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

Summary

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.

Abstract

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.

Introduction

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 mortality^1,2,3. The diagnosis of lymph node metastases is paramount as the presence of positive lymph nodes is a strong negative predictor of survival^4,5,6

Protocol

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’s Lab at the University Health Network.

1. Creation of in vitro VX2 cell line

1. Harvest the VX2 .......

Discussion

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 literature^35,37,53,56; however, thw 90% rate of pathologically confirmed lymph node metastases is consistent with previou.......

Materials

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