Method Article

Collection of Naturally Released Exosomes from Tumor Tissue

DOI:

10.3791/67739

⸱

June 10th, 2025

 ,  ,  ,  ,  ,  ,  , 
1Medicine and Engineering Interdisciplinary Research Laboratory of Nursing & Materials, West China Hospital, Sichuan University/West China School of Nursing, Sichuan University, 2Department of Occupational Diseases, The Second Affiliated Hospital of Chengdu Medical College Nuclear Industry 416 Hospital, 3Department of Plastic and Burn Surgery, West China School of Medicine, West China Hospital, Sichuan University

* These authors contributed equally

In This Article

Summary

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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

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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

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Exosomes are a subtype of extracellular vesicles secreted by cells, typically ranging in size from 30 nm to 200 nm1,2. 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 lipids1,3,4,5, to recipient cells to influence their physiological functions6,7. Initially identified in sheep reticulocytes8, exosomes have since been identified in various body fluids, including blood, saliva, urine, cerebrospinal fluid, ascites, amniotic fluid, and breast milk9,10,11,12,13,14,15,16. Moreover, exosomes can be secreted by many types of cells, including lymphocytes, macrophages, mast cells, fat cells, and tumor cells17. With their wide distribution, exosomes play a critical role in various pathological processes, especially cancer, and in various physiological processes, including embryonic development, tissue repair and regeneration, and immune regulation18,19,20.

In recent years, exosomes have been shown to promote tumor occurrence, invasion, metastasis, chemotherapy resistance, and anti- or pro-tumor immunity20,21,22. Cancer-derived exosomes can activate gene expression in receptors or neighboring cancer cells to change their biological phenotypes, and cancer cells can interact with stromal cells in the tumor microenvironment to promote cancer cell proliferation, invasion, and angiogenesis23,24,25. Exosomes released by tumor cells can help them excrete toxic drugs, and stromal cell-derived exosomes can promote the formation of cancer cells20. Cancer-derived exosomes are involved in various stages of cancer cell metastasis, including invasion of neighboring tissues, transfer through the circulatory system, and spread into distant organs20. Finally, exosomes perform both immunoactivation and immunosuppression functions in cancer, depending on the ligands, proteins, and miRNAs they carry20. Owing to the important functions of tumor tissue-derived exosomes, they show potential application value in early diagnosis, accurate treatment, and prognostic monitoring of tumors21,22.

Given the importance of tumor tissue-derived exosomes, many researchers have attempted to extract them to further study their properties and functions. Direct extraction of exosomes from tumor tissues not only reduces the need for in vitro cell culture but also more closely resembles the natural state of exosomes. Therefore, maintaining the natural state of exosomes in tumor tissue is the key to the extraction process. The physical properties of exosomes are the basis of common extraction methods, and rarely are the changes in exosome components caused by stimulation during the extraction process considered. In fact, the quality of exosomes is strongly affected by different exosome extraction procedures26,27. Currently, tumor tissues are digested by enzymes and subjected to methods such as density gradient centrifugation, ultracentrifugation, ultrafiltration, size exclusion chromatography (SEC), polymer precipitation, immunoaffinity chromatography, and microfluidic methods, before separation2,21,28,29. Here, we provide a method for extracting exosomes from tumor tissue. We used a serum-free medium to culture small tumor tissue blocks in a shaker, then collected and extracted exosomes naturally released by tumor tissue and identified the concentration and characteristics of the extracted exosomes, providing basic materials and experimental evidence for studying the function of exosomes in tumor development.

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Protocol

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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, then cut and weigh to obtain two pieces of equal weight.
    NOTE: One piece of tumor tissue is used for exosome extraction under this protocol, and another piece of tumor tissue of equal weight is used for exosome extraction by enzyme digestion30.

2. Preconditioning of tumor tissue

NOTE: Perform these steps on ice and in a sterile environment.

  1. Pipette 10 mL of precooled Phosphate Buffer Saline (PBS) (0.01 M) to wash the tumor tissue to remove surface blood and fat.
  2. Cut the tumor tissue (~400 mm3) into 1-2 mm pieces using ophthalmic scissors in 20 mL of PBS with 2 mM ethylene diamine tetraacetic acid (EDTA) in a 10 cm sterile Petri dish. Transfer the above mixture to a 50 mL conical tube.

