Advances in immunotherapy for triple-negative breast cancer

The chemotherapy drugs encapsulated in cholesterol liposomes generally have a limited effect on TNBC. This is mainly due to the formation of an impenetrable physical barrier by CAFs and their secreted products; these cells also secrete interleukin (IL)-10 and IL-6 and transforming growth factor-β (TGF-β), that collectively inhibit the expression of CD8 + T cells and have immunosuppressive effects within the TME [19, 20]. Notably, TGF-β signaling pathways inhibit CAFs, activate CD8 + T cells, enhance ECM degradation, increase blood perfusion and immune cell infiltration, and improve drug delivery, thereby improving the efficacy of immunochemotherapy [21, 22]. Ginsenoside Rg3, a component of ginseng, exhibits anti-tumor activity and inhibits growth and angiogenesis in human breast invasive ductal carcinoma xenografts in nude mice [23]. Rg3 has been used instead of cholesterol to develop a docetaxel-loaded Rg3 liposome, which can penetrate more deeply into the tumor, inhibit the CAFs ,TGF-β, and collagens to offer better chemosensitization in TNBC [24]. The combination of ginsenoside Rg3 with paclitaxel (PTX) can inhibit the nuclear factor-κB (NF-κB) pathway to enhance the cytotoxicity of chemotherapeutic agents in TNBC [25]. The liposomes based on Rg3 offer excellent encapsulation efficiency and drug-loading ability and can achieve tumor targeting and TME remodeling without any synthetic processes. They offer good clinical applicability and are expected to provide higher drug delivery efficiency for the treatment of TNBC.

Anacardic acid(6-Pentadecyl Salicylic Acid, 6SA) is found in the bark of dipteran plants and elicits the activation of macrophages by augmenting the MAPK signaling pathway, particularly ERK and NF-κB, as well as enhancing the release of nitric oxide (NO) and cytokines [26], which facilitates the activation of immune cells. 6SA reduces tumor volume without diminishing the number of TILs by inducing caspase-8 mediated cell apoptosis, and also concurrently augmenting the secretion of interferon-gamma (IFN-γ) and tumor necrosis factor-alpha (TNF-α), and improves the immune microenvironment of TNBC [26]. 6SA exhibits potent anti-tumor effects on TNBC in vivo with intact immune function, while also mitigating the myelosuppressive and leukopenic effects induced by paclitaxel [27]. Additionally, it enhances the tumor immune microenvironment in TNBC.

The response to ICIs is enhanced by the modulation of innate immunity, adaptive immunity, and the immunogenicity of tumor cells by gut microbiota. Specifically, the gut microbiota can influence the activity of DCs, mononuclear macrophages, and NK cells in terms of innate immune regulation. Additionally, the role of CD8 + T cells and CD4 + T cells could be influenced by the gut microbiota in terms of adaptive immune regulation. The metabolites, which are small molecules capable of disseminating from the intestinal primary site, promote the efficacy of ICI by influencing local and systemic anti-tumor immune responses [28]. Certain studies have identified subtypes of immunomodulatory organisms that induce a relatively active immune microenvironment in TNBC. The immunomodulatory (IM) subtype constitutes approximately 24% of TNBC cases and exhibits a more favorable prognosis in comparison to other subtypes. This particular subtype is more inclined to derive benefits from immunotherapy interventions, which involve the enhancement of immune signaling pathways and the augmentation of TILs. It has been found that the enrichment of Clostridium species and related metabolite trimethylamine oxide (TMAO) increases in tumor tissues of the IM subtype. TMAO can activate anti-tumor immunity and higher plasma TMAO levels are associated with better immune response. TMAO can induce tumor cell pyrosis mediated by GSDME protein through activation of endoplasmic reticulum kinase PERK, and secret inflammatory factors (IL-18 and IL-1β) into the TME, thereby enhancing the infiltration and cytotoxicity of CD8 + T cells. TMAO levels correlate positively with the amount of CD8 + T cells in the TME. In this context, TNBC patients with high CD8 + T cell counts demonstrate favorable IFN responses and higher immune cell infiltration [29]. In a clinical trial, choline (a precursor of TMAO) was employed to improve the efficacy of immunotherapy; this provided a new strategy for precision immunotherapy in TNBC. More metabolites of the gut microbiota have been shown to reshape the TME by regulating immune cells and affecting the efficacy of cancer immunotherapy, including ICIs. Therapeutic strategies for intestinal microbiome combined with ICI include FMT, probiotics, prebiotics, engineering bacteria, bacteriophage, and other strategies to enhance ICI response [30]. However, little is known regarding the potential molecular mechanisms related to gut microbiota and the impact of their metabolites on the efficacy of ICI in TNBC. Future studies are therefore needed to further explore the efficacy of microbiota in the treatment of TNBC, as this may help improve patient prognosis.

