This study was funded by the Terry Fox Research Institute (PPG#1075), the Canadian Institute of Health Research (Foundation Grant #154326), the Canadian Cancer Society Research Institute (704718), Natural Sciences and Engineering Research Council of Canada, &#1057;anada Foundation for Innovation and Princess Margaret Cancer Foundation.
I would like to thank Dr. Marguerite Akens for providing the initial VX2 cells for the establishment of the initial VX2 model and the frozen VX2 tumor blocks. I would like to thank Marco DiGrappa for helping to perform initial VX2 cell culture experiments and Lili Ding for helping with VX2 cell culture.

Deep abdominal wall closure: Identify the apex of the peritoneal incision and grasp the peritoneum, rectus muscle and fascia with tissue forceps.All animal studies were conducted in Animal Resource Center (ARC) approved facilities of the University Health Network and in accordance with approved animal use and care protocols (AUP #3994/#4299). VX2 cell line was obtained from Dr. Aken&#8217;s Lab at the University Health Network.
1. Creation of in vitro VX2 cell line
<ol>
	<li>Harvest the VX2 tumor from rabbit quadricep muscle and growth in mouse flank.
	<ol>
		<li>Thaw frozen VX2 tumor blocks (1 cm x 1 cm), mince on a culture plate using a scalpel blade and strain through a 70 &#181;m filter by adding small amounts of Hanks Balanced Salt Solution (HBSS) sequentially to ensure all cells are strained for a final volume of 1 mL.</li>
		<li>Count cells and dilute with 0.9% phosphate buffered saline solution to a concentration of 1 x 107/mL and place on ice in a sterile tube. 
		NOTE: Tumor blocks were obtained from a lab collaborator from previous propagation of VX2 tumors in rabbit quadriceps muscles.</li>
		<li>Anesthetize a female white New Zealand rabbit (weight 2.5-3.5 kg) with 2-5% inhaled isoflurane, remove the fur overlying the injection site and cleanse the injection site with povidone-iodine solution. Inject 500 &#181;L of the prepared VX2 cell suspension (5 x 106 cells/mL) into the quadriceps muscle of the rabbit. Monitor the rabbit clinically for tumor growth starting on day 10.</li>
		<li>Euthanize the rabbits after tumors reach 1-1.5 cm in diameter (measured externally with calipers) using&#160;a veterinarian approved method. Confirm euthanasia by checking vital signs including heart rate and respiratory rate. Excise the tumor using sterile instruments after cleaning the area with betadine solution.</li>
		<li>In a biological safety cabinet using sterile instruments, mince the tumor in to small pieces (0.2 cm) on a 10 cm tissue culture dish using a scalpel blade. Pass the tumor fragments through a 70 &#181;m cell strainer using 1 mL of HBSS as described in step 1.1.1. Count cells and dilute using 0.9% saline solution to obtain 7.5 x 105 cells in 200 &#181;L.</li>
		<li>Anesthetize male NOD scid gamma mice (weight 25 g) using 4% isoflurane at 1 L/min and maintain anesthesia using 2% isoflurane. Once mice are anesthetized, inject 200 &#181;L of the solution from step 1.1.5 into the subcutaneous tissue of the mouse flank using a 27 gauge needle.</li>
	</ol>
	</li>
	<li>Harvesting and passaging of VX2 cells in vitro
	<ol>
		<li>Monitor xenograft growth clinically twice weekly until tumors reach 1 cm in diameter using calipers.</li>
		<li>Euthanize mice by placing in a CO2 chamber or performing cervical dislocation after anesthetizing with 4% isoflurane gas. Excise tumors under sterile conditions in a biological safety cabinet.</li>
		<li>Mince tumors with a scalpel into 1-2 mm pieces in a 6 cm tissue culture dish containing 3 mL of Dulbecco&#8217;s Modified Eagle Medium (DMEM)/Ham&#8217;s F12+ 10% fetal bovine serum (FBS) media.</li>
		<li>Leave tumor fragments undisturbed in a 37 &#176;C incubator with 5% CO2 for two days until cells begin to grow out from the tumor pieces. Subsequently, check cultured plates daily by light microscopy for cell confluency. Upon reaching 70% confluency, trypsinize cells by adding 1 mL of 0.05% trypsin to the flask and placing back in the 37 &#176;C incubator for 5 min.</li>
		<li>Neutralize trypsin with 6 mL of DMEM/Ham&#8217;s F12 + 10% FBS medium, collect cells and centrifuge at 4 &#176;C for 5 min at 300 x g. Remove supernatant and re-suspend pellet in 3.5 mL of new DMEM/Ham&#8217;s F12 + 10% FBS media. Seed fresh 6 cm collagen I coated plates with 1.6 x 106 cells per plate to achieve approximately 50% cell seeding density. 
		&#8203;NOTE: 50% cell density refers to the cells taking up 50% of the surface area of the plate when attached.</li>
		<li>Early passaging: Repeat the above process (trypsinizing, seeding) until cells have been passaged 5 times and then assess cell line purity with PCR using a quantitative PCR assay to distinguish rabbit genomic DNA from mouse genomic DNA (see step 1.3.1-1.3.4). 
		NOTE: After 8 passages, cells can be propagated on non-collagen coated regular adherent tissue culture plates.</li>
		<li>Passage, trypsinize and freeze aliquots of cells in DMEM/HAM F12+30% FBS+10%DMSO at a concentration of 2 x 106 per vial. Store cells in liquid nitrogen until required for experiments.</li>
	</ol>
	</li>
	<li>Confirmation of VX2 origin of surviving cells:
	<ol>
		<li>Create a standard cure from purified genomic mouse and rabbit DNA (Step 1.3.2 and Step 1.3.3). Use primers which target species-specific sequences within the second open reading frame of the LINE-1 retrotransposon element. 
		NOTE: Primer sequences for CRPV E6 are: 5&#8217;- GATCCTGGACCCAACCAGTGand 5&#8217;-CCTGCCGGTCCCTGATTTAT. The LINE-1 retrotransposon elements were used. Primer sequences for rabbit LINE-1 are: 5&#8217;-TCAGGAAACCCCAGAAAGTATGC and 5&#8217;-TTTGATTTCTTGAATGACCCAGTGT Primer sequences for mouse LINE-1 are: 5&#8217;-AATGGAAAGCCAACATTCACGTG and 5&#8217;-CCTTCCTTGACCAAGGTATCATTG</li>
