This work was supported by Spanish Ministry of Economy and Competitiveness through the Miguel Servet Program [CP13/00077] cofounded by FEDER (European Regional Development Fund) and awarded to Dr R. G&#243;mez as well as by Carlos III Institute of Health grants awarded to Dr R G&#243;mez [PI14/00547 and PI17/02329] and to Prof A. Cano [PI12/02582].

The use of human tissue specimens was approved by the Institutional Review Board and Ethics Committee of the Hospital Universitario La Fe. All patients provided written informed consent. The study involving animals was approved by the Institutional Animal Care Committee at the Centro de Investigacion Principe Felipe de Valencia, and all procedures were performed following the guidelines for the care and use of mammals from the National Institutes of Health.
1. Endometrial Tissue Collection and Pre-processing
<ol>
	<li>Obtain a good quality biopsy of human endometrial aspirate by using a cannula attached to a suction device. Pour the biopsy into a flask containing 10 mL of sterile saline and wash with gentle manual agitation. 
	NOTE: Procedures on how to obtain good quality biopsies have been previously described17.</li>
	<li>Wash the biopsy of any remaining blood or mucus. Repeat the process by pouring the tissue to flasks containing fresh saline as many tines as required until tissue is observed clean.</li>
	<li>Transfer biopsy fragments with a healthy appearance into a solution containing 10 mL of complete Dulbecco&#39;s Modified Eagle&#39;s medium (DMEM) medium with 10% Fetal Bovine Serum (FBS) and 1% antibiotic-antimycotic solution.</li>
	<li>Pour the content in a 10 cm Petri dish and proceed to chop the tissue into 5&#8211;10 mm3 pieces with a pair of scalpels.</li>
</ol>
2. Adenoviral Transfection of Endometrial Fragments
NOTE: All the materials that are going to be employed in the process should be introduced in the hood in advance. Take out everything that is not going to be used in the process and place a flask with bleach. All material that comes into contact with the adenoviral vector must be disinfected with the bleach before discarding it in the biohazard container.
<ol>
	<li>Once the biopsy has been chopped, take a new Petri dish. Pipette multiple 30&#8211;50 &#181;L DMEM drops of spread throughout the whole dish. Leave enough space between drops so they do not come into contact.</li>
	<li>Aided with a needle attached to syringe, place a single piece of fragment inside each of the medium drops. 
	NOTE: Each drop should contain a single piece of endometrium.</li>
	<li>Prepare the Ad-mCherry working solution by diluting 1:20 of the mCherry adenoviral stock solution [1 x 1010 pfu/mL]) into DMEM medium WITHOUT antibiotics (DMEM + 10% filtered FBS).</li>
	<li>Dispense 100&#8211;200 &#956;L of the Ad-mCherry solution per well on a 96-well plate, filling as many wells as fragments are available in the Petri dish drops (step 2.2). In addition, fill at least 3 wells with adenovirus free DMEM medium as a negative control.</li>
	<li>Aided with a needle attached to syringe, transfer each of the fragments in the Petri dish (step 2.2) to each of the wells containing ad-mCherry solution in the 96-well plate.</li>
	<li>Place the 96 well-plate containing the endometrial fragments with Ad-mCherry solution into an incubator at 37 &#176;C with 5% CO2 for 16 h.</li>
	<li>Wash out the remaining adenoviruses in the medium by transferring tissues into a new 96-well plate filled with complete DMEM medium (WITH antibiotics-antimycotics, free of adenovirus) and incubate for 37 &#176;C with 5% CO2 for 24&#8211;48 h. 
	NOTE: Remember to cover with bleach all well plates and tips that came in contact with Ad-virus before discarding into the biohazard container.</li>
	<li>Take out the plate from the incubator and place it under a fluorescence microscope at 568 nm (red channel) to test for optimal labeling.</li>
	<li>Choose the most brilliant fragments and place them into a new 96-well plate with fresh complete DMEM medium (WITH antibiotics-antimycotics, free of adenovirus).</li>
	<li>Seal the plate well with a plastic paraffin film and transport it to into the specific-pathogen-free animal area for implantation of endometrial fragments into recipient animals.</li>
</ol>
3. Generation of the Endometriosis Mouse Model
NOTE: Use 6&#8211;8 week-old athymic nude (or similar immunocompromised strains) female mice housed in specific pathogen&#8211;free conditions, as recipient animals. To avoid hormonal cycle-dependent variations and simultaneously fuel lesion growth with estradiol, animals are ovariectomized and placed with 60-day release capsules containing 18 mg of 17 &#946;eta-Estradiol (17&#946;-E2). Oophorectomy and pellet placement have to be performed at least one week in advance of grafting endometrial fragments into the recipient animals.
<ol>
	<li>Oophorectomy 
