The Application in Cancer Diagnosis of Exosomes

As we know, exosomes are small vesicles secreted by cells. Therefore, exosomes have the characteristics of donor cells and have certain cell specificity. In a large number of studies on exosomes in cancer patients, it has been found that exosomes secreted by cancer cells have characteristic molecules that can be used to distinguish other exosomes, and can be used as molecular markers for cancer diagnosis. At present, the common molecular detection markers in exosomes that can be applied to cancer diagnosis mainly include two types: proteins and nucleic acids.

 

 

. Exogenous protein markers for cancer diagnosis

Based on a large number of studies, it is shown that exosomes in cancer patients are generally enriched in two types of protein molecules, one is the protein highly expressed in cancer cells, such as a proto-oncoprotein; the other is the protein that is specifically secreted to the outside by cancer cells, such as tumor suppressor proteins. Different from the characteristics of normal exosomes, these protein molecules in exosomes can be used as markers for cancer diagnosis. Many exosome protein markers have been found to be useful in the diagnosis of cancer, including early diagnosis of cancer and risk assessment for prognosis.

 

Exosomal protein markers can be used for early diagnosis of cancer. The earlier the cancer is discovered, the greater the chance of cure and the higher 5-year survival rate. Exogenous protein marker detection can be applied to the early diagnosis of many cancers, such as Fibronectin, EDIL3 for early diagnosis of breast cancer; early marker of ovarian cancer Claudin-4; Survivin can be used for early diagnosis of prostate cancer.

 

Exogenous protein marker detection can also be used as a good prognostic risk assessment tool. The exosome protein Glypican-1 is found in pancreatic cancer for early diagnosis of cancer and is also a good prognostic tool. The exosome protein NY-ESO-1 marker in patients with non-small cell lung cancer can be used to prognosis the 5-year survival rate of patients.

 

Exogenous protein marker detection improves the accuracy of cancer diagnosis. In the diagnosis of prostate cancer, exosome Survivin protein marker detection is more accurate than traditional blood PSA protein detection; in the diagnosis of ovarian cancer, the exosome protein marker Claudin-4 is more accurate than the traditional CA125 protein.

 

The combination of exogenous polyprotein markers can significantly improve the accuracy of cancer diagnosis. Sorensen used a multiprotein marker (CD151, CD171, TSPAN8) in exosomes in lung cancer detection and found that more accurate cancer diagnosis results were obtained.

 

. Exosomal nucleic acid markers for cancer diagnosis

The presence of proteins in exosomes is verified with the discovery of exosomes, such as iron transporters. But after more than 20 years, Valadi was first discovered in 2007 that the presence of RNA and miRNA in mouse and human mastocytosis and is biologically active and can be absorbed by recipient cells, affecting the expression of recipient cells. One year later, Taylor et al found that exosome miRNAs can be used as markers for cancer diagnosis. They analyzed EpCAM-positive exosomes isolated from ovarian cancer patients and non-cancer sera. By analyzing the difference in miRNA content in exosomes, it was found that 8 miRNAs can be used as diagnostic markers for various stages of ovarian cancer, opening up the application of nucleic acid markers in cancer diagnosis.

 

A large number of exosome miRNAs have been discovered and can be used as markers for cancer diagnostic testing. Seven miRNAs in colorectal cancer exosomes can be used as diagnostic markers for colorectal cancer at various stages; miR-21 and miR-1246 in exosomes can be used as markers for breast cancer detection. In addition, ovarian cancer, prostate cancer, pancreatic cancer, non-small cell lung cancer and other cancers have been found to be exogenous miRNA markers for cancer diagnosis.

 

Detection of exosome miRNA markers can be used as a risk assessment tool for cancer prognosis. Exosome miR-19a can be used as a low cure rate for colorectal cancer. It is found that patients with colorectal cancer with high miR-19a content in exosomes are prone to liver metastasis, with high prognosis rate and low 5-year survival rate. And miR-4772-3p can be used as a marker for recurrence assessment in patients with stage 2 and 3 colorectal cancer after FOLFOX chemotherapy. Patients with colorectal cancer with low miR-4772-3p content in exosomes have high recurrence rate and low survival rate after FOLFOX chemotherapy.

