Breast cancer (BC) is the most prevalent type of cancer and the most common cause of cancer-related deaths among women worldwide (1). TNBC is a subtype of breast cancer histologically defined by the lack of estrogen receptor (ER), progesterone receptor (PR), and HER-2 overexpression (2). Although this subtype of BC accounts for approximately 15%–20% of BC cases worldwide, it is associated with higher incidence of local recurrence and metastasis. In the last decades, the treatment of TNBC has been limited to surgery, chemotherapy, and radiotherapy (3). More recently, biomarker-driven therapies and immune checkpoint inhibitors are demonstrated as promising selections for a subset of TNBC treatment (4). Additionally, as a class of potent anticancer drugs, antibody-drug conjugates are approved for TNBC by FDA (5). Nevertheless, numbers of TNBC patients experience disease progression (DP) within 2–5 years from initial diagnosis (6). Hence, better understanding of the underlying mechanisms involved in TNBC progression and effective treatments against TNBC are urgently needed.

As a type of extracellular vesicles (EVs), exosomes are homogenous membrane vesicles (ranging from 30 to 150 nm), derived from the multivesicular bodies (MVBs), formed by the budding of the endosomal membranes and released in the extracellular space upon fusion with the plasma membrane (7, 8). In 1983, exosomes were discovered and considered to operate as cellular garbage disposal (9, 10). With the investigation of biological function, angiogenesis, immunity, and metastasis have been demonstrated to be regulated by exosomes from cancer cells, making a critical effect in facilitating tumorigenesis (8, 11, 12). Interestingly, some studies indicated that one part of the human epidermal growth factor receptor (HER) family was exosome associated in breast, gastric, and pancreatic cancers (8, 13–15). Moreover, other studies identified higher expression levels of serum exosomal-annexin A2 (exo-AnxA2) in female with breast cancer against noncancer, especially for TNBC rather than luminal and HER2-positive BC (16). Consequently, further searches for exosomes in breast cancer are emerging as a highly potential method for diagnosis and treatment of breast cancer.

In 1996, immunologists discovered that Epstein-Barr virus-transformed B lymphocytes were able to secrete exosomes via fusion of MVBs with the plasma membrane (17).

On the other hand, exosomes released by some tumors also contain a variety of immunosuppressive molecules (18), which can reduce proliferation of CD4 and CD8 T lymphocytes (19–22) or natural killer (NK) cells (23, 24) or promote the differentiation of regulatory T lymphocytes, myeloid cells, and immunosuppressive cells in vitro (25, 26). Consequently, exosomes are closely related to immunotherapy of malignant tumor. Recently, immunotherapy has made appreciable progress in antitumor practice. Based on the immune regulation between the tumor microenvironment (TME) and cancer cells, the clinical benefits of immunotherapy are achieved compared with the traditional treatments by stimulating a sustained antitumor immune response (27). As a vital part of the TME, infiltrating immune cells are considered closely related to tumor progression and the immunotherapy efficacy (28, 29). Previous studies in early stage TNBC and HER2-positive BC suggested potential benefit of immune activation to improve prognosis (30). Immune checkpoint blockade monotherapy and immunotherapy in combination with chemotherapy have been reported to achieve encouraging results in treatment of some subtypes of breast cancer (31, 32). Hence, it is critical to identify novel biomarkers to identify immunotherapy responsive subtypes of breast cancer.

This study aimed to explore the relationship between exosome-associated genes and the immune microenvironment of TNBC. In the present study, the gene profile data of TNBC patients were extracted from the TCGA and GEO databases and genetic data related to exosomes were downloaded from ExoBCD database to analyze and construct an exosome risk model. The prognostic prediction for TNBC patients was conducted according to this risk model. Subsequently, the exosome risk score was used as the entry point to investigate distinction in the infiltration rate of immune cells. On this basis, the interrelation between exosome risk score and tumor immune microenvironment was further searched and four genes (33–35) previously reported associated to immune checkpoint inhibitors were also analyzed. In the future, this crucial treatment method will be employed to develop numbers of interesting combination therapy strategies.

