Name: non-invasive ultrasound assessment of endometrial cancer progression in pax8-directed deletion of the tumor suppressors arid1a and pten in mice
Uploaded: 2023-02-17T13:45:00
Description: 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

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. Inducti...

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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. 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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>

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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagents and Equipment Used for Animal Care</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>Reagents and Equipment Used for Ultrasound Imaging</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>10 mL injectable 0.9% NaCl&#160;</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, &#38; 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 &#38; 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&#34; 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&#34;</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>Reagents and Equipment Used for Immunohistochemistry</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>&#160;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>&#160;C9999-1000ML</td>
			<td>Epitope Retrival Buffer (pTEN)</td>
		</tr>
		<tr>
			<td>Cytoseal &#8211; 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>&#34;Blueing&#34; 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>&#160;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 ...

This study shows that manual guided ultrasound is well-suited to monitor the development of uterine pathology in an induced mouse model of endometrial cancer. Ultrasound imaging is noninvasive and thus allows for longitudinal studies of individual mice across days and weeks. This allows for the capture of individual variation and ultimately for fewer animals to be used for study.This method can be readily applied to monitor pathologic changes and other mirroring models, including those for neoplasia and chronic disease of the urinary and digestive tract. Begin setting up the ultrasound machine for imaging the uterus or ovary by selecting a transducer or probe with a range of 32 to 56 megahertz. Go to the Applications tab from the home screen and select Mouse Small Abdomen Mode.Then click on Scan to return to the home screen and wait for a live image to be displayed. Next, select B Mode from the options on the left toolbar before clicking on More Controls option 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. Once the image settings are selected, return to the home screen by clicking on B Mode.Place a three to 10-milliliter syringe filled with a sterile isotonic fluid solution between a heating pad and an absorbent pad for several minutes to preheat it to 35 to 40 degrees Celsius. If the machine does not have a warmer, place a bottle of ultrasound gel on the heating pad. To inject one to two milliliters of solution into the peritoneal cavity of a mouse, secure the mouse by the scruff in one hand, exposing the ventrum.Then hold the mouse at approximately a 20-degree angle with the nose pointed to the floor. Insert a 25-gauge tuberculin syringe needle through the skin and abdominal wall of the caudal right quadrant of the abdomen. To avoid injection into the vasculature or the GI tract, pull back on the syringe plunger with minimal pressure to look for blood.Position the mouse in ventral recumbency on the absorbent pad over a heating pad before securely placing a rodent nose cone over the mouse's nose and muzzle. Place a small amount of pre-warmed ultrasound gel abaxial to the spine on either side of the anesthetized mouse between the last rib and pelvis. Put a small amount of gel on the ultrasound probe.Next, place the probe parallel to the vertebrae with the front of the probe on the cranial side. With a mouse in ventral recumbency, slowly scan the area for the kidney landmark. With the kidney in view, pull the probe caudal to find the ovary, which is a slightly hyperechoic oval to round structure within a very hyperechoic ovarian fat pad, bordered cranial-ventrally by the kidney and dorsal-laterally by the dorsal abdominal wall.Improve the image contrast by adjusting the signal gain using the slider at the bottom of the control screen. To improve the imaging of the kidney, apply pressure to the contra-lateral abdomen using a finger. Vary the pressure and angle from parallel to the spine to approximately 20 degrees ventral.Once the organ of interest is in view, capture a video by clicking on Save Clip or Start Recording. Once done, click Stop Recording to retain images at a preset number of frames. Hold the probe caudally until the ovary is in the most cranial aspect of the field of view.Vary the probe pressure and angle to view and image the uterus. Repeat video and frame collection for each organ of interest. Find the uterus running longitudinally along the dorsal abdominal wall with the lateral leg musculature in view.Monitor the tissue for a peristaltic motion to differentiate the intestinal loops from the uterine stationary horns. Place the mouse in dorsal recumbency to collect the images from a ventral approach. Ensure the eye lubrication is sufficient and the muzzle is secured in the nose cone.Apply a small amount of pre-warmed ultrasound gel to the ventral abdomen before applying the probe at the midline and cranial of the pubis to locate the bladder as a hypoechoic landmark. To find the uterine horns, pull the probe lateral to the bladder. Apply light digital pressure from either or both sides of the mouse.Once the uterine horns are in the field of view, hold the probe perpendicular to the mouse and scan both sides of the abdomen to capture transverse views of both horns. Rotate the probe to capture sagittal views. After the ultrasound, wipe the mouse with a paper towel before returning it to its cage to recover.Once the mouse is fully awake after two to five minutes, return the mouse to the animal room. The ultrasound image comparison of the mouse abdominal cavity before and after the saline injection showed the improved visualization of the abdominal organs, including the uterus and ovaries after the saline injection. A ventral approach was used to study the cross-sections of the uterine horns adjacent to the hypoechoic bladder.Immunohistochemistry with antibodies directed against Arid1a and Pten was used to study the adequate tumor suppressor protein loss accomplished in the uterine epithelial cells compared to the neighboring stromal tissue. Ultrasound images of a mouse uterus with early pathology changes were detected after three weeks of doxycycline treatment, showing the hypoechoic and dilated lumen of the uterus. In the gross pathology and histopathology study of the same uterus, a smaller but dilated thin-walled uterus and early endometrial hyperplasia with the preservation of the distinct lumen were observed respectively.The mice euthanized at later time points showed larger uterus with a smaller lumen and a thickened wall. When learning to use manual placement of probes, excessive pressure or rapid movement can make organ identification difficult. Use gentle, deliberate motions and focus on identifying the organs which are landmarks first.At study endpoint, mice are sacrificed and tissue is collected to confirm gene knockdown and characterize tissue changes. This technique allows investigators to monitor intraabdominal lesions over time. And in our case, we used it to monitor the uterine cancer progression in the iPad model, which we then confirmed by histopathology.

