Tracking Tumor Cell Dissemination from Lung Metastases Using Photoconversion

Summary

We present a method for studying tumor cell redissemination from lung metastases involving a surgical protocol for the selective photoconversion of lung metastases, followed by the identification of redisseminated tumor cells in tertiary organs.

Abstract

Metastasis - the systemic spread of cancer - is the leading cause of cancer-related deaths. Although metastasis is commonly thought of as a unidirectional process wherein cells from the primary tumor disseminate and seed metastases, tumor cells in existing metastases can also redisseminate and give rise to new lesions in tertiary sites in a process known as "metastasis-from-metastases" or "metastasis-to-metastasis seeding." Metastasis-to-metastasis seeding may increase the metastatic burden and decrease the patient's quality of life and survival. Therefore, understanding the processes behind this phenomenon is crucial to refining treatment strategies for patients with metastatic cancer.

Little is known about metastasis-to-metastasis seeding, due in part to logistical and technological limitations. Studies on metastasis-to-metastasis seeding rely primarily on sequencing methods, which may not be practical for researchers studying the exact timing of metastasis-to-metastasis seeding events or what promotes or prevents them. This highlights the lack of methodologies that facilitate the study of metastasis-to-metastasis seeding. To address this, we have developed - and describe herein - a murine surgical protocol for the selective photoconversion of lung metastases, allowing specific marking and fate tracking of tumor cells redisseminating from the lung to tertiary sites. To our knowledge, this is the only method for studying tumor cell redissemination and metastasis-to-metastasis seeding from the lungs that does not require genomic analysis.

Introduction

Metastasis is the leading cause of cancer-related deaths¹. Metastatic cancer arises when cells from the primary tumor disseminate throughout the body and proliferate into clinically detectable tumors in distant organs^2,3.

Although metastasis is commonly thought of as a unidirectional process wherein tumor cells disseminate from the primary tumor and colonize distant organs⁴, increasing clinical and experimental evidence suggests that a more complex, multi-directional process is at play. It has been shown that circulating tumor cel....

Protocol

All procedures described in this protocol have been performed in accordance with guidelines and regulations for the use of vertebrate animals, including prior approval by the Albert Einstein College of Medicine Institutional Animal Care and Use Committee.

Prior to surgery, lung metastases are to be generated in mice using cancer cells that express a photoconvertible/photo-switchable protein; several protocols for lung metastasis generation have been published^26,

Discussion

In this paper, we describe a surgical protocol for the selective photoconversion of tumor cells in the lung. This technique enables researchers to selectively mark tumor cells in the lung and track their fate by reidentifying them throughout the body at a later time point, facilitating the study of metastasis from lung metastases. Using this protocol, it was possible to visualize photoconverted cells in the brain, liver, and non-photoconverted right lung of mice that had undergone the surgery with photoconversion, indica.......

Materials

Name	Company	Catalog Number	Comments
0-30 V, 0-3 A Power Supply	MPJA	9616 PS
12 VDC, 1.2 A Unregulated Plug Supply	MPJA	17563 PD
28 G 1 mL BD Insulin Syringe	BD	329410
400 nm light emitting diode array lamp	LedEngin Inc.	897-LZPD0UA00	Photoconversion lamp, custom-built (individual parts included below)
5-0 braided silk suture with RB-1 cutting needle	Ethicon, Inc.	774B
9 cm 2-0 silk tie	Ethicon, Inc.	LA55G
Baytril 100 (enrofloxacin)	Bayer (Santa Cruz Biotechnology)	sc-362890Rx	Antibiotic used in drinking water
Buprenorphine	Hospira	0409-2012-32	Analgesic
Cables (Cable Assemblies) 2.1 DC JACK-STRAIGHT 72"  BLACK/ZIP CORD	Mouser	172-7426-E
Cables (Cable Assemblies) 2.5 JK-ST 72" ZIP CD	Mouser	172-0250
Chlorhexidine solution	Durvet	7-45801-10258-3	Chlorhexidine Disinfectant Solution
Compressed air canister	Falcon	DPSJB-12
Extra Fine Micro Dissecting Scissors 4" Straight Sharp/Sharp 24 mm	Roboz Surgical	RS-5912	Sharp Micro Dissecting Scissors
Fiber-optic illuminator	O.C. White Company	FL3000	Used during mouse intubation
Gemini Cautery Kit	Harvard Apparatus	726067	Cautery pen
Germinator 500	CellPoint Scientific	GER 5287-120V	Bead Sterilizer
Graefe forceps	Roboz	RS-5135
High power LEDs - single color ultraviolet 90 watts	Mouser	LZP-D0UA00
Infrared heat lamp	Braintree Scientific	HL-1
Isoflurane SOL 250 mL PVL	Covetrus	29405	Anesthetic
Isoflurane vaporizer	SurgiVet	VCT302
Jacobson needle holder with lock	Kalson Surgical	T1-140
Labeling tape	Fisher Scientific	S68702
LED Lighting Reflectors CREE MP-L SNGL LENS REFLECTOR & LOC PIN	Mouser	928-C11395TM
Long cotton tip applicators	Medline Industries	MDS202055
Masscool / Soccket 478 / Intel Pentium 4/Celeron up to 3.4GHz / Ball Bearing / Copper Core / CPU Cooling Fan	CompUSA	#S457-1023
Micro Dissecting Scissors 4" Straight Blunt/Blunt	Roboz Surgical	RS-5980	Blunt Micro Dissecting Scissors
Murine ventilator	Kent Scientific	PS-02	PhysioSuite
Nair Hair Removal Lotion	Amazon	B001RVMR7K	Depilatory cream
Personnet mini retractor	Roboz	RS-6504	Retractor
Phosphate Buffered Saline 1x	Fisher Scientific	14190144	PBS
pLenti.CAG.H2B-Dendra2.W	Addgene	51005	Dendra2 lentivirus
Puralube	Henry Schein Animal Health	008897	Eye Lubricant
Rodent intubation stand	Braintree Scientific	RIS 100
Small animal lung inflation bulb	Harvard Apparatus	72-9083
SurgiSuite Multi-Functional Surgical Platform for Mice, with Warming	Kent Scientific	SURGI-M02	Heated surgical platform
Test Leads 48" TEST LEAD BANANA - Black	Mouser	565-1440-48-0
Test Leads 48" TEST LEAD BANANA - Red	Mouser	565-1440-48-2
Tracheal catheter	Exelint International	26746	22 G catheter
Wound closing system veterinary kit	Clay Adams	IN015	Veterinary surgical stapling kit