The involvement of IFNs in the immunotherapy response against tumors presents a dual nature. Initially, IFNs can stimulate dendritic cells, thereby facilitating the cross-activation of CD8 + T cells specific to the tumor. However, prolonged exposure to IFNs can elicit negative feedback mechanisms, which faciliates the depletion of T cells and the induction of immunosuppressive effects. By modulating the immune response of IFNs, it is possible to augment the body’s immune response to tumors [31]. Studies have demonstrated that IFN signaling of tumor cells can restrict immune responses, whereas IFN signaling of adaptive and innate immune cells can enhance the immune response [32]. IFN-α and IFN-β, known as Type I IFN, have a wide range of impacts on different immune cells. such as augmenting the cytotoxic potential of NK cells and their ability to secrete IFN-γ, as well as facilitating the differentiation, maturation, and migration of antigen-presenting cells. On the other hand, IFN-γ, classified as a type II IFN, possesses antiviral, anti-tumor, and immunomodulatory properties [33], which are capable of activating other immune cells and fostering the immune response. Previous studies have found that MYC is overexpressed in some cases of TNBC and it promotes immunosuppression by inhibiting the IFN signaling pathway [34]. The MYC signaling pathway is related to low PD-L1 expression in cancer cells. Some studies suggest that patients with TNBC who demonstrate high MYC expression have lower major histocompatibility complex class I (MHC-I) expression [35]. MYC inactivation may therefore restore MHC-I expression, CD8 + T cell infiltration, and the anti-PD-L1 response. As MYC protein expression is slightly downregulated after treatment with IFN-γ, IFNs may increase MHC-I expression in tumor cells that demonstrate high MYC expression.

Unmethylated cytosine-phosphorothioate-guanine (CpG) oligodeoxynucleotides are synthetic toll-like receptor 9 (TLR9) agonists that stimulate plasmacytoid dendritic cells (DCs) to produce IFN α and β. They also activate T and B cells, recruit NK cells, induce humoral and cellular immunity, and upregulate IFN release; these promote the infiltration of antigen-specific CD8 + T cells [36]. Based on their chemical composition and in-vitro activity, CpG-oligodeoxynucleotides are mainly divided into three categories: A, B, and C. Studies have found that low doses of CpG-B can significantly inhibit tumor growth and offer synergistic effects with PD-1 inhibitors. Although higher doses of CpG-C may achieve the same anti-tumor effect as CpG-B, CpG-C offers more efficacy than CpG-B when combined with anti-PD-1 inhibitors [37]. The challenge of TNBC treatment interventions lies in effectively targeting IFNs to reshape the immune microenvironment, to guide rational combination therapy.

Numerous researches have shown that treatment with PD-1/PD-L1 inhibitors is more effective in cancers with more CD8 + T cells which may be used as predictive and therapeutic biomarkers for anti-PD-1 therapy [39, 40]. Notably, certain advanced TNBC cases lacking CD8 + infiltrating T cells demonstrate resistance to PD-1/PD-L1 inhibitors. The mutation frequency of tumor suppressor protein p53 may reach up to 80% in TNBC [41]; this may therefore be used as a potential biomarker. As the deletion or mutation of p53 can promote immune escape of cancer cells, restoring P53 activity in deficient tumors may potentially overcome resistance to PD-1/PD-L1 therapy. In this context, the naturally formed Pos3Aa protein crystal in Bacillus thuringiensis can be used as a carrier. Protein crystal-mediated p53 delivery can restore function in p53 deficient cancer cells, inducing significant apoptosis and cell cycle arrest. It is worth noting that Pos3Aa-p53 also significantly enhances the sensitivity of p53- deficient TNBC cells to the chemotherapy drug, 5-fluorouracil, and provides a new combined approach for the treatment of TNBC [42]. The strategy of Pos3Aa-p53 crystals combined with anti-PD-1 antibodies is safe, and significantly increases the number of IFNs, CD4+, CD44+, and CD8 + CD44 + memory T cells, thereby enhancing the curative effect to anti-PD-1 therapy [43]. In summary, these data confirm that intracellular p53 transmission may be an efficacious approach for improving the immunogenicity of p53 mutant tumors. The combination therapy of p53 restoration and ICIs in TNBC has considerable potential in overcoming resistance to ICIs. However, further studies are needed regarding the mechanisms involved; high-level evidence-based clinical trials are also needed for future clinical application.