		<li>To create a standard curve for CRPV E6, purify VX2 tumor cell lysate (Step 1.1.1) using a commercial kit following manufacturer&#8217;s protocol. Quantify this DNA using a commercial assay. Perform 6 serial dilutions from 225 pg/&#181;L to 0.925 pg/&#181;L in low EDTA-TE buffer. To create a standard curve for mouse genomic DNA, dilute 100 &#181;g of commercial mouse genomic DNA in low EDTA-TE buffer in 5 dilutions from 125 pg/&#181;L down to 0.0125 pg/&#181;L.</li>
		<li>Place 4 &#181;L from each diluted solution from the samples from step 1.3.2 in wells and run samples in a PCR thermocycler. Use the Cq values from each run along with the DNA amounts per well to calculate a standard curve using the thermocycler software auto-threshold settings.</li>
		<li>Use standard qPCR procedures to perform PCR with 40 cycles of 2-step cycling (98 &#176;C and 60 &#176;C) on a thermocycler. Establish threshold values, and set delta Ct values at &#62;35 to ensure there is minimal mouse contamination in the rabbit VX2 cell line. The total volume of each reaction was 10 &#181;L, consisting of 5 &#181;L of 2x Mastermix, 1 &#181;L of forward + reverse primers with final primer concentration in reaction at 250 nM, and 4 &#181;L of DNA sample. 
		NOTE: Please see references54,55 for details on performing PCR. Threshold values are established, and delta Ct values are set at &#62;35 to ensure there is minimal mouse contamination in the rabbit VX2 cell line. The recommended level of VX2 DNA for reliable detection by qPCR is 1-10 pg.</li>
	</ol>
	</li>
</ol>
2. VX2 cell culture and creation of cell suspension
<ol>
	<li>Thaw vials containing VX2 cells (frozen in step 1.2.7) in a water bath at 37 &#176;C for 1 minute and transfer cells to a conical centrifuge tube with 10 mL of culture medium (1:1 DMEM/F-12 + 10% FBS and 1% Penicillin-streptomycin). Centrifuge for 8 min at 107 x g, discard the supernatant and re-suspend the cell pellet in 9 mL of media.</li>
	<li>Transfer re-suspended cells to a large culture flask. Incubate cells without shaking at 37 &#176;C. Check cells daily for confluence using light microscopy and change culture media every three days.</li>
	<li>Creation of cultured VX2 cell suspension for injection
	<ol>
		<li>Trypsinize cells by adding 3 mL of 0.25% trypsin to flask once cells have reached 80% confluence. Place flask in incubator (37 &#176;C) for 5 min. Transfer solution to a conical centrifuge tube and centrifuge for 8 min at 107 x g and then remove supernatant.</li>
		<li>Wash the cell pellets 3 times with 9 mL of phosphate buffered saline, centrifuge and remove supernatant as above. Count the cells and dilute with 0.9% phosphate buffered saline solution to a concentration of 4 x 107 cells/mL and place in a sterile conical centrifuge tube on ice.</li>
	</ol>
	</li>
	<li>Creation of in vivo propagated VX2 cell suspension for injection
	<ol>
		<li>Thaw frozen VX2 tumor blocks (1 cm x 1 cm), mince on a culture plate using a scalpel blade and strain through a 70 &#181;m filter as in step 1.1.1. Add small amounts of HBSS sequentially to ensure all cells are strained for a final volume of 1 mL. Count cells and dilute with 0.9% phosphate buffered saline to a concentration of 1 x107/mL and place on ice in a sterile tube. 
		NOTE: Tumor blocks were obtained from a lab collaborator from previous propagation of VX2 tumors in rabbit quadriceps muscles.</li>
	</ol>
	</li>
</ol>
3. Surgical Model Establishment
<ol>
	<li>Establishment of surgical anesthesia and pre-operative preparation
	<ol>
		<li>Pre-medicate female white New Zealand rabbits (2.5-3.5 kg) 1 h&#160;prior to the planned surgical procedure using injections of acepromazine (1 mg/kg IM) and meloxicam (0.2 mg/kg SQ).&#160;Anesthetize a female white New Zealand rabbit (weight 2.5-3.5 kg) with 2-5% inhaled isoflurane, remove the fur overlying the injection site and cleanse the injection site with povidone-iodine solution.</li>
		<li>Intubate anesthetized rabbits using a laryngeal mask airway (LMA) and secure in place with tape, maintaining deep anesthesia by titrating the inhaled isoflurane dose between 2-5%. Monitor surgical anesthesia regularly throughout the procedure by checking vital signs (respiratory rate, capillary refill, oxygen saturation if available) and monitoring for signs suggestive of pain (movement, withdrawal from stimuli, noises) or light anesthesia (movement, chewing on LMA tube). 
		NOTE: This should be done by someone with experience monitoring surgical anesthesia.</li>
		<li>Place a 22-gauge ear-vein catheter in the marginal dorsal vein. Administer cefazolin (20 mg/kg) intravenously 10 min prior to surgical skin incision.</li>
		<li>Clip hair over the pelvis and abdomen prior to placing the rabbit on the operating table, then place rabbits in a dorsal position on the operating table. Clean the surgical field using a 3-step surgical skin prep (betadine soap, chlorhexidine solution, betadine solution). Drape the surgical field with laparotomy drapes after land-marking the top of the pubic bone, leaving a 5 cm x 5 cm area of the lower abdomen exposed.</li>
	</ol>
	</li>
	<li>Creation of the laparotomy incision and identification of the uterine horns
	<ol>
		<li>Put on a surgical cap and face mask. Scrub hands using chlorhexidine or betadine surgical scrub solution. Using sterile technique, put on a sterile gown and sterile surgical gloves.</li>
		<li>Using a # 11-blade scalpel, make a 2.5 cm long incision 1 cm cranial to the symphysis pubis through skin and subcutaneous tissue of the rabbit abdomen. Incise the rectus fascia and dissect the rectus muscles laterally to expose the underlying peritoneum. Enter the peritoneum sharply after ensuring the undersurface is clear of bowel or other abdominal organs.</li>
		<li>Locate the uterine horns identifying the urinary bladder and sweeping a gloved finger superiorly, posteriorly and laterally over the apex of the bladder. 
