Isolatie en Voortplanting van circulerende tumorcellen uit een muis Cancer

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 biology can contribute to the search for targeted therapy. Furthermore, CTCs may have diagnostic applications. To achieve these potential clinical applications, one needs to overcome some current challenges to studying CTCs. One challenge is related to the fact that CTCs may be present as single cells or as clusters and they may even be able to change their phenotype in response to the blood microenvironment². Moreover, detection can be very challenging, in part, due to the low count of CTCs (a few to hundreds per milliliter) among one billionhematologic cells per milliliterin the blood⁶. Nevertheless, in recent years, research into the potential clinical applications of CTCs from solid organ cancers has intensified.

Despite these efforts, the challenges of studying and understanding the role of CTCs persist due to the rarity of CTCs and the inadequacy of the technological tools currently available. Despite these challenges, the tremendous potential for clinical applications continues to be an incentive to pursue research into the role of CTCs in cancer metastasis.

We were recently successful in isolating and propagating in cell culture CTCs from an orthotopic syngeneic mouse model of hepatocellular carcinoma (HCC) metastasis⁵. The purpose of the current paper is to describe in detail all aspects of the successful methodology. The significance of this methodology lies in the fact that this approach may be modified in order to successfully isolate and propagate in culture human CTCs, thus enhancing the possibility of in vitro studies of CTC biology.

There are multiple potential clinical applications for the use of CTCs. CTCs may be useful for prognosis, response monitoring, screening, dynamic monitoring of tumor molecular alterations, and personalized therapy⁴. Therefore, a better understanding of the biology of CTCs has high potential for clinical impact.

Protocol

Ethiek Verklaring: Alle dierproeven werden goedgekeurd door de Institutional Animal Care en gebruik Comite (IACUC) van Hunter College van de City University van New York. 1. Pre-experiment Procedures Bij aankomst in het dierverblijf aanvankelijk huis 3 BALB / c muizen een kooi met een gefilterde top en houtsnippers beddengoed. Zorg ervoor dat de muizen hebben gratis toegang tot voedsel, Purina knaagdieren voer en water. Beddengoed en voedsel moet tweemaal per week worden gewijzigd…

Representative Results

De hier beschreven methode heeft aangetoond dat CTCs worden geïsoleerd. Muizen werden op humane wijze gedood bij experimentele eindpunt. Het proces betrokken kooldioxide stikken, intra-cardiale leegbloeden en cervicale dislocatie. Een schema van de belangrijkste stappen van de procedure zijn weergegeven in figuur 2. Volbloedmonsters door intra-cardiale leegbloeden verzameld werden verwerkt volgens de hierboven beschreven protocol. Daarna werd centrifugeren toegepast om de buffy coat laag die werd onder…

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

Heparin Sodium Salt 1G	VWR	89508-852
BTX Tube Micro 1.5mL Clear NS	VWR	89511-254	1.5mL  pyrogen-free eppendorf Tubes
Needle Sterile Disp BD 25GX1IN	VWR	BD305125	25G Needle
Slp Tip SRNG 1ml 200 each per pack	VWR	BD309659	1mL syringe tip
Syringe 1ml leur lok Pk 100	VWR	BD309628	1 mL syringe
VWR Forceps Tissue 6	VWR	82027-446	Forceps
Cyromold intrm 15X15X5MM PK 100	VWR	25608-924	Cyromold
Cryo-oct compund 4oz	VWR	25608-930	Oct compound
VWR Slide sprfrst 25X75MM PK72	VWR	48311-703	Slides
VWR Cover Glass #2 22X5oMM OZ	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 2L	VWR	101454-204	Nuclease Free Water
Buffer Tris Ultra Pure Grade 500G	VWR	97061-796	Tris Buffer
Ammonium Chloride ACS Grade 2  5KG	VWR	97062-048	Ammonium Chloride Buffer
Falcon Tissue Culture Dish 60 x 15mm Style polystyrene	VWR	353002	Tissue Culture Dish
Clorox® Germicidal Bleach, Regular	VWR	89501-620	Clorox Bleach
PBS, 1X (Phosphate-Buffered Saline) without calcium & magnesium (500mL)	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 Seru8m	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	13000rpm Centrifuge