	NOTE: Prepare surgical sterilized material in a hood. Prepare anesthesia equipment and a post-surgical recovery zone ready in the same room.
	<ol>
		<li>Perform a subcutaneous injection of morphine derivative at a dose of 5 mg/kg per mouse. Let the mice rest for 30 min after injection so as analgesics effects of drug can be manifested.</li>
		<li>Connect the inhalation anesthesia equipment and let oxygen and isoflurane (2% mg/kg) flow for a few min into a sealed anesthesia chamber.</li>
		<li>Introduce the animal into the isoflurane anesthesia chamber. Wait for 3&#8211;5 min and check that animals are fully anesthetized by pressing one of its paws. Transfer the animal to the surgery area and maintain anesthesia by placing a mask with continuous flow of isoflurane gas covering the respiratory airways.</li>
		<li>After disinfecting the area with chlorohexidine, perform a transverse 0.5 cm costal incision approximately at the height of the hip with sharp scissors.</li>
		<li>Separate the skin from the muscle to get access to the abdominal cavity, identify the white fat pad that surrounds the ovary and retract the ovary with dissection forceps.</li>
		<li>Tie a knot around the oviduct with absorbable suture and tighten it to ensure appropriate hemostasis before excising the ovary.</li>
		<li>Close the muscular layer with 6-0 absorbable suture, and then close the skin with 6-0 non-absorbable suture. Clean the area again with antiseptic solution.</li>
		<li>Repeat the procedure to remove the contra lateral ovary.</li>
	</ol>
	</li>
	<li>Estradiol pellet implant 
	NOTE: Take care that the animal is anesthetized during the oophorectomy surgery to place pellets at that point.
	<ol>
		<li>Immediately after the completion of the oophorectomy procedure, clean the skin with antiseptic solution surrounding the neck and make a transverse subcutaneous small (0.5 cm) incision with sharp scissors in the nape.</li>
		<li>Use the scissors to dissect the skin from the muscle, making a pocket large enough to allow placing the pellets.</li>
		<li>Insert the pellet containing 18 mg of 17&#946;-E2 and suture the skin with a 6-0 non-absorbable suture. Clean the area again with antiseptic solution.</li>
		<li>Place the animal in the recovery zone and administer an optimal dose of long-lasting analgesia to ease the recovery period.</li>
	</ol>
	</li>
	<li>Endometrial implant surgery 
	NOTE: Allow at least a period of seven days quarantine to allow full recovery of animals after oophorectomy before starting endometrial fragment implantation surgery. For optimal synchronization with labeling of tissue, collect the biopsy 2&#8211;3 days before implantation surgery so as to avoid long-term culture of explants.
	<ol>
		<li>Get the surgical room in the specific pathogen free zone ready in advance. Prepare the hood with all required surgical material, the anesthesia equipment and the post-surgical recovery zone also.</li>
		<li>Bring the animals to the room, perform a subcutaneous injection of a morphine derivative at a dose of 5 (mg/kg) in each mouse. Let the mice rest for 30 min after injection so analgesics effects of drug can be manifested.</li>
		<li>Connect the inhalation anesthesia equipment and let oxygen and isoflurane (2% mg/kg) flow for a few min into a sealed anesthesia chamber.</li>
		<li>Before starting surgery, move the plate containing fluorescently labeled fragments (from step 2.10) into the hood, unseal it and pour the fragments into a Petri dish for easier handling.</li>
		<li>Introduce the animal into the isoflurane anesthesia chamber. Wait for 3&#8211;5 min and check that animals are fully anesthetized by pressing one of its paws. Transfer the animal to the surgery area and maintain anesthesia by placing a mask with continuous flow of isoflurane gas covering the respiratory airways.</li>
		<li>Place the anesthetized animal face up. Disinfect the ventral area. Perform a longitudinal 1.5 cm incision in abdomen with sharp scissors and separate the skin from the muscle. Then, perform a longitudinal 1.5 cm incision in the muscle to access the peritoneal cavity.</li>
		<li>Hold the left edge of the abdomen muscular wall with mini-forceps and fold it trying to expose the inner face of the peritoneum on the outside.</li>
		<li>Take an endometrial implant with mini tweezers, soak it briefly in an n-butyl-ester cyanoacrylate adhesive and place it to the peritoneum where it will get attached. Let it dry for a few seconds. Repeat steps 3.3.6 and 3.3.7 to place an implant on the contralateral side of the peritoneum.</li>
		<li>Close the muscular layer with an absorbable 6-0 suture, and then close the skin with a non-absorbable 6-0 suture. Clean the area again with antiseptic solution.</li>
		<li>Place the animal in the recovery zone and administer an optimal dose of long-lasting analgesia.</li>
	</ol>
	</li>
</ol>
4. In Vivo Fluorescent Imaging with an In Vivo Imaging System
