What is the relationship between Tumor-derived exosomes and NK cell dysfunction?

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What is the relationship between Tumor-derived exosomes and NK cell dysfunction?

Cancer cells actively release a variety of soluble biomolecules, such as cytokines, chemokines, and growth factors, to establish the tumor microenvironment. In the past few decades, extracellular vesicles, especially exosomes, have been recognized as an important tool for cancer cells to communicate with stromal cells and distant cells.

New evidence suggests that tumor-derived exosomes ( TDEs ) contain multiple molecular components, including lipids, membrane-associated proteins, long noncoding RNAs ( lncRNAs ), and miRNAs, which are involved in several processes of tumor formation and invasion, including blood vessels Generation, proliferation and growth, metastasis and immune escape.

Numerous studies have shown that exosomes derived from tumor cells disrupt anti-tumor immunity by impairing the functions of DCs, NK cells, and T cells.

Among them, NK cells are considered to be the first layer of defense against malignant cell transformation, while tumor cells can inhibit the function of NK cells through different mechanisms, among which tumor exosomes play a key role in NK cell dysfunction.

TDEs can be taken up by NK cells or induce downstream signaling through receptor-ligand interactions, thereby downregulating their antitumor activity.

In addition, tumor exosomes contain a variety of surface ligands and biomolecules that interfere with NK cell recruitment, cytokine production, molecular expression, and cytolytic activity.

Therefore, understanding the role of TDEs in NK cell dysfunction has important clinical implications for NK cell-based tumor therapy.

Overview of Exosomes

In 1981 , exosomes were first described as nanovesicles secreted in vitro by different cell types with a lipid composition different from that of the plasma membrane, suggesting a different and more complex origin than simple membrane budding.

Exosomes can be secreted by almost all types of cells, including immune cells, blood cells, neuronal cells, epithelial cells, and cancer cells. They contain proteins that have been retained from primitive cells through the process of biogenesis.

Exosomes can eventually enter the circulation and have been detected in a variety of biological fluids, such as blood, urine, saliva, or breast milk , and in malignant effusions, such as pleural and ascites .

Studies have shown that exosomes can also contain miRNAs, messenger RNAs, and even long non-coding RNAs, which are significantly involved in the regulation of exosomes.

*What is the relationship between Tumor-derived exosomes and NK cell dysfunction?*

The bioactive substances of exosomes can affect the metabolism of recipient cells in two main ways. The first is the direct interaction of exosome surface proteins with target cell receptors. The second is internalization of the contents, after fusion with the plasma membrane of the target cell, or by endocytosis, phagocytosis by macrophages. Exosomes have important clinical implications, especially in tumors.

Effects of TDEs on NK cell function

TDEs can be taken up by various cells, preferably immune cells , through plasma membrane fusion, endocytosis, phagocytosis, micropinocytosis, and lipid raft-mediated internalization .

Multiple studies have shown that tumor exosomes can transport their contents into NK cells by fusing with cell membranes, thereby hindering their antitumor functions.

TDEs regulate NK cell recruitment and migration

There is evidence that TDE positively affects the recruitment and migration of cancer and immune cells to establish the tumor microenvironment and metastatic niche.

A previous study showed that tumor exosomes mediate the migration of MDSCs and promote the metastasis of mouse breast cancer cells ( 4T1 cells ) to the lung in a CCL2-dependent manner.

A recent study showed that exosomes isolated from AML patients can significantly reduce the migration of NK-92 cells to tumor cells.

Further proteomic analysis revealed that AML exosomes were highly enriched for CXCL4, CXCL7 and CCL5 compared to exosomes obtained from healthy donors.

TDEs affect NK cell proliferation and survival

Multiple studies support that TDEs promote tumor progression mainly by downregulating the proliferation and survival of immune cells.

In a pioneering study exploring the effects of tumor exosomes on NK cell proliferation and survival, pretreatment with mouse breast cancer ( TS/a cells ) exosomes was found to reduce the number and percentage of NK cells in vitro.

Subsequent studies found that not only TS/a exosomes, but also exosomes from MDA231 ( human breast cancer ), A2058 ( human melanoma ) and 4T1 ( mouse breast cancer ) cell lines could significantly prevent IL-2 induction NK cell proliferation.