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Cancer Research

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the context of a whole organism. Sophisticated techniques to generate genetic mosaics facilitate induction of visually marked, genetically defined clones surrounded by normal tissue. The clones can be analyzed through diverse molecular, cellular and omics approaches. This study describes how to generate fluorescently labeled clonal tumors of varying malignancy in the eye/antennal imaginal discs (EAD) of Drosophila larvae using the Mosaic Analysis with a Repressible Cell Marker (MARCM) technique. It describes procedures how to recover the mosaic EAD and brain from the larvae and how to process them for simultaneous imaging of fluorescent transgenic reporters and antibody staining. To facilitate molecular characterization of the mosaic tissue, we describe a protocol for isolation of total RNA from the EAD. The dissection procedure is suitable to recover EAD and brains from any larval stage. The fixation and staining protocol for imaginal discs works with a number of transgenic reporters and antibodies that recognize Drosophila proteins. The protocol for RNA isolation can be applied to various larval organs, whole larvae, and adult flies. Total RNA can be used for profiling of gene expression changes using candidate or genome-wide approaches. Finally, we detail a method for quantifying invasiveness of the clonal tumors. Although this method has limited use, its underlying concept is broadly applicable to other quantitative studies where cognitive bias must be avoided.

The Drosophila Imaginal Disc Tumor Model: Visualization and Quantification of Gene Expression and Tumor Invasiveness Using Genetic Mosaics

This protocol demonstrates how to generate fluorescently marked, genetically defined clonal tumors in the Drosophila eye/antennal imaginal discs (EAD). It describes how to dissect the EAD and brain from the third instar larvae and how to process them to visualize and quantify gene expression changes and tumor invasiveness.

Drosophila melanogaster has emerged as a powerful experimental system for functional and mechanistic studies of tumor development and progression in the ...

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic probes to provide quantitative information on the chemical tumor microenvironment (TME), including pO2, pH, redox status, concentrations of interstitial inorganic phosphate (Pi), and intracellular glutathione (GSH). In particular, an application of a recently developed soluble multifunctional trityl probe provides unsurpassed opportunity for in vivo concurrent measurements of pH, pO2 and Pi in Extracellular space (HOPE probe). The measurements of three parameters using a single probe allow for their correlation analyses independent of probe distribution and time of the measurements.

In Vivo EPR Assessment of pH, pO2, Redox Status, and Concentrations of Phosphate and Glutathione in the Tumor Microenvironment

Low-field (L-band, 1.2 GHz) electron paramagnetic resonance using soluble nitroxyl and trityl probes is demonstrated for assessment of physiologically important parameters in the tumor microenvironment in mouse models of breast cancer.

