Name: Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice (Video) | JoVE Journal
Uploaded: 2023-02-17T13:45:00.000+00:00
Duration: 7 min 44 s
Description: Learn noninvasive ultrasound monitoring of endometrial cancer progression in a Pax8-directed Arid1a and Pten deletion mouse model. This longitudinal method captures individual variation while reducing animal numbers.

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5 minutes

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/pubmed?term=((Staging+of+the+estrous+cycle+and+induction+of+estrus+in+experimental+rodents%3A+an+update%5BTitle%5D)+AND+%22Fertility+Research+and+Practice%22%5BJournal%5D)">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/pubmed?term=((Animal+models+closer+to+intrauterine+adhesive+pathology%5BTitle%5D)+AND+%22Annals+of+Translational+Medicine%22%5BJournal%5D)">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/pubmed?term=((Animal+models+of+polycystic+ovary+syndrome%3A+a+focused+review+of+rodent+models+in+relationship+to+clinical+phenotypes+and+cardiometabolic+risk%5BTitle%5D)+AND+%22Fertility+and+Sterility%22%5BJournal%5D)">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/pubmed?term=((Ultrasound+biomicroscopy+in+small+animal+research%3A+applications+in+molecular+and+preclinical+imaging%5BTitle%5D)+AND+%22Journal+of+Biomedicine+and+Biotechnology%22%5BJournal%5D)">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/pubmed?term=((Interobserver+agreement+of+transvaginal+ultrasound+and+magnetic+resonance+imaging+in+local+staging+of+cervical+cancer%5BTitle%5D)+AND+%22Ultrasound+in+Obstetrics+and+Gynecology%22%5BJournal%5D)">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/pubmed?term=((In+vivo+imaging+with+confirmation+by+histopathology+for+increased+rigor+and+reproducibility+in+translational+research%3A+A+review+of+examples%2C+options%2C+and+resources%5BTitle%5D)+AND+%22ILAR+Journal%22%5BJournal%5D)">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/pubmed?term=((Continuing+education+course+%231%3A+Non-invasive+imaging+as+a+problem-solving+tool+and+translational+biomarker+strategy+in+toxicologic+pathology%5BTitle%5D)+AND+%22Toxicologic+Pathology%22%5BJournal%5D)">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/pubmed?term=((Ultrasound+biomicroscopy%3A+A+non-invasive+approach+for+in+vivo+evaluation+of+oocytes+and+small+antral+follicles+in+mammals%5BTitle%5D)+AND+%22Reproduction%2C+Fertility+and+Development%22%5BJournal%5D)">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/pubmed?term=((Three-dimensional+ultrasound+biomicroscopy+for+xenograft+growth+analysis%5BTitle%5D)+AND+%22Ultrasound+in+Medicine+and+Biology%22%5BJournal%5D)">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/pubmed?term=((Complementarity+of+ultrasound+and+fluorescence+imaging+in+an+orthotopic+mouse+model+of+pancreatic+cancer%5BTitle%5D)+AND+%22BMC+Cancer%22%5BJournal%5D)">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/pubmed?term=((The+use+of+three-dimensional+ultrasound+micro-imaging+to+monitor+prostate+tumor+development+in+a+transgenic+prostate+cancer+mouse+model%5BTitle%5D)+AND+%22The+Tohoku+Journal+of+Experimental+Medicine%22%5BJournal%5D)">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/pubmed?term=((Ultrasound-guided+intrauterine+injection+of+lipopolysaccharide+as+a+novel+model+of+preterm+birth+in+the+mouse%5BTitle%5D)+AND+%22The+American+Journal+of+Pathology%22%5BJournal%5D)">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/pubmed?term=((Ultrasonography+in+experimental+reproductive+investigations+on+rats%5BTitle%5D)+AND+%22Journal+of+Visualized+Experiments%22%5BJournal%5D)">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/pubmed?term=((Inactivation+of+Arid1a+in+the+endometrium+is+associated+with+endometrioid+tumorigenesis+through+transcriptional+reprogramming%5BTitle%5D)+AND+%22Nature+Communications%22%5BJournal%5D)">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/pubmed?term=((Pre-existing+thyroiditis+ameliorates+papillary+thyroid+cancer%3A+Insights+from+a+new+mouse+model%5BTitle%5D)+AND+%22Endocrinology%22%5BJournal%5D)">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

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...

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.

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 ...

non-invasive-ultrasound-assessment-endometrial-cancer-progression

Research

JoVE Journal

Cancer Research

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.

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

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.

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

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.

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

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.

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

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.

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

Here, we present a protocol for live-imaging of fluorescently labeled human endometrial fragments grafted in mice. The method allows studying the effects of drugs of choice on endometriotic lesion size through monitoring and quantification of fluorescence emitted by the fluorescent reporter on real...

Noninvasive Monitoring of Lesion Size in a Heterologous Mouse Model of Endometriosis

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.

Detection of Lung Tumor Progression in Mice by Ultrasound Imaging

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.

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

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

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

This protocol describes an efficient, non-surgical method for the orthotopic implantation of breast cancer patient-derived xenografts in mice. The technique involves enzymatic tumor dissociation followed by direct injection into the mammary fat pads, enabling high-throughput implantation. Comprehensive validation ensures model fidelity, facilitating rigorous studies across various breast cancer subtypes.

High-Throughput Dissociation and Orthotopic Implantation of Breast Cancer Patient-Derived Xenografts

This protocol uses a multichamber bed for parallel magnetic resonance imaging (MRI) of up to four animals to detect pancreatic adenocarcinoma tumors in genetically engineered mouse models. This multianimal MRI protocol is fast and cost-effective for detecting and measuring tumors, facilitating animal selection for preclinical studies and longitudinal monitoring of tumor growth.

Multianimal Magnetic Resonance Imaging for Tumor Measurements in Pancreatic Cancer Mouse Models

Learn noninvasive ultrasound monitoring of endometrial cancer progression in a Pax8-directed Arid1a and Pten deletion mouse model. This longitudinal method captures individual variation while reducing animal numbers.

Non-Invasive Ultrasound Assessment of Endometrial Cancer Progression in Pax8-Directed Deletion of the Tumor Suppressors Arid1a and Pten in Mice (Video) | JoVE Journal

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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