<ol>
	<li>Turn on the in vivo imaging system device, initialize the program and allow the CCD camera to cool down for a few min.</li>
	<li>Prepare the inhalation anesthesia equipment. Open the isoflurane flow at 2% for a couple of min to fill the anesthetic chamber. Prepare the post-anesthesia recovery zone.</li>
	<li>Once the program has started and the CCD camera have cooled down, click the Imaging Wizard tool: A tutorial starts with a series of consecutive windows displayed, each one corresponding to a parameter of interest with several options available to be chosen by clicking in the corresponding box. Move forward through the tutorial by selecting the appropriate boxes in each window and click the OK button to move to the next set of parameters with the following sequence.
	<ol>
		<li>Select the Epiluminescence box for the fluorescence parameter, select the mCherry box for the filter pairs parameter. Check the Photograph mode and Confirm Focus boxes and select the Automatic box for the exposure parameter.</li>
	</ol>
	</li>
	<li>Once the instrument has been set up, move one animal inside the anesthesia chamber. When fully anesthetized, transfer the animal inside the in vivo imaging system cage and place it side up with its head inside a tubule connected to the anesthesia machine. Close the lid and click Acquire for monitoring.</li>
	<li>Acquire the images appearing (a total of five images, one image for each pair of filters selected) and save data by clicking the Save As button. Move the mouse to the area of post-anesthesia recovery. Repeat the process with the remaining animals.</li>
	<li>Repeat monitoring two or three times a week to follow-up the signal appropriately during the time course.</li>
	<li>Proceed to sacrifice the animal at the end of the time course by CO2 asphyxiation.</li>
</ol>
5. Quantification of In Vivo Fluorescence Images
<ol>
	<li>Segregation of actual fluorescence through image unmixing:
	<ol>
		<li>Open the In Vivo Imaging Analysis Coupled Software Program.</li>
		<li>Choose the &#8220;Sequenceinfo&#8221; file to start the analysis. Two windows will appear: &#8220;Sequence view&#8221; and &#8220;Tool Palette&#8221;. Choose Tool Palette and once the menu has been displayed select the following options.</li>
		<li>Select Corrections and click on the box Adaptive FL Background Subtraction to remove the undesirable fluorescent signals from the luminescent image data. Choose the threshold of greatest interest and click Set.</li>
		<li>Click Spectral Unmixing, select the wavelengths of interest, the method chosen for unmixing (library, guided, automatic or manual) and then click Start Unmix.</li>
		<li>Select the Unmixed image corresponding to mCherry signal and double click. A new window will appear with the final image of the signal of interest.</li>
		<li>Repeat step 5.1.3.</li>
		<li>OPTIONAL: If a representative image (JPEG) of the unmix result is needed, choose the desired settings in Tool Palette | Image Adjust (color table, binning, contrast, etc.), and then click on Export Graphics in the unmix window to export the current image view as an image.</li>
		<li>Save the unmixed file: File | Save As | Choose Folder and Ok.</li>
		<li>Repeat the process with the rest of the monitoring days and with all animals.</li>
	</ol>
	</li>
	<li>ROIs set up and signal quantification
	<ol>
		<li>Click Browse and select the Unmixed file (see step 5.1.8) of interest to be analyzed. A new window will appear.</li>
		<li>Click Add To List to include all the unmixed files from each animal at different time points and then click on Load As A Group. All images must appear as a single sequence.</li>
		<li>Go to Tool Palette window: Click off the box Individual scale to obtain all images on the same scale.</li>
		<li>Double click in one image of the sequence and create a ROI on the zone of interest using the following sequence. Go to ROI Tools and select Countour and Auto 1 option, click on the circle shape appearing, place it on the center the fluorescent signal and then click Create on the displayed window. 
		Note: This automatically highlights pixels with an intensity of fluorescence above background values (i.e., lesion) and generates a shape whose area embraces the outlined pixels.</li>
		<li>Copy the created ROI shape and paste on a background zone where there is no signal.</li>
		<li>Click on Measure ROIs | Select All to display values of fluorescence intensity. Proceed to select data values with the mouse, click right button, press copy and paste on a spreadsheet.</li>
	</ol>
	</li>
</ol>
6. Data (fluorescent signal) Normalization
<ol>
	<li>Select the initial time point at which signal intensity is maximal. Proceed to normalize signal at each time point by using the formula: 
	Signal intensity at each time point / Maximal signal intensity observed during the time course) x 100.</li>
</ol>