This protocol demonstrates the capability of low-field electron paramagnetic resonance (EPR)-based techniques in combination with functional paramagnetic ...

Radionuclide-fluorescence Reporter Gene Imaging to Track Tumor Progression in Rodent Tumor Models

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis remains incomplete. In vivo models are paramount for metastasis research, but require refinement. Tracking spontaneous metastasis by non-invasive in vivo imaging is now possible, but remains challenging as it requires long-time observation and high sensitivity. We describe a longitudinal combined radionuclide and fluorescence whole-body in vivo imaging approach for tracking tumor progression and spontaneous metastasis. This reporter gene methodology employs the sodium iodide symporter (NIS) fused to a fluorescent protein (FP). Cancer cells are engineered to stably express NIS-FP followed by selection based on fluorescence-activated cell sorting. Corresponding tumor models are established in mice. NIS-FP expressing cancer cells are tracked non-invasively in vivo at the whole-body level by positron emission tomography (PET) using the NIS radiotracer [18F]BF4-. PET is currently the most sensitive in vivo imaging technology available at this scale and enables reliable and absolute quantification. Current methods either rely on large cohorts of animals that are euthanized for metastasis assessment at varying time points, or rely on barely quantifiable 2D imaging. The advantages of the described method are: (i) highly sensitive non-invasive in vivo 3D PET imaging and quantification, (ii) automated PET tracer production, (iii) a significant reduction in required animal numbers due to repeat imaging options, (iv) the acquisition of paired data from subsequent imaging sessions providing better statistical data, and (v) the intrinsic option for ex vivo confirmation of cancer cells in tissues by fluorescence microscopy or cytometry. In this protocol, we describe all steps required for routine NIS-FP-afforded non-invasive in vivo cancer cell tracking using PET/CT and ex vivo confirmation of in vivo results. This protocol has applications beyond cancer research whenever in vivo localization, expansion and long-time monitoring of a cell population is of interest.

We describe a protocol for preclinical in vivo tracking of cancer metastasis. It is based on a radionuclide-fluorescence reporter combining the sodium iodide symporter, detected by non-invasive [18F]tetrafluoroborate-PET, and a fluorescent protein for streamlined ex vivo confirmation. The method is applicable for preclinical in vivo cell tracking beyond tumor biology.

Metastasis is responsible for most cancer deaths. Despite extensive research, the mechanistic understanding of the complex processes governing metastasis ...

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

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

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

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

Enrichment and Characterization of the Tumor Immune and Non-immune Microenvironments in Established Subcutaneous Murine Tumors

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for immunotherapies. Recent clinical advances in immunotherapy highlight the need for reproducible methods to accurately and thoroughly characterize the tumor and its associated immune infiltrate. Tumor enzymatic digestion and flow cytometric analysis allow broad characterization of numerous immune cell subsets and phenotypes; however, depth of analysis is often limited by fluorophore restrictions on panel design and the need to acquire large tumor samples to observe rare immune populations of interest. Thus, we have developed an effective and high throughput method for separating and enriching the tumor immune infiltrate from the non-immune tumor components. The described tumor digestion and centrifugal density-based separation technique allows separate characterization of tumor and tumor immune infiltrate fractions and preserves cellular viability, and thus, provides a broad characterization of the tumor immunologic state. This method was used to characterize the extensive spatial immune heterogeneity in solid tumors, which further demonstrates the need for consistent whole tumor immunologic profiling techniques. Overall, this method provides an effective and adaptable technique for the immunologic characterization of subcutaneous solid murine tumors; as such, this tool can be used to better characterize the tumoral immunologic features and in the preclinical evaluation of novel immunotherapeutic strategies.

Here we describe a method to separate and enrich components of the tumor immune and non-immune microenvironment in established subcutaneous tumors. This technique allows for the separate analysis of tumor immune infiltrate and non-immune tumor fractions which can permit comprehensive characterization of the tumor immune microenvironment.

The tumor immune microenvironment (TIME) has recently been recognized as a critical mediator of treatment response in solid tumors, especially for ...