In a majority of cell types, multivesicular endosomes release small membrane, such as exosomes, playing vital roles in cell-to-cell communications (18). Exosomes are investigated in various biological functions, including antigen presentation, immune regulation, apoptosis evasion, drug resistance, immune surveillance escape, and so on (8, 18). In addition, exosomes secreted from some malignant tumor cells were considered a key in regulating angiogenesis, immunity, and metastasis to promote tumorigenesis (11, 12). Some studies reported that exosomes were easily available and stable in vitro. Therefore, researchers suggested that exosomes would have huge potentiality in malignant tumor diagnosis and treatment in early stage (39). By way of example, the finds in pancreatic ductal adenocarcinomas by Bruno Costa-Silva et al. suggested that exosomal macrophage migration inhibitory factor (MIF) primed the liver for metastasis because of the high expression of MIF in PDAC-derived exosomes and the blockade of MIF could prevent liver premetastatic and metastatic niche formation (40). The study in melanoma by Peinado et al. found that the metastatic behavior of primary tumors was governed by the transfer of exosomes to bone marrow progenitor cells via MET receptor (11). Additionally, the exosome-related liquid biopsy approach has been applied to detect prostate and lung cancer markers (41, 42). Wang et al. discovered that exosomal tetraspanin CD82 was associated with BC progression and the high expression levels of CD82 were detected in BC patient serum (43). More recently, automated micro flow cytometer was employed by Kibria et al. to analyze expression status of exosomes isolated from tumor cells and blood of BC patients (44). A significant reduction in CD47 expression in circulating exosomes was observed in breast cancer patients (45). Furthermore, some studies in breast cancer indicate that exosomal proteins and microRNAs may also be used as cancer biomarkers. Gonzalez Villasana et al. confirmed the higher concentrations of exosomes in breast cancer patients by isolating miR-145, miR-155, and miR-382 in the exosomes from BC and noncancer patients, proving a correlation between the concentration of exosomes and the status of malignant breast tumors (46, 47). This phenomenon has been hotly discussed by many scholars. Anyway, the potential efficacy of exosomes to be a vital factor of microenvironment in TNBC diagnosis and treatment is always being researched.

With a deepening understanding of the mechanisms of exosomes, exosome-related gene expression status is associated with tumor progression. Therefore, three exosome-related genes were included in our exosome-related gene model by univariate Cox and LASSO Cox regression analysis. The prognosis of high-risk group and low-risk group patients were distinct different and patients in the low-risk group had a significantly higher survival ratio. Additionally, the ROC curve analysis confirmed that the established prognostic signature was powerful in predicting the OS for TNBC patients. Subsequently, the TNBC patients from the GEO cohort were categorized into high and low risk groups according to the median value of risk score and the group with lower risk score had a better prognosis rather than higher risk score. Hence, the three-gene signature along with covariates including race, age, and tumor stage were involved in the univariate and multivariate Cox regression models, proving that exosome-related gene risk score and tumor stage were independent prognostic factors for TNBC patients. Furthermore, R clusterProfiler package was applied to identify pathways enriched in the different risk groups. The result suggested that a majority of GO terms and KEGG pathways were related to lymphocyte activation and biological processes.

Immunologists discovered that exosomes play a vital role in antigen specific immune responses. Exosomes carry MHC-peptide complexes and antigens to increase the number of dendritic cells (DCs), which can present antigenic peptides to T cells (17). Notably, some studies indicated that exosomes released by tumors also bear various immunosuppressive molecules, for instance, CD4 and CD8 T lymphocytes (19, 21), NK cells (23, 24), regulatory T lymphocytes (25), and myeloid cells (26). Besides, some experiments in vivo mouse models obviously demonstrated that the antigen-shuttle function of exosomes overcame their inhibitory effects on immune cells in conditions of artificial overexpression of an antigen (24). By contrast, several groups favored the idea that, as for tumors, exosomes could inhibit anti-tumor immune responses and promote tumor progression such as migration and angiogenesis to form metastases (48). Recently, studies observed that patients with large malignant tumors had the increasing numbers of exosomes carrying tumor markers, which might only be the result of tumor expansion, instead of actively participating of vesicles in tumor progression of (49). Although, exosome secreted by tumors plays a vital role in immune system, the interaction mechanism is still much less explored.