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The authors have nothing to disclose.

The protocol herein detailed describes the implementation of an animal model of endometriosis in which the architecture of implanting lesions architecture is preserved whilst simultaneously allowing real time assessment of fluorescence emitted by mCherry labeled endometrial tissue. In this protocol, we describe the use of a specific in vivo imaging system and related software to non-invasively assess fluorescence emitted by the labeled lesion. Each user should adapt the protocol depending on the specific imaging device a...

Endometriosis is a chronic gynecologic disorder initiated by the implantation of the functional endometrium outside the uterine cavity. Ectopic lesions grow and induce inflammatory processes leading to chronic pelvic pain and infertility1. It is estimated that up to 10&#8211;15% of women of reproductive age are affected by endometriosis2, and it is present in approximately 40&#8211;50% of infertile women3. Current pharmacological treatments for endometriosis are unable to completely eradicate lesions and not free of side effects4,5. The research for more efficient therapies requires of the refinement of the existing animal models of endometriosis in such a way that human lesions can be appropriately mimicked, and the effects of compounds on lesion size among others can be closely assessed.
Primate models have been used to mimic endometriosis by implanting ectopic lesions histologically identical and at similar sites as in humans6,7,8; however, ethical concerns and the high economic costs related to experimentation with primates limit their use9. Consequently, the use of small animals, especially rodents, for the implementation of in-vivo models of endometriosis continues to be favored as it allows studies with larger numbers of individuals10,11. Endometriosis can be induced in these animals by transplanting either pieces of rodent uterine horns (&#34;homologous models&#34;)12,13 or human endometrial/endometriotic tissue to ectopic sites (heterologous models)14. In contrast to humans, rodents do not shed their endometrial tissue and thereby endometriosis can not be developed spontaneously in these species. Therefore, homologous mouse models of endometriosis have been criticized due to the fact that implanted ectopic mouse uterine tissue does not reflect the characteristics of human endometriotic lesions15.
Appropriate physiology of endometriosis can be mimicked in the heterologous models of endometriosis where fresh human endometrial fragments are implanted into immunodeficient animals. In conventional heterologous models, the therapeutic effects of compounds of interest are commonly assessed at the end point by the assessment of lesion size with the use of calipers16. An obvious limitation is that, as such, endpoint animal models do not allow studying implantation dynamics or endometriotic lesion development over time. An additional limitation is that the use of calipers does not allow accurate measurements of lesion size. Indeed, the standard error provided by calipers is in the same range (i.e., millimeters) as the size of the lesions implanted in mice, thus restricting the capacity of these tools to detect actual variations in size.
In order to overcome such limitations, herein, we describe the generation of a heterologous mouse model of endometriosis in which implanted human tissue is engineered to express a reporter m-Cherry fluorescent protein. Detection of the fluorescent signal with an appropriate image system enables non-invasive monitoring of lesion status with simultaneous quantification of its size in real time. Thus, our model provides clear advantages when compared to conventional endpoint models as it brings the opportunity of real-time non-invasive monitoring and the possibility to perform more objective and accurate estimation of variations in lesion size.