		NOTE: If necessary, the full bladder can be emptied using digital pressure. Once located, bring the uterine horns through the abdominal incision to rest on the abdominal wall.</li>
		<li>Using a 3-0 braided absorbable suture, perform a single suture ligation of each uterine horn approximately 1.5-2.0 cm distal to the cervices. Place the suture just medial to the uterine arteries, which run along the lateral aspect of each horn. Tie the sutures snugly to occlude the distal uterine horns (Figure 1).</li>
	</ol>
	</li>
	<li>Myometrial VX2 inoculation
	<ol>
		<li>Using a 27-gauge needle, inject 0.5 mL of the previously prepared VX2 cell suspension from either step 2.3.2 (for cultured VX2 model) or 2.4 (for in vivo propagated VX2 model) into the myometrium of each uterine horn proximal to the suture site (between the suture site and the cervix). Inject over 1 minute and ensure that cells are not being injected into the underlying uterine cavity.&#160;Anesthetize animals using inhaled isoflurane (2-5%) and an anesthetic machine with a Bain circuit.</li>
		<li>Apply pressure to the myometrial injection site for 30 s after injection to minimize leakage of cells. Inspect the injection and suture sites for hemostasis and place the uterine horns back into the abdomen.</li>
	</ol>
	</li>
	<li>Closing the surgical incision
	<ol>
		<li>Deep abdominal wall closure: Identify the apex of the peritoneal incision and grasp the peritoneum, rectus muscle and fascia with tissue forceps.
		<ol>
			<li>To do this, anchor the suture at one apex of the incision by suturing superficial to deep on one side of the incision and deep to superficial on the other. Tie a knot. Using the attached suture, make running stitches perpendicular to the incision through the layers of the abdominal wall working step-wise along the incision from side to side. Tie the suture and cut.</li>
		</ol>
		</li>
		<li>Superficial abdominal wall closure: Identify the apex of the skin incision and suture the abdominal skin using buried running subcuticular 3-0 absorbable poly-filament suture. Apply surgical glue to the closed incision to oppose the skin edges.
		<ol>
			<li>To do this, anchor the suture at one apex of the incision, by suturing deep to superficial on one side of the incision and superficial to deep on the other. Tie a knot. Using the attached sutures, make running stitches in the dermal layer parallel to the incision working step-wise along the incision from side to side. Tie the suture and cut.</li>
		</ol>
		</li>
	</ol>
	</li>
	<li>Post-operative care
	<ol>
		<li>Awakening from anesthesia: Turn off the isoflurane once the incision is closed and let the rabbit breath oxygen by mask while monitoring for signs of awakening (e.g., spontaneous movements, eye opening and chewing motions on the laryngeal mask airway). Extubate the rabbit once these signs are noted and provide&#160; oxygen by mask and a warm blanket until the rabbits are alert and able to sit independently.</li>
		<li>Post-operative monitoring: Monitor rabbits twice a day for 4 days and daily for an additional 10 days post-operatively. To monitor, assess their general condition, their food and water intake, urine and fecal output, pain assessment, weight, vital signs (heart rate, respiration rate) and their surgical site looking for swelling, erythema, discharge or dehiscence.</li>
		<li>Post-operative pain control: Administer Meloxicam daily (0.2 mg/kg SQ) for 48 h&#160;post-operatively and then as needed based on pain assessment.&#160;Administer buprenorphine immediately post-operatively and every 12 h&#160;(0.01-0.05 mg/kg SQ) for 24 h and then as needed based on pain assessment</li>
		<li>Antibiotics (enrofloxacin 5mg/kg IM) may be administered daily for seven days post-operatively to prevent wound infection as recommended by your veterinarian.&#160;Administer subcutaneous fluids (15 mL of SQ) twice a day as needed for dehydration and soft foods or other dietary supplements are provided daily as needed for weight loss.</li>
	</ol>
	</li>
</ol>
4. Tumor Growth Monitoring
<ol>
	<li>Clinical Monitoring: Monitor rabbits every 2 days for clinical signs of tumor growth starting on post-operative day 14. Clinical signs of tumor growth can include decreased oral intake, lethargy, abdominal tenderness, palpable abdominal mass and loss of cecotrophy.</li>
	<li>Imaging and invasive monitoring
	<ol>
		<li>CT imaging: On post-operative day 21-28, pre-medicate, anesthetize, and intubate rabbits as previously described in 3.1.1-3.1.2. Position rabbits in a dorsal position in a pre-clinical CT scanner and obtain images of the pelvis and abdomen after administering 10 mL of intravenous iohexol contrast agent.</li>
		<li>Surgical monitoring: Transfer rabbits to the operating room after imaging while still under general anesthetic. Position, prep and drape rabbits as previously described in step 3.1.4 and perform a repeat laparotomy incision approximately 1.5 cm in length through the previous incision. Identify the and the uterine horns and carefully examine for tumor. Close the incision and provide post-operative care as previously described in step 3.5.2-3.5.4.</li>
		<li>Rabbit model use: If rabbits are found to have tumors at the time of surgical monitoring, use rabbit endometrial cancer models for planned experiments. Use rabbits created from in vivo propagated cells for additional experiments at 4-weeks post-inoculation and use rabbit models created from cultured VX2 cells at approximately 5-6 weeks post-inoculation to account for slower tumor growth.&#160;Euthanize the rabbits after tumors reach 1-1.5 cm in diameter (measured externally with calipers) using a veterinarian approved method. Confirm euthanasia by checking vital signs including heart rate and respiratory rate. Excise the tumor using sterile instruments after cleaning the area with betadine solution.</li>
	</ol>
	</li>
</ol>

