Venous Thrombosis Assay in a Mouse Model of Cancer

Summary

This article aims to present an optimized method for assessing venous thrombosis in a mouse cancer model, using vascular clips to achieve venous ligation. Optimization minimizes variability in thrombosis-related measurements and enhances relevance to human cancer-associated venous thrombosis.

Abstract

This methodology paper highlights the surgical nuances of a rodent model of venous thrombosis, specifically in the context of cancer-associated thrombosis (CAT). Deep venous thrombosis is a common complication in cancer survivors and can be potentially fatal. The current murine venous thrombosis models typically involve a complete or partial mechanical occlusion of the inferior vena cava (IVC) using a suture. This procedure induces a total or partial stasis of blood and endothelial damage, triggering thrombogenesis. The current models have limitations such as higher variability in clot weights, significant mortality rate, and prolonged learning curve. This report introduces surgical refinements using vascular clips to address some of these limitations. Using a syngeneic colon cancer xenograft mouse model, we employed customized vascular clips to ligate the infrarenal vena cava. These clips allow residual lip space similar to a 5-0 polypropylene suture after IVC ligations. Mice with the suture method served as controls. The vascular clip method resulted in a consistent reproducible partial vascular occlusion and greater clot weights with less variability than the suture method. The larger clot weights, greater clot mass, and clot to the IVC luminal surface area were expected due to the higher pressure profile of the vascular clips compared to a 6-0 polypropylene suture. The approach was validated by gray scale ultrasonography, which revealed consistently greater clot mass in the infrarenal vena cava with vascular clips compared to the suture method. These observations were further substantiated with the immunofluorescence staining. This study offers an improved method to generate a venous thrombosis model in mice, which can be employed to deepen the mechanistic understanding of CAT and in translational research such as drug discovery.

Introduction

Cancer-associated venous thromboembolism (VTE)
Venous thromboembolism (VTE) risk is 4 to 7 times higher in cancer survivors compared to the general population^1,2,3. This condition proves fatal in one out of seven patients with cancer. The incidence of VTE varies depending on the type of cancer and the tumor burden and is highest among patients with pancreatic and gastric cancers⁴.

Cancer-associated VTE in cancer patients has prognostic significance. It is associated with unfavorable overall survival in....

Protocol

For this study, 16 female C57Bl6/J mice, 8-12 weeks in age, and a body weight of 20 to 25 g were used. The mice were housed under standard conditions and were fed with chow and water ad libitum. This study was performed with the approval of the Institutional Animal Care and Use Committee (IACUC) at Boston University. The open procedures described here were undertaken in a sterile condition.

1. Xenograft model

1. Cell culture
   1. Prior to heterotopic subcutaneous implantation, grow MC-38 cells as a monolayer to 80% confluency.
   2. Following trypsinization and resuspension in 10% FBS-containin....

Results

A group of female C57Bl6/J mice, 8-12 weeks of age, were injected with MC-38 cells at the logarithmic phase of the cell growth. The xenografts grew rapidly between the third- and fourth -weeks post-injection¹⁸. Once the tumors reached an average volume of 400 mm³, mice were randomized to the control and experimental groups. The control group underwent IVC ligation with suture, while the experimental mice were subjected to IVC ligation with vascular clip application. The tumor volumes in.......

Discussion

In a syngeneic xenograft colon cancer model, we observe higher thrombogenicity and expressions of coagulation markers in the experimental group compared to the control group. Importantly, the variance in all these parameters was lower in the experimental group compared to the control group. The modification involved introducing a vascular clip with a specific pressure profile at the confluence point of the IVC and the left renal vein. The clip was placed over a spacer, which was a 5-0 polypropylene suture. This modificat.......

Materials

Name	Company	Catalog Number	Comments
Buprenorphine 0.3 mg/mL	PAR Pharmaceutical	NDC 42023-179-05
C57BL/6J mice	The Jackson Lab	IMSR_JAX:000664
Caliper	VWR International, Radnor, PA	12777-830
CD31	Abcam	Ab9498
Cell Counter	MOXIE	MXZ000
Clamp	Fine Science Tools	13002-10
Clips ASSI.B2V Single Clamp, General Purpose,	Accurate Surgical & Scientific Instruments	PR 2 144.50 289.00
Dumont #5SF Forceps	Fine Science Tools	11252-00
Fibrin	Millipore	MABS2155-100UG
Fine Scissors - Large Loops	Fine Science Tools	14040-10
Forceps	Fine Science Tools	11002-12
Hill Hemostat	Fine Science Tools	13111-12
Isoflurane, USP	Covetrus	NDC 11695-6777-2
MC-38 cell	Sigma Aldrich	SCC172
Microscope	Nikon Eclipse Inverted Microscope	TE2000
Scissors	Fine Science Tools	14079-10
Suture- Vicryl	AD-Surgical	#L-G330R24
Suture-Nylon 2-0	Ethilon	664H
Suture-Prolene 5-0	Ethicon	8661G
Suture-Prolene 6-0	Ethicon	PDP127
VEV03100	VisualSonics	FujiFilm
Vitrogel Matrigel Matrix	The Well Bioscience	VHM01