Collection of Naturally Released Exosomes from Tumor Tissue

Summary

This protocol demonstrates a novel method for extracting exosomes from tumor tissues by releasing them naturally and collecting them repeatedly to provide an experimental basis for studying the function of exosomes in tumor development.

Abstract

As vesicles secreted by cells, exosomes enclose and transfer various nucleic acids, proteins, and lipids, and participate in communication between different cells. Exosomes in the tumor microenvironment play an important role in the biological characteristics of tumor cells and can strongly influence the states of other cells. Therefore, to further study the specific functions of exosomes in the development of tumors, it is undoubtedly necessary to extract and isolate exosomes from tumor tissues. Direct extraction of exosomes from tumor tissue reduces the need for in vitro cell culture, and exosomes from tumor tissue may be more similar to those in the natural state. Therefore, maintaining the natural state of exosomes in tumor tissue is the key to the extraction process. Enzymatic digestion is often combined with ultracentrifugation to extract exosomes from tumor tissues. Here, we provide a novel method to extract exosomes from tumor tissue. We used a serum-free medium to incubate small tumor tissue pieces on a shaking table, repeatedly collected and extracted exosomes naturally released from tumor tissue, and identified the extracted exosome structure, concentration, and characteristics, providing a new method for researchers studying the function of exosomes in tumor development.

Introduction

Exosomes are a subtype of extracellular vesicles secreted by cells, typically ranging in size from 30 nm to 200 nm^1,2. They participate in intercellular communication and deliver various signaling molecules, including nucleic acids (e.g., deoxyribonucleic acid (DNA), messenger RNA (mRNA), micro ribonucleic acid (miRNA), long non-coding RNA (lncRNA), circular RNA (circRNA)), proteins, and lipids^1,3,4,5, to recipient cells to influence their physiological functions⁶

Protocol

All tumor tissue exosome-related experiments were conducted according to the relevant guidelines and regulations. This study was approved and conducted by the guidelines of the Ethics Committee of West China Hospital, Sichuan University (20220301101). Four weeks after inoculation with SGC-7901 cells, one nude mouse (BALB/c, Male, 8 weeks old, 26 g) was euthanized via cervical dislocation.

1. Acquisition of tumor tissue

1. Soak in 75% alcohol for 5 min.
2. Move to a sterile operating table, use sterile surgical scissors to open the outer skin of the ectopic tumor.
3. Peel off the ectopic tumor tissue, ....

Results

TEM revealed that the exosomes derived from tumor tissue via this method had typical cup-like structures (Figure 1A), and there was no significant difference between the shape of exosomes extracted by this method and by enzyme digestion (Figure 1B). The NTA results revealed that the average exosome diameter was 172.8 nm, which was in the range of 30-200 nm and was consistent with the size of the exosomes; the particle concentration was 5.9 × 10⁶/mL (<.......

Discussion

In this method, exosomes derived from tumor tissue were extracted from the ectopic gastric cancer tissues of nude mice, and the key steps were the release, exudation, separation, and purification of the exosomes. Various exosome separation methods have been designed and verified according to the physical properties and composition of exosomes^2,21,28,29, each with advantages and disadvantages, b.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
α-MEM	HyClone	SH30265.01B
Anti-CD63 antibody	Bioss	bsm-43117M
Anti-CD9 antibody	Bioss	bs-2489R
Centrifuge tube	Thermo	339650
Constant temperature shaker	Thermo	MAXQ4450
Disposable sterile pipette-10 mL	JET	GSP01001
EDTA	Biosharp	BS107
Electric pipette	Thermo	9501
Horizontal controlled temperature centrifuge	Thermo	Sorvall ST1R plus
Nanoparticle tracking analyzer	Malvin	NanoSight Pro
Ophthalmic scissor	Jinzhong	Y3D010
PBS	HyClone	SH30256.01B
Phosphotungstic acid	Sigma	455970-10G
Surgical scissor	Jinzhong	J21010
Secondary antibody	Bioss	Bs-0295G	Goat Anti-Rabbit IgG H&L/FITC
Transmission Electron Microscope	Nikon	Plan Apo WF
Ultracentrifugal tube	Beckman	355618