<table><tbody><tr><td>Endosampler&amp;trade;</td><td>Medgyn</td><td>22720</td><td>Cannula for sampling the uterine endometrium</td></tr><tr><td>DMEM Medium</td><td>VWR</td><td>HYCLSH30285.FS</td><td>Medium</td></tr><tr><td>Ad-mCherry</td><td>Vector Biolabs</td><td>1767</td><td>Adenoviral vector expressing mCherry</td></tr><tr><td>PBS, 1x solution, sterile, pH 7.4</td><td>VWR</td><td>E504-500ML</td><td>Buffer for washes</td></tr><tr><td>Pellets 17-B-Estradiol 18 mg/60 days</td><td>Innovative Research of America</td><td>SE-121</td><td>Hormone pellets for rodents</td></tr><tr><td>Vetbond&amp;trade; Tissue Adhesive</td><td>3M</td><td>780-680</td><td>Tissue adhesive</td></tr><tr><td>Petri dishes in polystyrene crystal</td><td>Levantina</td><td>367-P101VR20</td><td>Petri dishes</td></tr><tr><td>Penicillin-Streptomicin</td><td>Sigma</td><td>P4333-100ML</td><td>Antibiotics</td></tr><tr><td>Syringes, medical 10 mL 0.5 ml</td><td>VWR</td><td>CODA626616</td><td>Syringes</td></tr><tr><td>Nitrile gloves, powder-free</td><td>VWR</td><td>112-2754</td><td>Gloves</td></tr><tr><td>Soft swiss nude mice</td><td>Charles River</td><td>SNUSSFE05S</td><td>Mice for animal experiment</td></tr><tr><td>Ivis Spectrum In vivo Imaging system</td><td>Perkin Elmer</td><td>124262</td><td>In vivo Monitoring equipment</td></tr><tr><td>Living Image&amp;reg; (Ivis software)</td><td>Perkin Elmer</td><td>---</td><td>In vivo monitoring software</td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>10082147</td><td>Enrichment serum</td></tr><tr><td>96-well cell culture treated plates</td><td>Life technologies</td><td>167008</td><td>Culture plates</td></tr><tr><td>Urine flasks</td><td>Summedical</td><td>4004-248-001</td><td>Flasks for washes</td></tr><tr><td>Sterile surgical blades</td><td>(Aesculap Division) Sanycare</td><td>1609022-0008</td><td>Surgical blades</td></tr><tr><td>Isovet 1,000 mg/g</td><td>B-BRAUN</td><td>---</td><td>Isoflurane (Anesthetic)</td></tr><tr><td>Buprex&amp;reg; 0.3 mg</td><td>Schering Plough S.A.</td><td>---</td><td>Buprenorphine (Analgesic solution)</td></tr><tr><td>Injectable morphine solution 10 mg/mL</td><td>B BRAUN</td><td>---</td><td>Morphine (Analgesic solution)</td></tr><tr><td>Monofyl&amp;reg; Absorbable Sutures</td><td>COVIDIEN</td><td>---</td><td>Sutures</td></tr><tr><td>Desinclor chlorhexidine</td><td>Promedic SA</td><td>---</td><td>Antiseptic solution</td></tr><tr><td>Microscopy DMi8</td><td>Leica Mycrosystems</td><td>---</td><td>fluorescence microscope</td></tr><tr><td>Hera Cell 150 Incubator</td><td>Thermo Scientific</td><td>51026282</td><td>Incubator</td></tr></tbody></table>

noninvasive monitoring of lesion size in a heterologous mouse model of endometriosis