The authors have nothing to disclose.

Herein, we have reported a standardized surgical method for the establishment of a VX2 endometrial cancer model and reported on the first use of cultured VX2 cells to create this model. The tumor take rate of 75% is lower than the 100% percent rate previously reported in the literature35,37,53,56; however, thw 90% rate of pathologically confirmed lymph node metastases is consistent with previou...

Endometrial cancer, or cancer of the lining of the uterus, is the second most common gynecologic malignancy worldwide and the most common malignancy in developed nations1. The incidence of endometrial cancer has steadily increased, rising by 2.3% per year between 2005-2013 with a corresponding 2.2% increase in mortality1,2,3. The diagnosis of lymph node metastases is paramount as the presence of positive lymph nodes is a strong negative predictor of survival4,5,6,7 and can guide the administration of adjuvant therapy8,9,10,11,12,13. Lymph node metastases are currently diagnosed by surgically removing the lymphatic tissue overlying the major blood vessels in the pelvis and abdomen. This procedure, known as a lymphadenectomy, is controversial due to conflicting survival data from two large trials14,15,16,17,18 and the known risk of intra-operative15,19,20 and post-operative morbidity21,22,23. As current non-invasive imaging modalities do not have the required sensitivity and specificity to replace lymph node dissection24, there has been a push to develop new diagnostic imaging techniques. To test these novel techniques in a pre-clinical setting, a reliable model of endometrial cancer with retroperitoneal lymph node metastases is required.
The rabbit VX2 tumor model is a well-established model which has been used extensively to study multiple human solid organ tumors25 including lung26, head and neck27,28, liver29, kidney30, bone31,32, brain33, pancreas34 and uterus35,36,37. The VX2 model was originally developed in 1940 by Kidd and Rous38 by successfully transplanting a cottontail rabbit papilloma virus discovered by Shope in 193339. Since that time, the VX2 model has been maintained in vivo, requiring serial passage in the quadriceps muscle of White New Zealand rabbits40. More recently however, multiple groups have successfully grown VX2 cells in vitro40,41,42 and demonstrated the retained tumorigenecity of the cultured cell line31,42,43. VX2 tumors are histologically defined as anaplastic squamous cell carcinomas44 and contain glandular features which resemble adenocarcinoma26. Tumors are characterized by ease of implantation, rapid growth and hyper-vascularity44,45 and reliably metastasize, most commonly to regional and distant lymph nodes45. Similarities in uterine vascular and lymphatic anatomy46 as well as the orthotopic growth site ensure that the metastatic pattern of rabbit VX2 carcinoma mimics that of human endometrial cancer, making the VX2 model a reliable model for studying human metastatic disease. Furthermore, histologic features such as abnormal microvascular proliferation47 , as well as immunological48 and genetic similarities49,50 between humans and rabbits suggest that the tumor microenvironment may reflect that of human endometrial cancer.
Multiple groups have reported on the use of VX2 to create a model of endometrial cancer with retroperitoneal metastases with a high reported rate of success36,51,52; however, significant variation exists within the current literature with respect the method of model creation. Cell suspension doses as low as 4 x 105 cells/uterine horn51 and as high as 5 x 109 cells/uterine horn37,53 have been reported with no standard consensus on the required VX2 cellular dose. As well, a variety of inoculation methods have been reported including micro-surgical implantation of tumor into the uterine myometrium36, injection of VX2 cell suspension37,44,52,53 and in some cases, the addition of uterine horn suturing prior to innoculation52. Finally, no groups have reported the use of cultured VX2 cells to create this model. Thus, the purpose of this study is to demonstrate a successful standardized method of VX2 model creation and to report the first use of cultured VX2 cells to create a model of endometrial cancer with retroperitoneal metastases in a rabbit.

