Advanced Animal Model of Colorectal Metastasis in Liver: Imaging Techniques and Properties of Metastatic Clones

Summary

The ability of metastatic clones to colonize distant sites depends on their proliferation capacity and/or their ability to survive in the host microenvironment without significant proliferation. Here, we present an animal model that allows quantitative visualization of both types of liver colonization by metastatic clones.

Abstract

Patients with a limited number of hepatic metastases and slow rates of progression can be successfully treated with local treatment approaches^1,2. However, little is known about the heterogeneity of liver metastases, and animal models capable of evaluating the development of individual metastatic colonies are needed. Here, we present an advanced model of hepatic metastases that provides the ability to quantitatively visualize the development of individual tumor clones in the liver and estimate their growth kinetics and colonization efficiency. We generated a panel of monoclonal derivatives of HCT116 human colorectal cancer cells stably labeled with luciferase and tdTomato and possessing different growth properties. With a splenic injection followed by a splenectomy, the majority of these clones are able to generate hepatic metastases, but with different frequencies of colonization and varying growth rates. Using the In Vivo Imaging System (IVIS), it is possible to visualize and quantify metastasis development with in vivo luminescent and ex vivo fluorescent imaging. In addition, Diffuse Luminescent Imaging Tomography (DLIT) provides a 3D distribution of liver metastases in vivo. Ex vivo fluorescent imaging of harvested livers provides quantitative measurements of individual hepatic metastatic colonies, allowing for the evaluation of the frequency of liver colonization and the growth kinetics of metastases. Since the model is similar to clinically observed liver metastases, it can serve as a modality for detecting genes associated with liver metastasis and for testing potential ablative or adjuvant treatments for liver metastatic disease.

Introduction

Patients with liver metastases from primary colorectal cancers (CRC) are characterized by a poor prognosis. The 5-year survival rate for primary nonmetastatic CRC (stages I - III) is estimated as 75 - 88%^3,4, while patients with liver metastases (stage IV) have a 5-year survival rate of only 8 - 12%^5,6. However, metastatic patients represent a heterogeneous group, presenting with different numbers of metastases and different recurrence times. Clinical observations indicate that the number of metastases (which may be proportional to colonizing ability or frequency of colonization) and the size of any single metastasis (proportional to the local gr....

Protocol

All animal procedures were approved by the Institutional Animal Care and Use Committee at the University of Chicago (Protocol # 72213-09) and performed under sterile conditions.

1. Preparations

1. Make 500 mL of medium for the culture of HCT116 tumor cells: Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS), 100 U/mL penicillin, and 100 mg/mL streptomycin.
2. Autoclave the instruments to be used for the spleen injection model, including 3 - 4 surgical towels, gauze, two small Adson pickups, a needle driver, two pairs of scissors, a small clamp, and 3 microclips, at 251 °F for 20 min.
3. Prepare....

Results

The goal of this experiment was to establish a consistent and easily reproducible animal model with the potential for the serial quantification of the in vivo metastatic tumor burden and for the estimation of the colonizing frequency and growth kinetics of developing liver metastases. Figures 2-6, with legends, are provided from our previous publication under a Creative Commons CC-BY license¹⁰.

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Discussion

The animal model presented in the current report is based on two major approaches. First, in order to ensure the ability to observe metastatic clones with different propensities to colonize and proliferate in the liver, a panel of highly heterogeneous monoclonal cell lines was established, rather than an established unfractionated cancer cell line^12,13. The monoclonal approach to metastasis development is justified by recent genomic data¹⁴ and was successfully used previously to model the metastatic.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
IVIS Spectrum In Vivo Imaging System	Caliper Life Sciences	124262	In Vivo imaging system
LivingImage 4.0 Software	Caliper Life Sciences	128165	Imaging software
VAD-MGX Research Anesthetic Machine	Vetamac	VAD-MGX	Inhalation anesthesia machine
DMEM	Gibco	11965-118	Cell culture reagents
DPBS	Gibco	14190250	Cell culture reagents
Penicillin/Streptomycin, liquid (10,000 units penicillin;10,000 μg streptomycin)	Invitrogen	15140163	Cell culture reagents
HBSS	ThermoFisher	24020117	Cell culture reagents
Buprenex Injection (0.3 mg/mL)	Reckitt Benckiser Healthcare Ltd.	12496-0757-5	Buprenorphine hydrochloride
Gemini Cautery System	Braintree Scientific	GEM 5917	Hand-held cautery for splenectomy
Micro Clip; Straight; 70 Grams Pressure; 1.5 mm Clip Width; 10 mm Jaw Length	Roboz Surgical Instrument	RS-5426	Hemoclip: Hemostasis instruments after spleen injection
D-luciferin, potassium salt	Goldbio Technology	LUCK-1G	Luciferin potassium salt
Opti-MEM I Reduced Serum Medium	Gibco	31985062	Reduced Serum Medium
TC20 Automated Cell Counter	BIO-RAD	1450102	Automatic cell counter
JMP10 software	SAS Institute		Data analysis software
BD FACSAria II cell sorter	BD Biocsiences		Cell sorter