Name: an orthotopic endometrial cancer model with retroperitoneal lymphadenopathy made from in vivo propagated and cultured vx2 cells
Uploaded: 2019-09-12T12:30:00
Description: 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

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 ...

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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