<table>
	<tbody>
		<tr>
			<td>11-blade scalpel, Sterile, Disposible</td>
			<td>Aspen Surgical (VWR)</td>
			<td>80094-086</td>
			<td></td>
		</tr>
		<tr>
			<td>22-gague ear vein catheter</td>
			<td>CDMV</td>
			<td>14332</td>
			<td></td>
		</tr>
		<tr>
			<td>3-0 absorbable poly-filament suture (Polysorb)</td>
			<td>Covidien</td>
			<td>356718</td>
			<td></td>
		</tr>
		<tr>
			<td>3-0 braided absorbable suture (Polysorb)</td>
			<td>Covidien</td>
			<td>356718</td>
			<td></td>
		</tr>
		<tr>
			<td>70uM cell strainer, Individually wrapped, Nylon</td>
			<td>Falcon</td>
			<td>352350</td>
			<td></td>
		</tr>
		<tr>
			<td>Acepromazine (Atravet)</td>
			<td>CDMV</td>
			<td>1047</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine soap (Poviodone iodine 7.5%)</td>
			<td>CDMV</td>
			<td>4363</td>
			<td></td>
		</tr>
		<tr>
			<td>Betadine solution (Poviodone iodine 10%)</td>
			<td>UHN Stores</td>
			<td>457955</td>
			<td></td>
		</tr>
		<tr>
			<td>Buprenorphine</td>
			<td>McGill University</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Cefazolin</td>
			<td>UHN in-patient pharmacy</td>
			<td>No Cat # Needed</td>
			<td></td>
		</tr>
		<tr>
			<td>Chlorhexidine solution</td>
			<td>CDMV</td>
			<td>119872</td>
			<td></td>
		</tr>
		<tr>
			<td>Corning BioCoatCellware, Collagen Type I, 100mm dishes</td>
			<td>Corning</td>
			<td>354450</td>
			<td>brand not important</td>
		</tr>
		<tr>
			<td>Corning BioCoatCellware, Collagen Type I, 24-well plates</td>
			<td>Corning</td>
			<td>354408</td>
			<td>brand not important</td>
		</tr>
		<tr>
			<td>Corning BioCoatCellware, Collagen Type I, 6-well plates</td>
			<td>Corning</td>
			<td>354400</td>
			<td>brand not important</td>
		</tr>
		<tr>
			<td>Corning Matrigel Basement Membrane Matrix, *LDEV-free, 10 mL</td>
			<td>Corning</td>
			<td>354234</td>
			<td></td>
		</tr>
		<tr>
			<td>DMEM/HAM F12 1:1</td>
			<td>Life Technologies</td>
			<td>11320</td>
			<td>brand not important</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>Caledon Lab Chem</td>
			<td>1/10/4100</td>
			<td></td>
		</tr>
		<tr>
			<td>Enrofloxacin (Baytril injectable)</td>
			<td>CDMV</td>
			<td>11242</td>
			<td></td>
		</tr>
		<tr>
			<td>Falcon Tube</td>
			<td>Corning Centri-Star</td>
			<td>430828</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Bovine Serum, Qualified, Canadian Origin, 500ml</td>
			<td>Life Technologies</td>
			<td>12483020</td>
			<td>brand/source not important</td>
		</tr>
		<tr>
			<td>Isoflurane</td>
			<td>UHN in-patient pharmacy</td>
			<td>No Cat # Needed</td>
			<td></td>
		</tr>
		<tr>
			<td>Isohexol contrast</td>
			<td>GE Healthcare</td>
			<td>407141210</td>
			<td></td>
		</tr>
		<tr>
			<td>Meloxicam (Metacam 0.5%)</td>
			<td>CDMV</td>
			<td>104674</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal Saline</td>
			<td>House Brand (UofT Medstore)</td>
			<td>1011</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS</td>
			<td>Multicell or Sigma</td>
			<td>331-010-CL or D8537-500mL</td>
			<td></td>
		</tr>
		<tr>
			<td>Penicillin/Streptomycin (100mL; 10000U Penicillin, 10000ug Streptomycin)</td>
			<td>Corning-Cellgro</td>
			<td>CA45000-652</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterile Hanks Balanced Salt Solution (-Ca++, -Mg++, -Phenol Red)</td>
			<td>T.C.M.F (Dr Bristow)</td>
			<td>28-Jan-11</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgical Glue (Tissue Adhesive)</td>
			<td>3M Vetbond</td>
			<td>14695B</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin (0.25%), Proteomics Grade</td>
			<td>Sigma</td>
			<td>T-6567-5X20UG</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA, 0.05%, 100ml</td>
			<td>Wisent Inc</td>
			<td>325-542-EL</td>
			<td>brand not important</td>
		</tr>
	</tbody>
</table>

an orthotopic endometrial cancer model with retroperitoneal lymphadenopathy made from in vivo propagated and cultured vx2 cells

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or without adjuvant therapy depending on lymph node involvement as determined by lymphadenectomy. Lymphadenectomy is a morbid procedure, which has not been shown to have a therapeutic benefit in many patients, and thus a new method to diagnose lymph node metastases is required. To test novel imaging agents, a reliable model of endometrial cancer with retroperitoneal lymph node metastases is needed. The VX2 endometrial cancer model has been described frequently in the literature; however, significant variation exists with respect to the method of model establishment. Furthermore, no studies have reported on the use of cultured VX2 cells to create this model as only cells propagated in vivo have been previously used. Herein, we present a standardized surgical method and post-operative monitoring method for the establishment of the VX2 endometrial cancer model and report on the first use of cultured VX2 cells to create this model.