 

In addition to miRNA-like markers, other nucleic acid molecules in exosomes have also been found to be useful markers for cancer diagnosis. Manterola et al found that RNU6-1, a small non-coding RNA molecule derived from serum exosomes, can be used to diagnose glioblastoma multiforme; Nilsson et al found PCA-3, TMPRSS2 in exosomes derived from urine: ERG transcribed RNA molecules are potential diagnostic markers for prostate cancer; Melo et al found that DNA molecules with KRAS mutations in GPC-positive exosomes can be used for the diagnosis and personalized treatment of pancreatic cancer.

 

 

. Application of exosome “liquid biopsy” in cancer diagnosis

The exocrine secreted by the cells can be directly obtained from various body fluids, blood, urine, saliva, ascites, pleural effusion, etc., and the body fluid of cancer patients often contains a large amount of exosomes, which is used for liquid biopsy of exosomes and provides a material basis for cancer diagnosis. A large number of studies have shown that a variety of cancers can be diagnosed using proteins or nucleic acid markers in exosomes, such as breast cancer, ovarian cancer, prostate cancer, colorectal cancer, etc. Therefore, it provides a theoretical basis for the application of exogenous liquid biopsy in cancer diagnosis. Exosomes can be directly purified from a variety of body fluids, purification and identification techniques are becoming more and more perfect, providing a technical basis for the application of exogenous liquid biopsy for cancer diagnosis.

 

The advantages of exosome liquid biopsy for cancer diagnosis can be summarized as three points: easy to sample, good stability, and coverage of cancer heterogeneity. Compared with tissue biopsy, exudate liquid biopsy has the advantages of non-invasive sampling and convenient sampling. With the improvement of exosome purification and identification technology, it is easy to obtain from blood and urine, and the demand for samples is also reduced.

 

The stability advantages of exosome liquid biopsy are mainly reflected in two aspects: on the one hand, exosomes can maintain the stability of internal molecular markers. Exosomes can protect internal nucleic acids or proteins from various enzymes in body fluids. Valadi et al. found that RNA molecules in exosomes are not degraded by RNase in the external environment. On the other hand, exosomes are still stable in long-term in vitro storage samples, and the activity is maintained very well. Taylor et al found that exosome miRNA markers in blood samples stored at 4 degrees for 24hr were still available for ovarian cancer detection. Exosomes obtained from a two-year sample stored at -80 degrees still have good stability.

 

Exogenous fluid biopsy covers the heterogeneity of cancer and reflects the full picture of cancer compared to traditional tissue biopsy. Cancer heterogeneity is the basis for cancer development and metastasis, as well as a barrier to cancer diagnosis and treatment. Traditional tissue biopsy is limited by sampling and does not completely cover the heterogeneity of cancer, especially in patients with advanced or metastatic cancer, while exogenous fluid biopsy can well cover the heterogeneity of cancer. At the same time, in the course of cancer treatment, exosome molecular markers can change with treatment. Therefore, exogenous liquid biopsy can dynamically monitor patients.

 

Exosomes obtained from body fluids of cancer patients can be used for cancer diagnosis and prognosis recurrence assessment by protein or nucleic acid markers, which significantly improves the application of liquid biopsy in cancer diagnosis. At present, exogenous liquid biopsy has gradually been applied to the diagnosis of cancer. For example, ExoDx Lung (ALK) testing products were launched by Exosome Diagnostics in 2016, which is effectively applied to the diagnosis of non-small cell cancer, and the exosome liquid biopsy product will be used in the diagnosis of various cancers more and more in the future.

 

The Application in cancer treatment

Exosomes can not only provide molecular detection markers for cancer diagnosis, but also can be used in cancer treatment. At present, most of the research on exocytosis cancer is used as a tool for cancer treatment. Of course, exosomes can be directly used as targets for cancer treatment.

 

 

. Exosomes can be used as a tool for cancer treatment

Exosomes serve as a tool for cancer immunotherapy. The more mature research is to isolate dendritic cell exosomes (DEX) from cancer patients, then introduce the antigen polypeptide (MAGE) in vitro, and then reintroduced the modified exosomes into the patient, which activate the body’s immune system, thereby promoting immune clearance of cancer cells. The technology is initially applied to the treatment of some cancer patients, such as melanoma, non-small cell lung cancer.