Several immune activities are closely related to exosomes in tumors. Exosomes released by tumors can transfer antigens to DCs to activate specific T cells and make the activating ligands of NK cells and macrophages exposed to further promote immune responses (18). Oppositely, they carry different signals that can suppress various immune cells. Recently, a study about exosomal miRNA in breast cancer suggested that the transmission of miR-138-5p via exosomes could led to downregulation of KDM6B expression, inhibition of M1 polarization and stimulation of M2 polarization. Therefore, the relationship between exosomes and immune system was further detected (50). It has been reported that ESTIMATE scores could be used to predict survival time of patient with cancer to further clarify the facilitating effect of the microenvironment to tumor cells infiltration (51). In addition, stromal and immune cells from the TME play a crucial role in the tumorigenesis and tumor progression, related to the prognosis of patients with malignant tumors. In our study, low exosome-related risk group indicated a higher immune and ESTIMATE scores rather than high risk group. Subsequently, CIBERSORT algorithm was employed to calculate TIIC proportions and establish 22 kinds of TIIC profiles for high and low exosome-related risk groups. The result implicated that CD4⁺-activated memory T cells and M1 macrophages were both upregulated in low-risk group in patients from the TCGA and GEO databases, whereas M2 macrophages was downregulated in the low-risk group in patients from the TCGA and GEO databasses, respectively. As important regulators of the tumor microenvironment, exosomes have been suggested to play vital roles both in promoting immune response, as well as in inhibiting immune responses (52). Some studies implicated that exosome derived from tumor cell contained a lot of DNA, mRNAs, miRNAs, and enzymes, which shaped innate immune responses in tumor microenvironment. The exchange between immune cells and other cell types may be accomplished through the packaging of RNAs and DNAs (both single and double stranded) into exosomes that are selectively targeted and internalized with specific cell surface motifs. Fabbri et al. proposed that oncogenic genes miR-21 and miR-29a excreted by exosomes derived from lung cancer cells were able to combine TLR with mouse (TLR7) and human (TLR8), resulting in TLR-mediated NF-κB activation and secretion of the prometastatic inflammatory cytokines TNF-α and IL-6 (53). The mechanisms of tumor-immune system communication are of great significance for investigating the TIME regulatory factors. NK cells are known to kill tumor cells and produce cytotoxic cytokines, which can be trained through tumor-derived exosomes (54). Additionally, the status of DCs in tumor immune microenvironment (TIME) can also be influenced by exosomes released from tumor cells. Some other membrane and immune-related molecules involved in the recruitment and activation of immune cells in TIME were detected in exosomes derived from DCs (55). Moreover, exosomes secreted from tumor cells were demonstrated as one of stimuli to regulate macrophages. M2 was reported to be closely associated with the progression and prognosis of malignant tumor. Compared with M2, exosomes released by M1 could enhance antigen-specific cytotoxic T-cell responses to further enhance the activity of lipid calcium phosphate nanoparticle-encapsulated Trp2 vaccine. While, exosomes derived from M2 were verified to promote the growth and invasion of BC cells (56, 57). As for neutrophils, some cytokines and mediators were reported to be loaded via exosome-related neutrophils to modulate tumor progression. However, the stimuli in the TIME can in turn regulate the status of neutrophils to further shape tumor immune responses and influence tumor development (55). The accumulation of mast cells in tumor sites accounts for the construction of TIME. The regulator signals and other components released by mast cells were delivered to B cells, T cells, DCs, and tumor cells by exosomes (55). Exosomes also mediate the crosstalk between tumor cells and adaptive immune cells, which is one of major mediators for intercellular communications among adaptive immune cells, tumor cells. The dysfunction of T cell can be impaired because of tumor-derived exosomes. Some in vitro studies indicated that exosomes from tumor cells can induce the apoptosis of antigen-specific CD8⁺ T cells to suppress their functions (58). T-cell receptor and IL-2 receptor were also reported to be negatively modulated via tumor-related exosomes, resulting to the inhibition of T-cell proliferation (59). In addition to diverse categories of immune cells, other cellular constituents of the tumor microenvironment, involving mesenchymal stem cells, fibroblasts, and endothelial cells, play an aggressive role in tumor inception, promotion, proliferation, and metastasis. Tumor cell-derived exosome could educate normal MSCs with a protumor phenotype. Exosomes derived from MSCs with inflammatory cytokine stimulation include various mediators to suppress not only the progression of B cells, T cells, and NK cells but also the differentiation and antibody production of plasma cells, as well as to induce Tregs (60–62).