Twenty-eight rabbits were used for the creation of the endometrial cancer model. Rabbits had an average weight of 2.83 kg (2.71-3.58 kg) at the time of experiment. Uterine tumors successfully grew in 21 rabbits for an overall model success rate of 75%. Prior to the inclusion of uterine suturing in the protocol, the success rate was 57% compared to 81% after uterine suturing was added. Uterine suturing was added to the protocol after the 7th rabbit in response to the initial low...

Watch this Scientific Journal Video about An Orthotopic Endometrial Cancer Model with Retroperitoneal Lymphadenopathy Made From In Vivo Propagated and Cultured VX2 Cells at JoVE.com

An Orthotopic Endometrial Cancer Model with Retroperitoneal Lymphadenopathy Made From In Vivo Propagated and Cultured VX2 Cells

This protocol presents a standardized method to grow VX2 cells in culture and to create an orthotopic VX2 model of endometrial cancer with retroperitoneal lymph node metastases in rabbits. Orthotopic endometrial cancer models are important for the pre-clinical study of novel imaging modalities for the diagnosis of lymph node metastases.

Endometrial cancer is the most common gynecologic malignancy in North America and the incidence is rising worldwide. Treatment consists of surgery with or ...

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8.0K Views.  University of Toronto. This protocol presents a standardized method to grow VX2 cells in culture and to create an orthotopic VX2 model of endometrial cancer with retroperitoneal lymph node metastases in rabbits. Orthotopic endometrial cancer models are important for the pre-clinical study of novel imaging modalities for the diagnosis of lymph node metastases.

Video: An Orthotopic Endometrial Cancer Model with Retroperitoneal Lymphadenopathy Made From In Vivo Propagated and Cultured VX2 Cells

Fluorescent Orthotopic Mouse Model of Pancreatic Cancer

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will survive longer than five years. In order to understand and mimic the microenvironment of pancreatic tumors, we utilized a murine orthotopic model of pancreatic cancer that allows non-invasive imaging of tumor progression in real time. Pancreatic cancer cells expressing green fluorescent protein (PANC-1 GFP) were suspended in basement membrane matrix, high concentration, (e.g., Matrigel HC) with serum-free media and then injected into the tail of the pancreas via laparotomy. The cell suspension in the high concentration basement membrane matrix becomes a gel-like substance once it reaches room temperature; therefore, it gels when it comes in contact with the pancreas, creating a seal at the injection site and preventing any cell leakage. Tumor growth and metastasis to other organs are monitored in live animals by using fluorescence. It is critical to use the appropriate filters for excitation and emission of GFP. The steps for the orthotopic implantation are detailed in this article so researchers can easily replicate the procedure in nude mice. The main steps of this protocol are preparation of the cell suspension, surgical implantation, and whole body fluorescent in vivo imaging. This orthotopic model is designed to investigate the efficacy of novel therapeutics on primary and metastatic tumors.

A procedure to implant green fluorescent protein-expressing pancreatic cancer cells (PANC-1 GFP) orthotopically into the pancreas of Balb-c Ola Hsd-Fox1nu mice to assess tumor progression and metastasis is presented here.

Pancreatic cancer remains one of the cancers for which survival has not improved substantially in the last few decades. Only 7% of diagnosed patients will ...

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is important to control peritoneal dissemination. However, it is still unclear how tumor cells detach from primary lesions and attach to the mesothelium. The establishment of an appropriate animal model is needed to gain an understanding of the mechanism of peritoneal dissemination in vivo. In the current study, we introduce the process from the local injection of EOC cells into the murine ovarian surface to the development of metastasis, including the peritoneum and distant organs. Female nude mice (BALB/c nu/nu) at 8 weeks of age were used. Under a microscopic field of view, EOC cells (1 x 105 cells/&#181;l of medium-extracellular matrix (ECM)-based hydrogel/unilateral ovary/mouse) were injected into murine ovaries through a retroperitoneal approach from the dorsal flank. This proposed method is a less invasive procedure for the mouse and minimizes damage to the ovary. Here, we describe the methodological steps in the development of the original and metastatic tumor formation of EOC.

Murine Experimental Model of Original Tumor Development and Peritoneal Metastasis via Orthotopic Inoculation with Ovarian Carcinoma Cells

To clarify the multistep process of peritoneal dissemination of ovarian carcinoma, the current study presents a murine experimental model of original tumor development and peritoneal metastasis via orthotopic inoculation with these tumor cells.

Epithelial ovarian carcinoma (EOC) is associated with a poor prognosis because it shows peritoneal dissemination. To improve the prognosis, it is ...

Isolation of Circulating Tumor Cells in an Orthotopic Mouse Model of Colorectal Cancer

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate tumor biology and tumor cell dissemination. Orthotopic mouse models have been introduced to overcome these limitations; however, such models are technically demanding, especially in hollow organs such as the large bowel. In order to produce uniform tumors which reliably grow and metastasize, standardized techniques of tumor cell preparation and injection are critical. 
We have developed an orthotopic mouse model of colorectal cancer (CRC) which develops highly uniform tumors and can be used for tumor biology studies as well as therapeutic trials. Tumor cells from either primary tumors, 2-dimensional (2D) cell lines or 3-dimensional (3D) organoids are injected into the cecum and, depending on the metastatic potential of the injected tumor cells, form highly metastatic tumors. In addition, CTCs can be found regularly. We here describe the technique of tumor cell preparation from both 2D cell lines and 3D organoids as well as primary tumor tissue, the surgical and injection techniques as well as the isolation of CTCs from the tumor-bearing mice, and present tips for troubleshooting.