CXCR2 chemokine receptor 2 (CXCR2) is mainly expressed by neutrophils. Notably, TNBC demonstrates higher levels of CXCR2 expression than other types of breast cancer [44]. In this context, CXCR2 participates in the treatment of drug resistance by maintaining of stemness of cancer stem cells (CSCs). The combination of the CXCR2 antagonist, AZD5069, with doxorubicin, has been found to inhibit doxorubicin-mediated CXCR2 overexpression and reduce chemoresistance and TGF-β expression in TNBC. An in vitro study found the combination of 30 nM of AZD5069 and 200 nM of atezolizumab to induce a notable cytotoxic effect (p = 0.0065); this indicates that in addition to the reduction of chemoresistance, CXCR2 inhibition enhances the efficacy of PD-L1 inhibitor [45]. Clinical trials and in vivo experiments are needed to evaluate CXCR2 inhibitor-based combination therapy in specific patient cohorts. This may provide breakthroughs and improve survival rates in TNBC patients.

CD44 and PD-L1, which have overexpression in TNBC, play a important role in CSC stemness immune escape [1].In this context, a methoxy polyethylene glycol-covered CD44 × PD-L1/CD3 three specific T cell nano adapter (TriTNE) was loaded with c-di-AMP (CDA), the agonist of STING pathway, by using nanotechnology to develop PmTriTNE@CDA. Methoxy polyethylene glycol coverage blocks the antigen-binding of TriTNE; this dual targeting strategy (of CD44 and PD-L1) may improve selectivity and binding affinity to tumors. STING agonists can upregulate the expression of type I IFNs in TME, promote DCs maturation, activate T cells via tumor antigen presentation, and recruit CD8 + T cells and NK cells to the tumor lesion, thereby improving the immunogenicity of TNBC [46]. PmTriTNE@CDA targets CD3 and PD-L1 while inherently activating IFN signals; CSCs are suppressed and eliminated, and they interact synergistically with CDA to overcome the heterogeneity of TNBC and immunotherapeutic resistance.

PD-1 is an important immunosuppressive molecule that is mainly found on immune cells [48] and is typically expressed on the surface of T, B, NK, and myeloid cells. PD-L1 may be expressed by tumor and activated T cells, macrophages, and CAFs [49]. The binding of PD-1 to its ligand PD-L1 trigger SH2 protein tyrosine phosphatase 2 (SHP2), this downregulates the PI3K/AKT axis, inhibits the proliferation of T and B cells in peripheral tissue, induces programmed T cell death, and prevents autoimmune diseases, thereby maintaining immune homeostasis. However, this also provides the possibility of immune escape in tumor cells [50]. The Food and Drug Administration (FDA) of United States has approved the following PD-1/PD-L1 inhibitors: nivolumab, pembrolizumab, atezolizumab, cemiplimab, avelumab, and durvalumab [51]. Certain studies have reported that PD-L1 is present in around 20–30% of cases of TNBC, and is associated with lymphocyte infiltration and histological grades. In combination with extracellular signal-regulated kinase 1/2 inhibitors, PD-1/PD-L1 inhibitors show greater effects in TNBC cell lines than in non-TNBC cells [52]. Phase I clinical trial KEYNOTE-012 showed that pembrolizumab has good safety and object response rate (ORR) in TNBC patients [53]. Studies have confirmed that monotherapy of pembrolizumab offers sustained anti-tumor activity in patients with early and advanced PD-L1-positive TNBC (with a combined positive score [CPS] of ≥ 1) [54‐56]. In phase I clinical trial (NCT02838823), PD-1 inhibitor JS001 shows good safety and effectiveness in metastatic TNBC patients who failed in multi-line treatment [57]. In the Phase I clinical trial (NCT01375842), the monotherapy of PD-L1 inhibitor atezolizumab provides long-lasting clinical benefits in mTNBC patients [58]. Phase I JAVELIN study showed that Avelumab, a PD-L1 inhibitor, had an ORR of 44.4% and 2.6% in 58 TNBC patients with PD-L1 ≥ 10% and < 10%, respectively [59].