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time through noninvasive monitoring of fluorescence emitted by implanted ectopic human endometrial tissue. For this purpose, biopsies of human endometrium are obtained from donor women ongoing oocyte donation. Human endometrial fragments are cultured in the presence of adenoviruses engineered to express cDNA for the reporter fluorescent protein mCherry. Upon visualization, labeled tissues with an optimal rate of fluorescence after infection are subsequently chosen for the implantation in recipient mice. One week prior to the implantation surgery, recipient mice are oophorectomized, and estradiol pellets are placed subcutaneously to sustain the survival and growth of lesions. On the day of surgery mice are anesthetized, and peritoneal cavity accessed through a small (1.5 cm) incision by the linea-alba. Fluorescently labeled implants are tweezed, briefly soaked in glue and attached to the peritoneal layer. Incisions are sutured, and animals left to recover for a couple of days. Fluorescence emitted by endometriotic implants is usually non-invasively monitored every 3 days for 4 weeks with an in vivo imaging system. Variations in the size of endometriotic implants can be estimated in real time by quantification of the mCherry signal and normalization against the initial time-point showing maximal fluorescence intensity.
Traditional preclinical rodents of models of endometriosis do not allow non-invasive monitoring of lesion in real time but rather allow evaluation of the effects of drugs assayed at the end point. This protocol allows one to track lesions in real time and is more useful to explore the therapeutic potential of drugs in preclinical models of endometriosis. The main limitation of the model thus generated is that non-invasive monitoring is not possible over long periods of time due to the episomal expression of Ad-virus.

Here, we describe the process for creating a heterologous model of endometriosis in which the architecture of lesions is preserved by implanting fluorescently labeled pieces of human endometrium into immunocompromised mice, thus allowing non-invasive monitoring of lesion progression. Labeling of endometrial fragments is achieved by infection with adenovirus engineered to express mCherry, a protein emitting fluorescence in the near infrared region. In Figure 1...

Watch this Scientific Journal Video about Noninvasive Monitoring of Lesion Size in a Heterologous Mouse Model of Endometriosis at JoVE.com

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

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 time

Here, we describe a protocol for the implementation of a heterologous mouse model in which progression of endometriosis can be assessed in real time ...

noninvasive-monitoring-lesion-size-heterologous-mouse-model

Research

JoVE Journal

Biology

8.1K Views.  Instituto de Investigación Sanitaria. 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 time

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

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all gynecologic malignancies among US women 1,2,3. Serous tumors are the most widespread forms of ovarian cancer and 4,5 the Tg-MISIIR-TAg transgenic represents the only mouse model that spontaneously develops this type of tumors. Tg-MISIIR-TAg mice express SV40 transforming region under control of the Mullerian Inhibitory Substance type II Receptor (MISIIR) gene promoter 6. Additional transgenic lines have been identified that express the SV40 TAg transgene, but do not develop ovarian tumors. Non-tumor prone mice exhibit typical lifespan for C57Bl/6 mice and are fertile. These mice can be used as syngeneic allograft recipients for tumor cells isolated from Tg-MISIIR-TAg-DR26 mice.
Objective: Although tumor imaging is possible 7, early detection of deep tumors is challenging in small living animals. To enable preclinical studies in an immunologically intact animal model for serous ovarian cancer, we describe a syngeneic mouse model for this type of ovarian cancer that permits in vivo imaging, studies of the tumor microenvironment and tumor immune responses.
Methods: We first derived a TAg+ mouse cancer cell line (MOV1) from a spontaneous ovarian tumor harvested in a 26 week-old DR26 Tg-MISIIR-TAg female. Then, we stably transduced MOV1 cells with TurboFP635 Lentivirus mammalian vector that encodes Katushka, a far-red mutant of the red fluorescent protein from sea anemone Entacmaea quadricolor with excitation/emission maxima at 588/635 nm 8,9,10. We orthotopically implanted MOV1Kat in the ovary 11,12,13,14 of non-tumor prone Tg-MISIIR-TAg female mice. Tumor progression was followed by in vivo optical imaging and tumor microenvironment was analyzed by immunohistochemistry.
Results: Orthotopically implanted MOV1Kat cells developed serous ovarian tumors. MOV1Kat tumors could be visualized by in vivo imaging up to three weeks after implantation (fig. 1) and were infiltrated with leukocytes, as observed in human ovarian cancers 15 (fig. 2).
Conclusions: We describe an orthotopic model of ovarian cancer suitable for in vivo imaging of early tumors due to the high pH-stability and photostability of Katushka in deep tissues. We propose the use of this novel syngeneic model of serous ovarian cancer for in vivo imaging studies and monitoring of tumor immune responses and immunotherapies.