Digital Polymerase Chain Reaction Assay for the Genetic Variation in a Sporadic Familial Adenomatous Polyposis Patient Using the Chip-in-a-tube Format

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as tumors. Because digital polymerase chain reactions (PCR) enable the precise quantification of DNA copy number variants, they are becoming an essential tool for detecting rare genetic variations, such as drug-resistant mutations. It is expected that molecular diagnoses using digital PCR (dPCR) will be available in clinical practice in the near future; thus, how to efficiently conduct dPCR with human genetic material is a hot topic. Here, we introduce a method to detect Adenomatous polyposis coli (APC) somatic mosaicism using dPCR with the chip-in-a-tube format, which allows eight dPCR reactions to be simultaneously conducted. Care should be taken when filling and sealing the reaction mixture on the chips. This article demonstrates how to avoid the over- and underestimation of positive partitions. Furthermore, we present a simple procedure for collecting the dPCR product from the partitions on the chips, which can then be used to confirm the specific amplification. We hope that this methods report will help promote the dPCR with the chip-in-a-tube method in genetic research.

Digital polymerase chain reaction (PCR) is a useful tool for the high-sensitivity detection of single nucleotide variants and DNA copy number variants. Here, we demonstrate key considerations for measuring rare variants in the human genome using digital PCR with the chip-in-a-tube format.

The quantitative analysis of human genetic variation is crucial for understanding the molecular characteristics of serious medical conditions, such as ...

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

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

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

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

In Vivo Inhibition of MicroRNA to Decrease Tumor Growth in Mice

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ever-increasing number of studies have identified miRNAs as potential biomarkers for cancer diagnosis and prognosis, and also as therapeutic targets, adding an extra dimension to cancer evaluation and treatment. In the context of thyroid cancer, tumorigenesis results not only from mutations in important genes, but also from the overexpression of many miRNAs. Accordingly, the role of miRNAs in the control of thyroid gene expression is evolving as an important mechanism in cancer. Herein, we present a protocol to examine the effects of miRNA-inhibitor delivery as a therapeutic modality in thyroid cancer using human tumor xenograft and orthotopic mouse models. After engineering stable thyroid tumoral cells expressing GFP and luciferase, cells are injected into nude mice to develop tumors, which can be followed by bioluminescence. The in vivo inhibition of a miRNA can reduce tumor growth and upregulate miRNA gene targets. This method can be used to assess the importance of a determined miRNA in vivo, in addition to identifying new therapeutic targets.

This protocol describes xenograft and orthotopic mouse models of human thyroid tumorigenesis as a platform to test microRNA-based inhibitor treatments. This approach is ideal to study the function of non-coding RNAs and their potential as new therapeutic targets.

MicroRNAs (miRNAs) are important regulators of gene expression through their ability to destabilize mRNA and inhibit translation of target mRNAs. An ...

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 ...

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

T cell-mediated immunity plays a crucial role in immune responses against tumors, with cytotoxic T lymphocytes (CTLs) playing the leading role in eradicating cancerous cells. However, the origins and replenishment of tumor antigen-specific CD8+ T cells within the tumor microenvironment (TME) remain obscure. This protocol employs the B16F10-OVA melanoma cell line, which stably expresses the surrogate neoantigen, ovalbumin (OVA), and TCR transgenic OT-I mice, in which over 90% of CD8+ T cells specifically recognize the OVA-derived peptide OVA257-264 (SIINFEKL) bound to the class I major histocompatibility complex (MHC) molecule H2-Kb. These features enable the study of antigen-specific T cell responses during tumorigenesis.
Combining this model with tumor transplantation surgery, tumor tissues from donors were transplanted into tumor-matched syngeneic recipient mice to precisely trace the influx of recipient-derived immune cells into transplanted donor tissues, allowing the analysis of the immune responses of tumor-inherent and periphery-originated antigen-specific CD8+ T cells. A dynamic transition was found to occur between these two populations. Collectively, this experimental design has provided another approach to precisely investigate the immune responses of CD8+ T cells in TME, which will shed new light on tumor immunology.

Tumor Transplantation for Assessing the Dynamics of Tumor-Infiltrating CD8+ T Cells in Mice

Here, we present a tumor transplantation protocol for the characterization of tumor-inherent and periphery-derived tumor-infiltrated lymphocytes in a mouse tumor model. Specific tracing of the influx of recipient-derived immune cells with flow cytometry reveals the dynamics of the phenotypic and functional changes of these cells during antitumor immune responses.