography scanning, ultrasound has advantages in observing the ATC murine model, such as being noninvasive, portable, in real-time, and without radiation exposure. High-resolution ultrasound is suitable for dynamic and multiple measurements. However, ultrasonographic examination of the thyroid in mice requires relevant anatomical knowledge and experience. This article provides a detailed procedure for utilizing high-resolution ultrasound to scan tumors in the transgenic ATC model. Meanwhile, ultrasonic parameter adjustment, ultrasound scanning skills, anesthesia and recovery of the animals, and other elements that need attention during the process are listed.

The present protocol describes high-frequency ultrasonography for visualizing the entire mouse thyroid gland and monitoring the growth of anaplastic thyroid carcinoma.

Anaplastic thyroid carcinoma (ATC) is associated with a poor prognosis and short median survival time, but no effective treatment improves the outcomes ...

Murine Model for Non-invasive Imaging to Detect and Monitor Ovarian Cancer Recurrence

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of care consisting of surgical debulking and combination chemotherapy consisting of platinum and taxane compounds, almost 90% of patients recur within a few years. In these patients the development of chemoresistant disease limits the efficacy of currently available chemotherapy agents and therefore contributes to the high mortality. To discover novel therapy options that can target recurrent disease, appropriate animal models that closely mimic the clinical profile of patients with recurrent ovarian cancer are required. The challenge in monitoring intra-peritoneal (i.p.) disease limits the use of i.p. models and thus most xenografts are established subcutaneously. We have developed a sensitive optical imaging platform that allows the detection and anatomical location of i.p. tumor mass. The platform includes the use of optical reporters that extend from the visible light range to near infrared, which in combination with 2-dimensional X-ray co-registration can provide anatomical location of molecular signals. Detection is significantly improved by the use of a rotation system that drives the animal to multiple angular positions for 360 degree imaging, allowing the identification of tumors that are not visible in single orientation. This platform provides a unique model to non-invasively monitor tumor growth and evaluate the efficacy of new therapies for the prevention or treatment of recurrent ovarian cancer.

We describe a non-invasive animal imaging platform that allows the detection, quantification, and monitoring of ovarian cancer growth and recurrence. This intra-peritoneal xenograft model mimics the clinical profile of patients with ovarian cancer.

Epithelial ovarian cancer is the most lethal gynecologic malignancy in the United States. Although patients initially respond to the current standard of ...

Medicine

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States. Mortality is due to diagnosis of 75% of women with late stage disease, when metastasis is already present. EOC is characterized by diffuse and widely disseminated intra-peritoneal metastasis. Cells shed from the primary tumor anchor in the mesothelium that lines the peritoneal cavity as well as in the omentum, resulting in multi-focal metastasis, often in the presence of peritoneal ascites. Efforts in our laboratory are directed at a more detailed understanding of factors that regulate EOC metastatic success. However, quantifying metastatic tumor burden represents a significant technical challenge due to the large number, small size and broad distribution of lesions throughout the peritoneum. Herein we describe a method for analysis of EOC metastasis using cells labeled with red fluorescent protein (RFP) coupled with in vivo multispectral imaging. Following intra-peritoneal injection of RFP-labelled tumor cells, mice are imaged weekly until time of sacrifice. At this time, the peritoneal cavity is surgically exposed and organs are imaged in situ. Dissected organs are then placed on a labeled transparent template and imaged ex vivo. Removal of tissue auto-fluorescence during image processing using multispectral unmixing enables accurate quantitation of relative tumor burden. This method has utility in a variety of applications including therapeutic studies to evaluate compounds that may inhibit metastasis and thereby improve overall survival.

Ovarian cancer metastasis is characterized by numerous diffuse intra-peritoneal lesions, such that accurate visual quantitation of tumor burden is challenging. Herein we describe a method for in situ and ex vivo quantitation of metastatic tumor burden using red fluorescent protein (RFP)-labeled tumor cells and optical imaging.

Epithelial ovarian cancer (EOC) is the leading cause of death from gynecologic malignancy in the United States.  Mortality is due to diagnosis of 75% of ...

Using Micro-computed Tomography for the Assessment of Tumor Development and Follow-up of Response to Treatment in a Mouse Model of Lung Cancer

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse models of lung cancer are being used to study the mechanism of cancer development and to experiment with various therapeutic strategies. However, the absence of a real-time technique to identify the development of tumor nodules in mice lungs and to monitor the changes in their size in response to various experimental and therapeutic interventions hampers the ability to obtain an accurate description of the course of the disease and its timely response to treatments. In this study, a method using a micro-computed tomography (CT) scanner for the detection of the development of lung tumors in a mouse model of lung adenocarcinoma is described. Next, we show that monthly follow-up with micro-CT can identify dynamic changes in the lung tumor, such as the appearance of additional nodules, increase in the size of previously detected nodules, and decrease in the size or complete resolution of nodules in response to treatment. Finally, the accuracy of this real-time assessment method was confirmed with end-point histological quantification. This technique paves the way for planning and conducting more complex experiments on lung cancer animal models, and it enables us to better understand the mechanisms of carcinogenesis and the effects of different treatment modalities while saving time and resources.

We describe a method for the detection of tumor nodule development in the lungs of an adenocarcinoma mouse model using micro-computed tomography and its use for monitoring changes in nodule size over time and in response to treatment. The accuracy of the assessment was confirmed with end-point histological quantification.

Lung cancer is the most lethal cancer in the world. Intensive research is ongoing worldwide to identify new therapies for lung cancer. Several mouse ...

A Syngeneic Murine Model of Endometriosis using Naturally Cycling Mice

Endometriosis is a leading cause of pelvic pain and infertility. It is defined by the presence of endometrial tissue in extrauterine locations. The development of novel therapies and diagnostic tools for endometriosis has been limited due in part to challenges in studying the disease. Outside of primates, few mammals menstruate, and none develop spontaneous endometriosis. Rodent models are popular but require artificial induction of endometriosis, with many utilizing either immunocompromised mice or surgically induced disease. Recently, more attention has been given to models involving intraperitoneal injection. We present a murine model of endometriosis that integrates several features of existing endometriosis models into a novel, simplified system that relies on microscopic quantification in lieu of subjective grading. In this model, we perform hormonal stimulation of donor mice, intraperitoneal injection, systematic abdominal survey and tissue harvest, and histologic quantification that can be performed and verified at any time after necropsy. This model requires minimal resources and training; does not require expertise by lab technicians in murine survival surgery or in the identification of gross endometriotic lesions; can be used in immunocompromised, immunocompetent, and/or mutant mice; and reliably creates endometriotic lesions that are histologically consistent with human endometriotic disease.

Many rodent models of endometriosis are limited by technical complexity, reproducibility, and/or need for immunocompromised animals or special reporter mice. We present a simplified system of lesion induction using any experimental mouse with an independently verifiable, objective scoring system and with no requirement for ovariectomy or survival surgery.

Endometriosis is a leading cause of pelvic pain and infertility. It is defined by the presence of endometrial tissue in extrauterine locations. The ...

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice

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