 

Exosomes can be used as carriers for cancer therapeutics and can carry various types of drugs, including nucleic acids, proteins, lipids or other small molecule chemicals. Exosomes can be transported and delivered through body fluids such as blood, and are absorbed by cancer cells to achieve targeted therapy for cancer. The exosomes are the cell’s own secretions. Compared with the currently used liposomes and polymeric nano-microparticles, it is not easy to cause immune rejection, has small toxicity and good stability. Exosomes are capable of maintaining internal drug stability, especially for nucleic acids or proteinaceous drugs. Exosomes can increase the solubility of hydrophobic drugs, increase the content of drugs in body fluids, and improve the efficiency of drug absorption by cancerous tissues. In addition, as a drug carrier, exosomes can promote tissues that are not easily targeted by body fluid drugs to effectively absorb drugs, such as brain, exosomes can improve the absorption of drugs from blood to brain tissue, and effectively treat cancer in the brain.

 

. Exosomes may serve as direct targets for cancer treatment

A large number of studies have found that exosomes are directly involved in the occurrence and deterioration of cancer, including carrying carcinogenic factors (proto-oncoproteins, RNA, etc.), leading to canceration of recipient cells, metastasis and spread of cancer cells. At the same time, exosomes can inhibit the activity of immune cells (immune cell apoptosis, etc.), so that cancer cells can escape from the immune system.

 

Therefore, Aethlon Medical has developed a device called Hemopurifier that specifically removes cancer-derived exosomes and relieves their immune system to achieve cancer treatment. Some studies directly target exosomes, such as inhibiting the synthesis and secretion of exosomes, such as amiloride; blocking exosomes from being absorbed by recipient cells, such as diannexin. However, there are few patients who have benefited from the treatment of targeted exosomes, the problem of toxic side effects is not clear, and more research is still needed.

 

In conclusion, exosome treatment strategies for cancer are still under investigation and some of the problems in application need to be overcome: for example, the acquisition of high-purity exosomes still has technical limitations and high costs; whether there are side effects during exosome treatment cannot be completely determined; whether the in vitro synthesis of exosomes for cancer treatment still requires a comprehensive understanding of the structure of the exosomes. However, exosomes have made initial clinical advances as a therapeutic tool for cancer, and the application of exosomes may be an effective means of treating cancer in the future.

 

Conclusion

The exosome is like a “double-edged sword” and has a close connection with cancer. Exosomes can promote the proliferation and spread of cancer cells, and play an important role in the occurrence and deterioration of cancer. At the same time, exosomes can be applied to the diagnosis and treatment of cancer. Exogenous fluid biopsy improves the convenience and accuracy of cancer diagnosis, and the characteristics of exosomes that achieve intercellular substance transport are used in the treatment of cancer. Of course, the application of exosomes in the diagnosis and treatment of cancer is still in its infancy, and there is still a need for a deeper understanding of exosomes in the future, so that it can be better applied to medical treatment and improve the survival rate of cancer patients.

 

Exosomes and microvesicles are vesicular membrane structures that are secreted by cells, but they are different in structural composition and biological function. The main differences can be summarized into three points:

. Size and shape: the exosomes are small, about 30-120 nm, and the size and shape are uniform; while the microvesicles are generally larger, 50-100

. Production mechanism: Exosome production requires cell membrane invagination, fusion with intracellular bodies to form MVB, and then released in fusion with the plasma membrane. Microvesicles generally refer to direct extracellular vesicle structures. There are also plasma membrane features, but the exosomal membrane also has intracellular membrane characteristics.

. Membrane-encapsulated substances: due to the difference in the mechanism of production, the substances in the exosomes are related to the intracellular bodies, while the microvesicles are generally not available.

 

However, exosomes and microvesicles have not been well distinguished or even distinguished in many studies (called extracellular vesicles EV); one is limited by purification techniques and it is difficult to thoroughly separate exosomes from small microvesicles (less than 100 nm); second, there is a lack of uniform standards, and even for the size range of exosomes, different researchers choose different standards. Therefore, the above mentioned is only a standard that is generally accepted now. With the further research and technological innovation, it is possible to have a more precise understanding of various vesicle structures in the future.

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