Studies on the downstream signaling pathways of IL-2R showed that the activities of p42/p44 and Akt (a substrate of PI3K) in NK cells were not altered after treatment with TDEs.

However, a decrease in phosphorylated STAT5 levels was observed, and exosomes were able to inhibit Jak3 expression, and the decrease in STAT5 phosphorylation was dependent on the concentration of TDEs.

Likewise, a dose-dependent reduction in phosphorylation levels of cyclin D3 and its substrate Rb was also observed in NK cells treated with tumor exosomes.

TDEs alter the cytolytic activity of NK cells

Recent studies have shown that, in addition to reducing NK cell numbers, tumor exosomes are also candidates for reducing NK cell cytolytic activity in the tumor environment. It has been demonstrated that treatment of NK-92 cells with AML exosomes can significantly inhibit their cytotoxicity against K562 cells.

Likewise, NK cells pretreated with pancreatic cancer-derived exosomes showed reduced cytotoxicity to pancreatic cancer stem cells.

Other results investigating oral cancer-derived exosomes showed that oral cancer killing of NK cells may be increased after co-incubation with NK cells for a short time, but the cytotoxicity of NK cells was significantly reduced by increasing the incubation time.

These findings suggest that although TDEs may stimulate NK cell cytotoxicity for a short period of time, prolonged exposure to TDEs inhibits their cytolytic function, leading to immune escape and cancer progression.

TDEs regulate cytokine production by NK cells

TNF-α and IFN-γ are two major cytokines produced by activated NK cells, which coordinate antitumor immune responses.

Exosomes from cholangiocarcinoma were found to significantly inhibit the secretion of TNF-α by NK cells and reduce their antitumor function.

Similarly, NK cells co-cultured with pancreatic cancer-derived exosomes had a significant reduction in TNF-1 and IFN-γ production, suggesting that tumor exosomes can affect NK cell cytokine production.

TDEs alter receptor and molecular expression patterns of NK cells

NK cell function is known to be tightly regulated by activating and inhibitory receptors. NKG2D, NKp30, NKp44, and NKp46 are important activating receptors on NK cells, and their expression levels determine the antitumor ability of NK cells.

On the other hand, the expression of NKG2A, one of the most important inhibitory receptors of NK cells, was negatively correlated with its antitumor activity.

In fact, tumor cells utilize different mechanisms to manipulate the expression of these molecules on NK cells, thereby altering the antitumor ability of NK cells and leading to tumor progression. It is highly likely that TDEs play a role in altered receptor and molecular expression, leading to tumor-associated NK cell dysfunction.

Recently, researchers investigated the effect of oral cancer-derived exosomes ( OCEX ) on the expression of NK cells NKG2D, NKp30, NKp44, NKp46 and NKG2A and found that OCEX significantly upregulated the activating receptor ( NKG2D ) on NK92MI cells within 24 hours of administration , NKp30, NKp44 and NKp46 ), while the expression of NKG2A was significantly decreased at the same time.

Further experiments revealed that the expression of activating receptors on NK cells gradually decreased over a period of 7 days, while no significant changes in NKG2A expression were observed during this time period.

Since tumors continuously release exosomes to the surrounding microenvironment and circulation, NK cells are more likely to be continuously exposed to tumor exosomes, ultimately leading to the loss of NK cell cytotoxic function.

Molecular basis of TDEs-mediated NK cell dysfunction

Tumor exosomes can actively induce immunosuppression through multiple mechanisms. Various immunosuppressive proteins on its surface and lipid and RNA ( miRNA, lncRNA, etc. ) content of TDE can affect immune cells, including NK cells.

Ligands for NKG2D (MICA/B and ULBP1-6)

Continued exposure of NK cells to ligands expressed on the surface of tumor cells has been shown to contribute to NK cell abnormalities.

Studies have shown that NKG2D-activating ligands MHC class I-associated chain ( MIC ) A and MICB can be shed from tumor cells, and their presence in patient plasma is strongly associated with impaired NK cell responses and disease progression.

The presence of MICA and MICB in tumor exosomes has been demonstrated, therefore, exosomal MICA/B may be able to alter NKG2D expression, and AML exosomes have been found to be enriched in membrane-associated MICA/MICB, which is thought to be responsible for NKG2D downregulation and concomitant Causes of decreased NK cytotoxicity.