We describe the establishment of orthotopic colorectal tumors via injection of tumor cells or organoids into the cecum of mice and the subsequent isolation of circulating tumor cells (CTCs) from this model.

Despite the advantages of easy applicability and cost-effectiveness, subcutaneous mouse models have severe limitations and do not accurately simulate ...

Development of a Human Preclinical Model of Osteoclastogenesis from Peripheral Blood Monocytes Co-cultured with Breast Cancer Cell Lines

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We developed an in vitro fully human preclinical model of a co-culture of breast cancer cells and monocytes undergoing differentiation towards osteoclasts. We optimized a model of osteoclastogenesis starting from a sample of peripheral blood collected from healthy donors. Peripheral blood mononuclear cells (PBMCs) were first separated by density gradient centrifugation, seeded at a high density and induced to differentiate by adding two growth factors (GFs): receptor activator of nuclear factor-&#954;B ligand (RANKL) and macrophage colony-stimulating factor (MCSF). The cells were left in culture for 14 days and then fixed and analyzed by downstream analysis. In osteolytic bone metastases, one of the effects of cancer cell arrival in bone is the induction of osteoclastogenesis. We thus challenged our model with co-cultures of breast cancer cells to study the differentiation power of cancer cells with respect to GFs. A straightforward way of studying cancer cell-osteoclast interaction is to perform indirect co-cultures based on the use of conditioned medium collected from breast cancer cell cultures and mixed with fresh medium. This mixture is then used to induce osteoclast differentiation. We also optimized a method of direct co-culture in which cancer cells and monocytes undergoing differentiation share the medium and exchange secreted factors. This is a significant improvement over the original indirect co-culture method as researchers can observe the reciprocal interactions of the two cell types and perform downstream analyses for both cancer cells and osteoclasts. This method enables us to study the effect of drugs on the metastatic bone microenvironment and to seed cell lines other than those derived from breast cancer. The model can also be used to study other diseases such as osteoporosis or other bone conditions.

This protocol describes development of an in vitro human preclinical model of osteoclastogenesis from peripheral blood monocytes cultured with breast cancer cell lines to mimic the cancer cell-osteoclast interaction. The model could be used to further our understanding of bone metastasis formation and improve therapeutic options.

The crosstalk between tumor cells and bone cells in the bone microenvironment is crucial to understanding the mechanism of bone metastasis formation. We ...

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve microenvironment. This type of invasion is exhibited by a variety of cancer types, and very frequently is found in pancreatic cancer. The microscopic size of nerve fibers within mouse pancreas renders the study of PNI difficult in orthotopic murine models. Here, we describe a heterotopic in vivo model of PNI, where we inject syngeneic pancreatic cancer cell line Panc02-H7 into the murine sciatic nerve. In this model, sciatic nerves of anesthetized mice are exposed and injected with cancer cells. The cancer cells invade in the nerves proximally toward the spinal cord from the point of injection. The invaded sciatic nerves are then extracted and processed with OCT for frozen sectioning. H&amp;E and immunofluorescence staining of these sections allow quantification of both the degree of invasion and changes in protein expression. This model can be applied to a variety of studies on PNI given its versatility. Using mice with different genetic modifications and/or different types of cancer cells allows for investigation of the cellular and molecular mechanisms of PNI and for different cancer types. Furthermore, the effects of therapeutic agents on nerve invasion can be studied by applying treatment to these mice.

An In Vivo Murine Sciatic Nerve Model of Perineural Invasion

We describe an in vivo murine model of perineural invasion by injecting syngeneic pancreatic cancer cells into the sciatic nerve. The model allows for quantification of the extent of nerve invasion, and supports investigation of the cellular and molecular mechanisms of perineural invasion.

Cancer cells invade nerves through a process termed perineural invasion (PNI), in which cancer cells proliferate and migrate in the nerve ...

Development and Angiographic Use of the Rabbit VX2 Model for Liver Cancer

The rabbit VX2 tumor is an animal model commonly utilized for translational research regarding hepatocellular carcinoma (HCC) in the field of Interventional Radiology. This model employs an anaplastic squamous cell carcinoma that is easily and reliably propagated in the skeletal muscle of donor rabbits for eventual harvest and allograft implantation into the liver of na&#239;ve recipients. This tumor graft rapidly grows within the liver of recipient rabbits into an angiographically identifiable tumor characterized by a necrotic core surrounded by a viable hypervascular capsule. The physical size of the rabbit anatomy is sufficient to facilitate vascular instrumentation allowing for the application and testing of various interventional techniques. Despite these benefits, there exists a paucity of technical resources to act as a concrete reference for researchers working with the model. Herein, we present a comprehensive visual outline for the technical aspects of development, growth, propagation, and angiographic utilization of the rabbit VX2 tumor model for use by novice and experienced researchers alike.

The goal of this article is to provide a primer for the development and use of the VX2 carcinoma rabbit model for liver cancer.

The rabbit VX2 tumor is an animal model commonly utilized for translational research regarding hepatocellular carcinoma (HCC) in the field of ...

