Isolation and Propagation of Circulating Tumor Cells from a Mouse Cancer Model

Summary

Circulating tumor cells (CTCs) have been shown to play an important role in tumor metastasis. Here, a method for the isolation and propagation of CTCs from the whole blood of a syngeneic mouse tumor model of hepatocellular carcinoma (HCC) metastasis is described.

Abstract

Cancer metastasis is the foremost cause of cancer-associated deaths. Recent studies have shown that circulating tumor cells (CTCs) are important in cancer metastasis. Indeed, the number of CTCs correlates with tumor size. Here, a detailed description is provided of a methodology for isolation and propagation of CTCs from a syngeneic mouse model of hepatocellular carcinoma (HCC) which allows for downstream analysis of potentially important molecular mechanisms of solid organ tumor metastasis. This method is efficient and reproducible. It is a non-invasive technique and, therefore, has potential to replace the invasive biopsy of tissues from humans which may be associated with complications. Therefore, the method discussed here allows for the isolation and propagation of CTCs from whole blood samples such that they can be examined and characterized. This has potential for future adaptation for clinical applications such as diagnosis, and personalized targeted therapy.

Introduction

The cancer research community has known of the existence of circulating tumor cells (CTCs) since first being observed by Thomas Ashworth in 1869¹. Since then, CTCs have been shown to be important in tumor metastasis and disease progression^2-5. Today, solid tumors are a major cause of morbidity and mortality worldwide. CTCs are rare cells that originate from primary tumors and travel through the blood stream to different organs of which only a small fraction ultimately develop into metastasis^2-5. Notably, there is positive correlation between tumor size and CTC number^3,4.

An understanding of CTC b....

Protocol

Ethics Statement: All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) of Hunter College of The City University of New York.

1. Pre-experiment Procedures

1. Upon arrival to the animal facility, initially house 3 BALB/c mice to a cage with a filtered top and wood chip bedding. Ensure that the mice have free access to food, purina rodent chow, and water. Bedding and food should be changed twice a week while cages should be cleaned once a week.
2. Induce general anesthesia in mice using an isoflurane vaporizer along with oxygen at a constant flow rate of 1 L per min. The system is self-rel....

Results

The methodology described here has shown that CTCs can be isolated. Mice were humanely euthanized at experimental end-point. The process involved carbon dioxide asphyxiation, intra-cardiac exsanguination, and cervical dislocation. A schematic of key steps of the procedure are illustrated in Figure 2. Whole blood samples collected by intra-cardiac exsanguination were processed using the protocol described above. After which, centrifugation was used to determine the buffy coat layer that was subjected to t.......

Discussion

In this study, how to isolate and propagate CTCs from the whole blood of a syngeneic mouse with HCC were described. The objective of this work is to enhance the ongoing studies of the mechanisms of cancer metastasis.

A factor that contributes to the poor prognosis of many cancers is the lack of timely detection and consequent widespread dissemination of the malignancy³. CTCs originate from primary cancers and spread via the blood stream to distant organs. As such, CTCs are importan.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Heparin Sodium Salt 1 g	VWR	89508-852
BTX Tube Micro 1.5 ml Clear NS	VWR	89511-254	1.5 ml  pyrogen-free eppendorf Tubes
Needle Sterile Disp BD 25 G x 1 in	VWR	BD305125	25 G Needle
Slp Tip SRNG 1 ml 200 each per pack	VWR	BD309659	1 ml syringe tip
Syringe 1 ml leur lok Pk 100	VWR	BD309628	1 ml syringe
VWR Forceps Tissue 6	VWR	82027-446	Forceps
Cyromold intrm 15 x 15 x 5 mm PK 100	VWR	25608-924	Cyromold
Cryo-oct compund 4 oz	VWR	25608-930	Oct compound
VWR Slide sprfrst 25 x 75 mm PK72	VWR	48311-703	Slides
VWR Cover Glass #2 22 x 50 mm	VWR	48-382-128	Cover Glass
VWR Slide Box True North Fm Pu	VWR	89140-278	Slide Box
Super HT PAP Pen	VWR	89427-058	PAP pen
Water RNASe and DNAse free 2 L	VWR	101454-204	Nuclease Free Water
Buffer Tris Ultra Pure Grade 500 g	VWR	97061-796	Tris Buffer
Ammonium Chloride ACS Grade 2, 5 Kg	VWR	97062-048	Ammonium Chloride Buffer
Falcon Tissue Culture Dish 60 x 15 mm Style polystyrene	VWR	353002	Tissue Culture Dish
Clorox Germicidal Bleach, Regular	VWR	89501-620	Clorox Bleach
PBS, 1x (Phosphate-Buffered Saline) without calcium & magnesium (500 ml)	Thermo Fischer Scientific	21-040-CV	PBS, 1x
DMEM, 1x  with 4.5 g/L glucose & L-glutamine without sodium pyruvate	Thermo Fischer Scientific	10-017-CV	DMEM 1x media for BNL 1ME A.7R.1 cells
Fetal Bovine Serum	Thermo Fischer Scientific	35-010-CV	FBS
Penicillin Streptomycin Solution, 100x	Thermo Fischer Scientific	30-002-Cl	Penicillin Streptomycin
Sorvall Biofuge pico	Thermo Fischer Scientific	75002411	13,000 rpm Centrifuge