However, the monotheraputic efficacy of PD-1/PD-L1 inhibitors is limited in TNBC and influenced by numerous factors. The resistance of breast cancer to ICIs also hinders their clinical application.

The CD28 and CTLA-4 receptors on T cells bind to CD80/CD86 to provide positive and negative feedback, respectively, for T cell activation. CTLA-4 is considered to be a necessary suppressor of the adaptive immune response, and is capable of maintaining peripheral tolerance, binding to CD80/CD86 on activated T cells, competitively inhibiting CD28, and binding to B7 co-stimulatory molecules expressed on antigen-presenting or tumor cells; this reduces the binding of B7 to CD28 and downregulates the expression of T cells [60]. CTLA-4 hinders the growth of T cells, progression of the cell cycle, production of IL-2, and differentiation of T cells. It is therefore a specific regulator of the CD4 + T cell response, and thereby maintains T cell homeostasis [61]. Some studies have analyzed the CTLA-4 expression among different types of breast cancer in The Cancer Genome Atlas (TCGA), it is demonstrated that the expression of CTLA-4 is found to be the highest in TNBC. Notably, it may be controlled by hsa-mir-92a to form a competing endogenous ribonucleic acid (RNA) network, which affects the prognosis of TNBC patients via the leukocyte and T cell activation pathways [61]. In this context, CTLA-4 expression is negatively associated with the T-cell activation score and survival rate (P = 0.0061) [62]. Notably, a study found CTLA-4 levels to be significantly higher in tissue samples from lymph node metastases than in those from the primary breast tumor. Additionally, the patients with high expression of CTLA-4 demonstrated a significantly increasing in axillary lymph node metastases [63]. CTLA-4 blocking may therefore activate or restore T cell function. In addition to inhibiting axillary lymph node metastasis, it can also enhance immune defense mechanisms and tumoricidal effects in breast cancer. In a study, combined preoperative tumor cryoablation and ipilimumab therapy demonstrated potential intratumoral and systemic immune effects with better safety; this suggests the possibility of a synergistic anti-TNBC immunity effect [64]. None of the CTLA-4 inhibitors has been currently approved for sole use in TNBC. However, combined pembrolizumab and chemotherapy with subsequent pembrolizumab monotherapy has been used for the adjuvant treatment of early high-risk TNBC with a PD-L1 CPS of ≥ 20.

Both PD-1 and CTLA-4 are highly expressed in regulatory T cells (Treg). In this context, PD-1 can promote Treg cell proliferation in the presence of ligands. Numerous tumors demonstrate high infiltration by Treg cells (including CD8 + and NK cells), which inhibit the immune response. Blocking the PD-1 pathway may therefore enhance the immune response by reducing the inhibitory activity of Treg cells [65]. As CTLA-4 and PD-1 prohibit T cell function via different mechanisms, it may be reasonable to use CTLA-4 and PD-1 inhibitors to treat TNBC. Some studies have shown that the combination of two types of ICIs or ICIs with chemotherapy drugs offers a better response rate than monotherapy with either agent [66, 67]. However, 55% of grade 3 or 4 side effects occur in patients receiving ipilimumab and nivolumab; the corresponding incidence is 16.3% and 27.3% in those who receive nivolumab and ipilimumab, respectively. Notably, dual blockade with durvalumab (anti-PD-L1 monoclonal antibody) and tremelimumab (anti-CTLA-4 monoclonal antibody) activates and effectively expands T cell populations. PD-L1 and CTLA4 inhibitors may influence the crosstalk between B and T cells, inducing plasma cells to produce specific antibodies in the TME. These findings suggest that patients with TNBC are prone to benefit from combined treatment with durvalumab and tremelimumab [66]. Although this study had an exploratory design and a small sample size for verification of reproducibility, it confirmed the feasibility and effectiveness of combining two ICIs to a certain extent. The findings may be validated by future preclinical or clinical studies including larger sample sizes.