Spatial and Temporal Control of Murine Melanoma Initiation from Mutant Melanocyte Stem Cells

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented with increasing depth, the incidence rate of cutaneous melanoma has shown a rapid and continuous increase during recent decades. In order to find effective preventative methods, it is important to understand the early steps of melanoma initiation in the skin. Previous data has demonstrated that follicular melanocyte stem cells (MCSCs) in the adult skin tissues can act as melanoma cells of origin when expressing oncogenic mutations and genetic alterations. Tumorigenesis arising from melanoma-prone MCSCs can be induced when MCSCs transition from a quiescent to active state. This transition in melanoma-prone MCSCs can be promoted by the modulation of either hair follicle stem cells&#39; activity state or through extrinsic environmental factors such as ultraviolet-B (UV-B). These factors can be artificially manipulated in the laboratory by chemical depilation, which causes transition of hair follicle stem cells and MCSCs from a quiescent to active state, and by UV-B exposure using a benchtop light. These methods provide successful spatial and temporal control of cutaneous melanoma initiation in the murine dorsal skin. Therefore, these in vivo model systems will be valuable to define the early steps of cutaneous melanoma initiation and could be used to test potential methods for tumor prevention.

The following procedure describes a method for spatial and temporal control of melanocytic tumor initiation in murine dorsal skin, using a genetically engineered mouse model. This protocol describes macroscopic as well as microscopic cutaneous melanoma initiation.

Cutaneous melanoma is well known as the most aggressive skin cancer. Although the risk factors and major genetic alterations continue to be documented ...

Studying Triple Negative Breast Cancer Using Orthotopic Breast Cancer Model

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less aggressive breast tumors, the 5-year survival rate of TNBC patients is 77% due to their characteristic drug-resistant phenotype and metastatic burden. Toward this end, murine models have been established aimed at identifying novel therapeutic strategies limiting TNBC tumor growth and metastatic spread. This work describes a practical guide for the TNBC orthotopic model where MDA-MB-231 breast cancer cells suspended in a basement membrane matrix are implanted in the fourth mammary fat pad, which closely mimics the cancer cell behavior in humans. Measurement of tumors by caliper, lung metastasis assessment via in vivo and ex vivo imaging, and molecular detection are discussed. This model provides an excellent platform to study therapeutic efficacy and is especially suitable for the study of the interaction between the primary tumor and distal metastatic sites.

This work presets an advanced protocol to accurately assess tumor loading by detection of green fluorescent protein and bioluminescence signals as well as the integration of quantitative molecular detection technique.

Triple-negative breast cancer (TNBC) is an aggressive breast cancer subtype with limited therapeutic options. When compared to patients with less ...

An Orthotopic Resectional Mouse Model of Pancreatic Cancer

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer (PC). To address this deficiency, we describe a mouse model involving orthotopic implantation of PC followed by distal pancreatectomy and splenectomy. The model has been demonstrated to be safe and suitably flexible for the study of various therapeutic approaches in adjuvant and neo adjuvant settings.
In this model, a pancreatic tumor is first generated by implanting a mixture of human pancreatic cancer cells (luciferase-tagged AsPC-1) and human cancer associated pancreatic stellate cells into the distal pancreas of Balb/c athymic nude mice. After three weeks, the cancer is resected by re-laparotomy, distal pancreatectomy and splenectomy. In this model, bioluminescence imaging can be used to follow the progress of cancer development and effects of resection/treatments. Following resection, adjuvant therapy can be given. Alternatively, neoadjuvant treatment can be given prior to resection.
Representative data from 45 mice are presented. All mice underwent successful distal pancreatectomy/splenectomy with no issues of hemostasis. A macroscopic proximal pancreatic margin greater than 5 mm was achieved in 43 (96%) mice. The technical success rate of pancreatic resection was 100%, with 0% early mortality and morbidity. None of the animals died during the week after resection.
In summary, we describe a robust and reproducible technique for a surgical resection model of pancreatic cancer in mice which mimics the clinical scenario. The model may be useful for the testing of both adjuvant and neoadjuvant treatments.

In the clinical context, patients with localized pancreatic cancer will undergo pancreatectomy followed by adjuvant treatment. This protocol reported here aims to establish a safe and effective method of modelling this clinical scenario in nude mice, through orthotopic implantation of pancreatic cancer followed by distal pancreatectomy and splenectomy.

There is a lack of satisfactory animal models to study adjuvant and/or neoadjuvant therapy in patients being considered for surgery of pancreatic cancer ...

Generation of an Orthotopic Xenograft of Pancreatic Cancer Cells by Ultrasound-Guided Injection

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant behavior and high resistance to therapeutic treatments. Indeed, despite many efforts, both standard chemotherapy and innovative target therapies have substantially failed when moved from preclinical evaluation to the clinical setting. In this scenario, the development of preclinical mouse models better mimicking in vivo characteristics of PCa is urgently needed to test newly developed drugs. The present protocol describes a method to generate a mouse model of PCa, represented by an orthotopic xenograft obtained by ultrasound-guided injection of human pancreatic tumor cells. Using such a reliable and minimally invasive protocol, we also provide evidence of in vivo engraftment and development of tumor masses, which can be monitored by ultrasound (US) imaging. A noteworthy aspect of the PCa model described here is the slow development of the tumor masses over time, which allows precise identification of the starting point for pharmacological treatments and better monitoring of the effects of therapeutic interventions. Moreover, the technique described here is an example of implementation of the 3Rs principles since it minimizes pain and suffering and directly improves the welfare of animals in research.

We present a protocol to generate a minimally invasive orthotopic pancreatic cancer model by ultrasound-guided injection of human pancreatic cancer cells and the subsequent monitoring of tumor growth in vivo by ultrasound imaging.

Pancreatic cancer (PCa) represents one of the deadliest cancer types worldwide. The reasons for PCa malignancy mainly rely on its intrinsic malignant ...