Other studies have found that tumor cells also release exosomal ULBP3, which is more effective in down-regulating NKG2D compared with soluble ULBP2, limiting the cytotoxic activity of NK cells.

These results may partially explain the clinically observed NK cell dysfunction in leukemia/lymphoma patients, suggesting that exosomal ligands of NKG2D play a key role in tumor-associated NK cell abnormalities.

TGF-beta

It is well known that TGF-β is involved in a wide range of processes in the regulation of immune responses, and it has inhibitory effects on immune cells including NK cells.

Recent studies have shown that tumor exosomes also carry high levels of membrane-associated TGF-β, which can induce immunosuppressive effects similar to their soluble counterparts.

Multiple studies have now reported that exosomes isolated from AML and clear cell renal cell carcinoma ( ccRCC ) patients or mesothelioma cells contain significantly high levels of mature TGF-β.

In addition, since hypoxic tumor microenvironment has been shown to regulate TGF-β signaling and exosome content loading, we also investigated whether hypoxia affects NK ligands on non-small cell lung cancer ( NSCLC )-derived exosomes and TGF-β expression.

Although no significant changes in NK ligand expression were observed, interestingly, TDEs isolated from hypoxic cells had significantly higher levels of TGF-β compared with normoxic tumor cells.

Adenosine and Glucose Metabolism

Adenosine ( ADO ) is a recognized inhibitor of immune cell function. Studies have shown that NK cells are capable of producing ADO from exogenous ATP in the presence of CD39/CD73-bearing tumor exosomes.

And since NK-92 cells carry A2AR, ADO activation of A2AR autocrine signaling on NK-92 cells is likely to partially lead to the loss of function of NK-92 cells.

In addition, nutrient uptake and glucose metabolism are also important for the normal response of NK cells. CD71 ( transferrin receptor ), CD98 ( large neutral amino acid transporter ) and 2-NBDG are three commonly used metabolic parameters of NK cells.

However, pancreatic cancer cell-derived exosomes significantly reduced the expression of CD71 and CD98 and their glucose uptake capacity in NK cells.

Fas-L, survivin, B7-H3 and PD-L1

Accumulating evidence suggests that TDEs contain a large number of membrane-associated proteins that play critical roles in tumor immune evasion.

Some studies have shown that TDE carrying Fas-L can induce apoptosis of CD8+ T cells.

In a study on lymphoma, lymphoma-derived exosomes were found to be enriched in Fas-L and Survivin, and the results showed that the use of lymphoma-derived exosomes downregulated NKG2D expression on NK cells and reduced perforation The protein levels of cytokines, granzyme B, TNF-α and IFN-γ, which may be Fas-L/Survivin-dependent.

B7-H3, a member of the B7 family of proteins, is another checkpoint molecule that has been shown to have immunosuppressive effects in a variety of tumors.

It has been found that glioblastoma cells secrete B7-H3 via exosomes, thereby inhibiting NK-mediated tumor lysis.

In addition, most recent studies on TDE point out that exosomes also play an important role in tumor immune escape via the PD-L1/PD1 axis.

RNA

In addition to proteins, TDEs also contain mRNAs, microRNAs, and lncRNAs that can be taken up by other cells, including NK cells , and alter their function.

The study found that liver cancer-derived exosomes contain high levels of miR-92b, and exosomes containing miR-92b can significantly induce miR-92b expression in NK-92 cells, indicating that tumors can actively convert inhibitory organisms through exosomes.

Molecules, including miRNAs , are transferred to infiltrating NK cells. Transfer of miR-92b via TDEs significantly suppressed CD69 expression on NK-92 cells and reduced its cytotoxicity against parental Hep3B tumor cells.

Furthermore, overexpression of miR-92b was found to be associated with enhanced migration of liver cancer cell lines.

Summary

From the studies of the effects of TDEs on NK cells to date, it is clear that tumor exosomes are important players in tumor immunosuppression.

TDEs are involved in tumor NK cell dysfunction through multiple mechanisms and content molecules.

Therefore, by gaining an in-depth understanding of the pathological roles and mechanisms of TDE-mediated NK cell dysfunction, we will be able to further improve the therapeutic potential of NK cells, as well as other immunotherapies, in the treatment of cancer.

References:

1. Cancer exosomes and natural killer cellsdysfunction: biological roles, clinical significance and implications for immunotherapy. Mol Cancer. 2022; 21: 15.