3. Release of exosomes

  1. Centrifuge the tube from step 1.2 at 200 × g for 5 min at 4 °C.
  2. Discard the supernatant and resuspend the pellet with 20 mL of Minimum Essential Medium α (α-MEM) medium. Incubate at 37 °C at 100 rpm for 1 h in a constant temperature shaker.
  3. Centrifuge at 200 × g for 5 min at 4 °C.
  4. Discard the supernatant and resuspend the pellet with 10 mL of α-MEM medium. Incubate at 37 °C at 100 rpm for 6 h in a constant temperature shaker.
    NOTE: Be gentle with all steps. A short incubation and one wash with α-MEM medium is enough to remove intracellular components that leak due to cutting, including intracellular vesicles and organelles.
  5. Centrifuge at 200 × g for 5 min, 4 °C and collect the supernatant in a new tube.
  6. Resuspend the tumor tissue with 10 mL of α-MEM medium.
  7. Repeat the incubation from step 2.4 followed by step 2.5.
  8. Mix the medium supernatants collected in steps 2.5 and 2.7.
    NOTE: Be gentle with all steps. Two 6 h incubations induced the natural release of exosomes from the tumor tissue pieces.

4. Ultracentrifugation

  1. Filter the supernatant through a 0.45 µm microporous filter.
  2. Centrifuge the supernatant at 12,000 × g for 30 min at 4 °C to remove the large vesicles.
  3. Transfer the supernatant to a new tube and centrifuge at 120,000 × g for 90 min at 4 °C to collect exosomes.
  4. Wash the exosome precipitate with PBS (0.01 M) and centrifuge again at 140,000 × g for 60 min at 4 °C.
  5. Suspend the precipitated exosomes in PBS (0.01 M) for immediate use or storage at -80 °C.
    NOTE: Be gentle with all steps. Perform each step with the smallest possible centrifugal force and the shortest centrifugal time.

5. Presentation of exosome identification results

  1. Identify the morphology of exosomes from tumor tissue via Transmission electron microscopy (TEM)30.
  2. Determine the size and concentration of exosomes from tumor tissue via Nanoparticle tracking analysis (NTA)30.
  3. Identify the exosomal protein markers CD9 and CD63 via western blot (WB)30.
    NOTE: Recent guidelines for exosome research, including the use of CD63, CD9, as criteria for exosome markers31.

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Results

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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 (<...

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Discussion

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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 exosomes2,21,28,29, each with advantages and disadvantages, b...

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Disclosures

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The authors have no conflicts of interest to declare.

Acknowledgements

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This work was supported by the Sichuan Province Science and Technology Support Program (2024NSFSC0592, 2023YFS0064).

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Materials

List of materials used in this article
NameCompanyCatalog NumberComments
α-MEMHyCloneSH30265.01B
Anti-CD63 antibodyBiossbsm-43117M
Anti-CD9 antibodyBiossbs-2489R
Centrifuge tubeThermo339650
Constant temperature shakerThermoMAXQ4450
Disposable sterile pipette-10 mLJETGSP01001
EDTABiosharpBS107
Electric pipetteThermo9501
Horizontal controlled temperature centrifugeThermoSorvall ST1R plus
Nanoparticle tracking analyzerMalvinNanoSight Pro
Ophthalmic scissorJinzhongY3D010
PBSHyCloneSH30256.01B
Phosphotungstic acid  Sigma455970-10G
Surgical scissorJinzhongJ21010
Secondary antibodyBiossBs-0295GGoat Anti-Rabbit IgG H&L/FITC
Transmission Electron MicroscopeNikonPlan Apo WF
Ultracentrifugal tubeBeckman355618