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

To study in vivo tumor growth and tumor microenvironment, we used a syngeneic and orthotopic mouse model of ovarian cancer in immunocompetent animals. We transduced a mouse tumor cell line (MOV1) with Katushka fluorescent protein (MOV1KAT) and here we show its orthotopic implantation in ovary and in vivo imaging.

Background: Ovarian cancer is generally diagnosed at an advanced stage where the case/fatality ratio is high and thus remains the most lethal of all ...

Immunology and Infection

Mouse Model of Surgically-induced Endometriosis by Auto-transplantation of Uterine Tissue

Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of lesions, and/or chronic pharmaceutical management of pain and infertility symptoms. The cost associated with endometriosis has been estimated at 22 billion dollars per year in the United States1. To further our understanding of mechanisms underlying this enigmatic disease, animal models have been employed. Primates spontaneously develop endometriosis and therefore primate models most closely resemble the disease in women. Rodent models, however, are more cost effective and readily available2. The model that we describe here involves an autologous transfer of uterine tissue to the intestinal mesentery (Figure 1) and was first developed in the rat3 and later transferred to the mouse4. The goal of the autologous rodent model of surgically-induced endometriosis is to mimic the disease in women. We and others have previously shown that the altered gene expression pattern observed in endometriotic lesions from mice or rats mirrors that observed in women with the disease5,6. One advantage of performing the surgery in the mouse is that the abundance of transgenic mouse strains available can aid researchers in determining the role of specific components important in the establishment and growth of endometriosis. An alternative model in which excised human endometrial fragments are introduced to the peritoneum of immunocompromised mice is also widely used but is limited by the lack of a normal immune system which is thought to be important in endometriosis2,7. Importantly, the mouse model of surgically induced endometriosis is a versatile model that has been used to study how the immune system8, hormones9,10 and environmental factors11,12 affect endometriosis as well as the effects of endometriosis on fertility13 and pain14.

A description of the surgical induction of endometriosis in mice and rats by auto-transplantation of uterine tissue to the arterial cascade of the intestinal mesentery.

Endometriosis is a chronic, painful disease whose etiology remains unknown. Furthermore, treatment of endometriosis can require laparoscopic removal of ...

Medicine

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.

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.

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 ...

Cancer Research

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 ...

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic mechanisms that contribute to the onset and progression of intestinal diseases. The identification of new molecules that promote these pathologies has led to a flurry of activity focused on the development of potential new therapies to inhibit their function. As a result, various pre-clinical mouse models with an intact immune system and stromal microenvironment are now heavily used. Here we describe three experimental protocols to test the efficacy of new therapeutics in pre-clinical models of (1) acute mucosal damage, (2) chronic colitis and/or colitis-associated colon cancer, and (3) sporadic colorectal cancer. We also outline procedures for serial endoscopic examination that can be used to document the therapeutic response of an individual tumor and to monitor the health of individual mice. These protocols provide complementary experimental platforms to test the effectiveness of therapeutic compounds shown to be well tolerated by mice.

We describe methods for longitudinal monitoring of the efficacy of therapeutics for the treatment of colonic pathologies in mice using a rigid endoscope. This protocol can be readily used for the characterization of the therapeutic response of an individual tumor in live mice and also for monitoring potential disease relapse.

Animal models of inflammatory bowel disease (IBD) and colorectal cancer (CRC) have provided significant insight into the cell intrinsic and extrinsic ...

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Human cancer and response to therapy is better represented in orthotopic animal models. This paper describes the development of an orthotopic mouse model of ovarian cancer, treatment of cancer via oral delivery of drugs, and monitoring of tumor cell behavior in response to drug treatment in real time using in vivo imaging system. In this orthotopic model, ovarian tumor cells expressing luciferase are applied topically by injecting them directly into the mouse bursa where each ovary is enclosed. Upon injection of D-luciferin, a substrate of firefly luciferase, luciferase-expressing cells generate bioluminescence signals. This signal is detected by the in vivo imaging system and allows for a non-invasive means of monitoring tumor growth, distribution, and regression in individual animals. Drug administration via oral gavage allows for a maximum dosing volume of 10 mL/kg body weight to be delivered directly to the stomach and closely resembles delivery of drugs in clinical treatments. Therefore, techniques described here, development of an orthotopic mouse model of ovarian cancer, oral delivery of drugs, and in vivo imaging, are useful for better understanding of human ovarian cancer and treatment and will improve targeting this disease.

