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Method Article

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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DOI:

10.3791/64732

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February 17th, 2023

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In This Article

Summary

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.

Abstract

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.

Introduction

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 avera....

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Protocol

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

  1. Maintain 10 Pax8-Cre-<....

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Results

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.......

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Discussion

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.......

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Disclosures

The authors have no conflicts of interest to disclose.

Acknowledgements

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.

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
Reagents and Equipment Used for Animal Care
Rodent Diet (2018, 625 Doxycycline)EnvigioTD.01306Mouse Feed
Reagents and Equipment Used for Ultrasound Imaging
10 mL injectable 0.9% NaCl Hospira, IncRL-7302Isotonic Fluid
Absorbent Pad with Plastic BackingDaiggerEF8313Absorbant Pads
Anesthesia Induction ChambersHarvard Apparatus75-2029Induction Chamber
Anesthetic absorber kit with absorber canister, holder, tubing, & adaptersCWE, Inc13-20000Nose Cone and Tubing
Aquasonic Clear Ultrasound Gel (0.25 Liter)Parker Laboratoies08-03Ultrasound Gel
BD Plastipak 3 mL SyringeBD Biosciences309657Syringe
F/Air Scavenger Charcoal CanisterOMNICON80120Scavenging System for Anesthesia
Isoflurane, USPVet One502017Anesthesia Agent
M1050 Non-Rebreathing Mobile Anesthesia MachineScivena ScientificM1050Anestheic Vaporizer
MX550S, 25-55 MHz Transducer, 15mm, LinearVisualSonicsMX550SUltrasound Transducer (Probe)
Nair Hair Aloe & Lanolin Hair Removal Lotion - 9.0 ozNairDepilliating Cream
Philips Norelco Multigroomer All-in-One Trimmer Series 7000Philips North AmericaMG7750Clippers
PrecisionGlide 25 G 1" NeedleBD Biosciences305125Needle
Puralube Ophthalmic OintmentDechra17033-211-38Lubricating Eye Drops
Vevo 3100 Imaging SystemVisualSonicsVevo 3100Ultrasound Machine
Vevo LAB 5.6.1VisualSonicsVevo LAB 5.6.1Ultrasound Analysis Software
Vinyl Heating Pad with cover, 12 x 15"Sunbeam731-500-000RHeating Pad
Wd Elements 2TB Basic StorageWestern Digital ElementsWDBU6Y0020BBK-WESNData Storage
Reagents and Equipment Used for Immunohistochemistry
10% w/v FormalinFischer ScientificSF98-4Tissue Fixation Buffer
Animal-Free Blocker and Diluent, R.T.U.Vector Laboratories Inc. SP5035Antibody Blocker
Charged Super Frost Plus Glass SlidesVWR4831-703Tissue Mounting Slides
Citrate BufferMilliporeSigma C9999-1000MLEpitope Retrival Buffer (pTEN)
Cytoseal – 60Thermo Scientific8310-4Resin for Slide Sealing
Gold Seal Cover GlassThermo Scientific3322Coverslide
Harris Modified HematoxylinMilliporeSigmaHHS32-1LCounterstain Buffer
Hybridization Incubator (Dual Chamber)Fischer Scientific13-247-30QOven to Melt Parraffin
ImmPACT DAB Substrate, Peroxidase (HRP)Vector Laboratories Inc.SK-4105Signal Development Substrate
ImmPRESS HRP Goat Anti-Rabbit IgG Polymer Detection Kit, PeroxidaseVector Laboratories Inc.MP-7451Secondary IHC Antibody
Oster 5712 Digital Food SteamerOster5712Vegetable Steamer for Epitope Retrival
rabbit mAB anti-ARID1aabcamab182560Primary IHC Antibody (1:1,000)
rabbit mAB anti-PTENCell Signaling9559Primary IHC Antibody (1:100)
Scotts Tap Water SubstituteMilliporeSigmaS5134-100ML"Blueing" Buffer
Tissue Path IV CassetteFischer Scientific22272416Tissue Fixation Cassette
Trilogy BufferCell Marque 920P-10Epitope Retrival Buffer (ARID1a)

References

  1. Ajayi, A. F., Akhigbe, R. E. Staging of the estrous cycle and induction of estrus in experimental rodents: an update. Fertility Research and Practice. 6, 5(2020).
  2. Kim, S. W., Kim, Y. Y., Kim, H., Ku, S. Y. Animal models closer to intr....

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