References

$$\rightleftharpoonup{xx}$$ $$\longleftharp{xx}$$, $$\longrightharp{xx}$$,
  1. Yáñez-Mó, M., et al. Biological properties of extracellular vesicles and their physiological functions. J Extracell Vesicles. 4, 27066(2015).
  2. Zhou, B., et al. Application of exosomes as liquid biopsy in clinical diagnosis. Signal Transduct Target Ther. 5, 144(2020).
  3. Rani, S., O'Brien, K., Kelleher, F., Corcoran, C., Germano, S., Radomski, M., et al. Isolation of exosomes for subsequent mRNA, MicroRNA, and protein profiling. Methods Mol Biol. 784, 181-195 (2011).
  4. Zhou, L., et al. The biology, function and clinical implications of exosomes in lung cancer. Cancer Lett. 407, 84-92 (2017).
  5. D'Agostino, E., et al. Exosomal microRNAs: Comprehensive methods from exosome isolation to miRNA extraction and purity Analysis. MicroRNA Profiling. 2595, 75-92 (2022).
  6. Whiteside, T. Tumor-derived exosomes and their role in cancer progression. Adv Clin Chem. 74, 103-141 (2016).
  7. Ludwig, N., Whiteside, T., Reichert, T. Challenges in exosome isolation and analysis in health and disease. Int J Mol Sci. 20 (19), 4684(2019).
  8. Johnstone, R., Adam, M., Hammond, J., Orr, L., Turbide, C. Vesicle formation during reticulocyte maturation. Association of plasma membrane activities with released vesicles (exosomes). J Biol Chem. 262, 9412-9420 (1987).
  9. Li, B., Cao, Y., Sun, M., Feng, H. Expression, regulation, and of exosome-derived miRNAs in cancer progression and therapy. FASEB J. 35 (10), e21916(2021).
  10. Caby, M., Lankar, D., Vincendeau-Scherrer, C., Raposo, G., Bonnerot, C. Exosomal-like vesicles are present in human blood plasma. Int Immunol. 17, 879-887 (2015).
  11. Pisitkun, T., Shen, R. F., Knepper, M. A. Identification and proteomic profiling of exosomes in human urine. Proc Natl Acad Sci USA. 101 (36), 13368-13373 (2004).
  12. Street, J., et al. Identification and proteomic profiling of exosomes in human cerebrospinal fluid. J Transl Med. 10, 5(2012).
  13. Palanisamy, V., et al. Nanostructural and transcriptomic analyses of human saliva derived exosomes. PLoS ONE. 5, e8577(2010).
  14. Runz, S., et al. Malignant ascites-derived exosomes of ovarian carcinoma patients contain CD24 and EpCAM. Gynecol Oncol. 107, 563-571 (2007).
  15. Keller, S., et al. CD24 is a marker of exosomes secreted into urine and amniotic fluid. Kidney Int. 72, 1095-1102 (2007).
  16. Admyre, C., et al. Exosomes with immune modulatory features are present in human breast milk. J Immunol. 179, 1969-1978 (2007).
  17. Simpson, R., Lim, J., Moritz, R., Mathivanan, S. Exosomes: proteomic insights and diagnostic potential. Expert Rev Proteomics. 6 (3), 267-283 (2009).
  18. Sung, B. H., Parent, C. A. Extracellular vesicles: Critical players during cell migration. Dev Cell. 56, 1861-1874 (2021).
  19. Xie, Q., et al. Exosome-mediated immunosuppression in tumor microenvironments. Cells. 11 (12), 1946(1946).
  20. Zhang, L., Yu, D. Exosomes in cancer development, metastasis, and immunity. Biochim Biophys Acta Rev Cancer. 1871 (2), 455-468 (2019).
  21. Jiang, Z., Wang, H., Mou, Y., Li, L., Jin, W. Functions and clinical applications of exosomes in pancreatic cancer. Mol Biol Rep. 49 (11), 11037-11048 (2022).
  22. Pegtel, D. M., Gould, S. J. Exosomes. Annu Rev Biochem. 88 (1), 487-514 (2019).
  23. Al-Nedawi, K., et al. Intercellular transfer of the oncogenic receptor EGFR VIII by microvesicles derived from tumour cells. Nat Cell Biol. 10, 619-624 (2008).
  24. Nazarenko, I., et al. Cell surface tetraspanin Tspan8 contributes to molecular pathways of exosome-induced endothelial cell activation. Cancer Res. 70, 1668-1678 (2010).
  25. Skog, J., et al. Glioblastoma microvesicles transport RNA and proteins that promote tumour growth and provide diagnostic biomarkers. Nat Cell Biol. 10, 1470-1476 (2008).
  26. Macias, M., et al. Liquid biopsy: from basic research to clinical practice. Adv Clin Chem. 83, 73-119 (2018).
  27. Brennan, K., et al. A comparison of methods for the isolation and separation of extracellular vesicles from protein and lipid particles in human serum. Sci Rep. 10, 1039(2020).
  28. Hu, J., et al. Application exosomes as nanocarriers in cancer therapy. J Mater Chem B. 11 (44), 10595-10612 (2023).
  29. Wang, C., et al. A light-activated magnetic bead strategy utilized in spatio-temporal controllable exosomes isolation. Front Bioeng Biotechnol. 10, 1006374(2022).
  30. Crescitelli, R., et al. Subpopulations of extracellular vesicles from human metastatic melanoma tissue identified by quantitative proteomics after optimized isolation. J Extracell Vesicles. 9 (1), 1722433(2020).
  31. Théry, C., et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J Extracell Vesicles. 7 (1), 1535750(2018).
  32. Théry, C., Amigorena, S., Raposo, G., Clayton, A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol. 3 (Unit 3.22), (2006).
  33. Gardiner, C., et al. Techniques used for the isolation and characterization of extracellular vesicles: results of a worldwide survey. J Extracell Vesicles. 5, 32945(2016).
  34. Li, W., et al. Comparison of the yield and purity of plasma exosomes extracted by ultracentrifugation, precipitation, and membrane-based approaches. Open Chem. 20, 182-191 (2022).

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Tumor ExosomesExosome IsolationTumor TissueExosome ExtractionTumor MicroenvironmentUltracentrifugationEnzymatic DigestionCell CommunicationExosome CharacterizationSerum Free Medium
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