Immune checkpoints play a vital role in carcinogenesis by facilitating tumor immunosuppression. Stimulating the immune checkpoint targets can prevent tumors from attacking, including PD-L1, CTLA-4, LAG3, and TIM-3. The functions of these molecules are to inhibit T-cell receptor from activating downstream signals, thereby eliminating cytotoxic T lymphocytes and suppressing antitumor immunity (63). On the contrary, it was reported that PD-1 receptor and PD-L1 as a pair of T-cell immune response costimulatory molecules play a negative role in adoptive immunity by inhibiting T lymphocyte function. AiErken et al. reported that PD-L1 expression and tumor-infiltrating lymphocytes (TILs) were particularly biologically important in TNBC. The OS and DFS of patients with negative PD-L1 expression were shorter than PD-L1 expression (64). Notably, according to recent researches, TILs was able to become a basic marker in predicting treatment response (65). Additionally, previous studies reported that LAG3 positive intraepithelial tumor infiltrating lymphocytes (iTILs) were enriched in ER-negative breast tumors and considered an independent favorable prognostic factor, and the high expression of LAG3 in tissues was related to the good prognosis of triple negative breast cancer (66). In this research, PD-L1, LAG3, TIM-3, and CTLA-4 were upregulated in the low-risk group, as well. The high expression levels of PD-L1 might be associated with TIL-mediated antitumor inflammatory, indicating that cells in immune system are active (64). However, in some studies, the expression levels of CTLA-4 and TIM-3 were high in TNBC with poor clinical outcomes, which might be associated with the different TIL characters of different TNBC subtypes, supporting potential immune checkpoint blockade combination strategies to be a novel therapy for BC.

Neoadjuvant chemotherapy has been recognized to play better efficacy in BC patients without metastasis in lymph nodes, and therefore the indication for neoadjuvant chemotherapy were extended to patients with early TNBC. The patients in early-stage TNBC treated with neoadjuvant chemotherapy regimens based on paclitaxel and anthracyclines had a pathologic complete remission (PCR) rate of 30% to 40% (67), while those treated with carboplatin and paclitaxel had a PCR rate of 45% (68). Notably, patients treated with sequential dose-intensive combination of adriamycin, cyclophosphamide, paclitaxel, and carboplatin achieved a PCR rate in approximately 50% (69). The patients treated with chemotherapy regimen of docetaxel combined with carboplatin reached a PCR rate of 55% (70). Although the addition of carboplatin induces higher PCR rate, it also entails more complex adverse effects. Besides, targeted therapy and immunotherapy also play vital roles in TNBC treatment in recent years. Unfortunately, there are still no effective drugs for TNBC treatment, which is one of the reasons why TNBC has a high mortality rate. Eventually, we investigated whether the exosome-related risk model could predict chemosensitivity in TNBC. The results demonstrated that the IC₅₀ values were statistically higher in the low-risk group for some anticancer agents. However, mithramycin, pipobroman, and sonidegib have seldom reported to apply in breast cancer treatment. It is the limitation of our study that lacking experiments to verify the effects of the three kinds of medicine on TNBC.

This study was the first one to establish and validate an exosome risk model according to three exosome-related genes, serving as an independent prognostic factor in TNBC patients. Our findings indicated that three-exosomal-gene risk model played a vital role in immune infiltration and has a close relationship with the prognosis of TNBC. Besides, some limitations of our study should be considered. A part of a clinical data in the TCGA or GEO cohort is incomplete, and the missing data may not be completely random, causing the bias in the clinical correlation analysis. In addition, the risk model was only established by exosome-related genes, but some other hot biomarker genes were absent. We only identified the association between the exosome-related risk model and immune infiltration, while the correlation between exosomes and TIME was seldom involved. Consequently, it requires a wider range of multicenter clinical verification to support our hypothesis and further experiments are needed to validate the association between exosomes and immune cells to give novel insights in the immunotherapy of TNBC patients.

By combining bioinformatics tools and related algorithms, an exosome risk model that associated with immune infiltration was established and validated to predict the prognosis of TNBC patients. It can serve as an independent prognostic factor and bring new insights into the treatment of TNBC.

The datasets presented in this study can be found in online repositories. The names of the repository/repositories and accession number(s) can be found in the article/ Supplementary Material .

Conception and design: PQ, QG, and JL. Development of methodology: QG and PQ. Acquisition of data (acquiring databases, data processing, etc.): PQ and QG. Analysis and interpretation of data (e.g., statistical analysis, biostatistics, computational analysis): PQ, QG, and JC. Writing, review, and/or revision of the manuscript: QG, QY, PQ, and JL. Administrative, technical, or material support: PQ, QG, and JL. Study supervision: JL. All authors contributed to the article and approved the submitted version.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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