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Orthotopic animal models of ovarian cancer replicate better human disease and therefore enhance our understanding of cancer progression and tumor response to therapy. A mouse model receives an intrabursal injection of luciferase-expressing ovarian tumor cells. Treatment is administered via oral gavage. Tumor growth is monitored by in vivo imaging system.

Human cancer and response to therapy is better represented in orthotopic animal models.  This paper describes the development of an orthotopic mouse model ...

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 ...

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 ...

Development of a Novel Endometrioma-Simulating Mouse Model

Establishment of an Experimental Mouse Model of Endometrioma to Study its Related Infertility

Endometrioma (OMA), a subtype of endometriosis characterized by the formation of endometriotic cysts in the ovaries, affects 17-44% of individuals diagnosed with endometriosis. Women with OMA often experience compromised fertility, yet the exact mechanisms underlying OMA-associated infertility remain unclear. Notably, existing animal models simulate superficial peritoneal endometriosis (SUP) and deep infiltrating endometriosis (DIE), leaving a notable gap in research focused on OMA. In response to the gap of knowledge, this paper introduces a pioneering OMA-simulating mouse model and provides a comprehensive description of the techniques and procedures employed in the model. With a high success rate of 83% and ovarian lesion specificity, this model holds significant promise for advancing our understanding of OMA, particularly in the context of infertility. It offers a valuable platform for conducting targeted research into OMA-associated fertility challenges, potentially paving the way for improved diagnostic and therapeutic strategies in the field of reproductive medicine.

This study introduces a novel mouse model specifically designed to simulate endometrioma, a clinically significant subtype of endometriosis. By utilizing C57BL/6J mice, the model aims to elucidate the pathophysiological mechanisms underlying endometrioma-related infertility, offering a refined tool to bridge the current knowledge gap in reproductive medicine.

Endometrioma (OMA), a subtype of endometriosis characterized by the formation of endometriotic cysts in the ovaries, affects 17-44% of individuals ...

Optimized Thin Endometrium Mouse Model with Low Mortality and Minimal Adhesions

Establishing a Mouse Model of Thin Endometrium

Thin endometrium (TE) has been widely recognized as a critical cause of infertility. However, the pathogenesis of TE remains unclear, and satisfactory treatment options are still urgently needed. Several animal models of TE have been developed, but the mouse model involving abdominal surgery and injection of 95% ethanol presents a formidable challenge due to the high mortality rate and risk of intrauterine adhesions if not performed correctly. Here, we describe a detailed protocol that produces reliable and reproducible TE with a very low mortality rate and minimal intrauterine adhesions by injecting 95% ethanol into the mouse uterus with varying infusion times. The results showed that all of the mice successfully developed TE with infusion times ranging from 1-3 min, characterized by a typical reduction in endometrial thickness and the number of glands, as well as excessive endometrial fibrosis. These findings suggest that this mouse model is suitable for studying thin endometrium and can serve as a platform for developing future TE treatments.

This article describes a detailed protocol for producing a reliable and reproducible thin endometrium with a very low mortality rate and minimal intrauterine adhesions by injecting 95% ethanol into the mouse uterus within 1-3 min.

Thin endometrium (TE) has been widely recognized as a critical cause of infertility. However, the pathogenesis of TE remains unclear, and satisfactory ...

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

Summary

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Chapters in this video

An Orthotopic Model of Serous Ovarian Cancer in Immunocompetent Mice for in vivo Tumor Imaging and Monitoring of Tumor Immune Responses

Mouse Model of Surgically-induced Endometriosis by Auto-transplantation of Uterine Tissue

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

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

Non-invasive Assessment of the Efficacy of New Therapeutics for Intestinal Pathologies Using Serial Endoscopic Imaging of Live Mice

In vivo Imaging and Therapeutic Treatments in an Orthotopic Mouse Model of Ovarian Cancer

Quantitation of Intra-peritoneal Ovarian Cancer Metastasis

A Syngeneic Murine Model of Endometriosis using Naturally Cycling Mice

Establishment of an Experimental Mouse Model of Endometrioma to Study its Related Infertility

Establishing a Mouse Model of Thin Endometrium