A meta-analysis that included early-stage TNBC patients showed that the use of ICIs in combination with anthracyclines and taxanes significantly increased the pathological complete response rate (pCR); in addition, ICI-chemotherapy drug combinations offered a lower incidence of adverse events compared with platinum chemotherapy [68]. Metastatic TNBC (mTNBC) is usually treated using chemotherapy; however, the objective response rate (ORR) with single-agent chemotherapy is only 10 − 30% and that of multidrug combination therapy can reach 63% [69, 70]. Clinical trials have found that cisplatin and doxorubicin significantly increase T-cell infiltration in patients with TNBC who subsequently receive nivolumab. Based on the high response rate to anti-PD-1 treatment and the upregulation of immune-related genes, cisplatin or doxorubicin induction may be considered to initiate tumor responses to PD-1 therapy. Clinical data on TNBC indicate that these chemotherapy agents can induce the formation of a more favorable TME in the short term, thereby improving the efficacy of ICIs [71]. Several clinical trials show that multiple neoadjuvant chemotherapy combined with Pembrolizumab regimens showed a favorable PCR rate in the treatment of early TNBC [72, 73]. In a study on early-stage TNBC, the combination of chemotherapy with pembrolizumab in the neoadjuvant setting provided a higher pCR (64.8%) than that of placebo (51.2%) [74]. The combination of anthracycline and taxane chemotherapy with durvalumab as a new adjuvant treatment can improve the prognosis of early TNBC patients [75, 76]. In the phase III clinical trial NeoTRIPaPDL1, the addition of PD-L1 inhibitor atezolizumab to neoadjuvant chemotherapy haven’t significantly improved the pCR rate of TNBC patients compared to neoadjuvant chemotherapy alone [77]. In the IMpassion031 trial, which compared atezolizumab combined with albumin-bound PTX and anthracycline with placebo and chemotherapy, the pCR was higher in the atezolizumab group than in the placebo group (58% vs. 41%, respectively) [78].

In IMpassion130 trial, which compared the efficacy of atezolizumab combined with albumin-bound PTX (experimental group) with that of albumin-bound PTX monotherapy (control group), the experimental group exhibited a longer median progression-free survival (PFS) and the median overall survival (OS) compared to the control group. In PD-L1 positive patients, the median PFS was longer in the experimental group than in the control group [79]. The results showed that atezolizumab combined with albumin-bound PTX offered a better prognosis than single-drug therapy in advanced TNBC patients, especially in PD-L-positive patients. However, in Phase III clinical study IMpassion131, the combination of atezolizumab and paclitaxel in the treatment of metastatic TNBC did not significantly improved PFS in the PD-L1-positive population. In another trial that evaluated pembrolizumab with chemotherapy against placebo with chemotherapy for treating advanced TNBC, the subgroup with CPS of > 10 demonstrated a median OS of 23.0 months in the pembrolizumab group and 16.1 months in the placebo group. Chemotherapy combined with ICIs was, therefore, more effective than chemotherapy in patients with PD-L1-expressing (CPS ≥ 10) advanced TNBC [80]. The therapeutic effect and prognosis of immunotherapy may therefore be predicted before treatment in advanced TNBC patients (based on the expression of PD-L1). KEYNOTE-355 showed that the first-line treatment of pembrolizumab combined with chemotherapy significantly improved the OS for advanced TNBC with PD-L1 (CPS ≥ 10) [81].

An open-label, non-randomized, single-arm phase I/II clinical trial demonstrated favorable tolerability and efficacy of the combination therapy comprising eribulin and pembrolizumab for managing metastatic TNBC [82]. Similar clinical studies have achieved different results, such as in the IMpassion031 study and NeoTRIPaPDL1 study, which may be related to factors such as the pathological characteristics of the enrolled population, PD-L1 detection methods, and compatibility protocols.

Despite significant advances in chemotherapy, endocrine therapy, and targeted therapy for breast cancer, radiotherapy continues to play an important role in TNBC. Radiotherapy recruits immune cells into the TME in various ways. It induces dying tumor cells to release danger signals. DCs combine with these danger signals to ingest antigens from cancer cells and transmit the antigens to lymph nodes. These are then presented to the initial T cells, thereby activating CD8 + and CD4 + T cells. This in turn triggers chemokine-induced recruitment of effector T cells to tumors [83]. Preclinical data indicate that radiotherapy combined with ICIs can not only enhance anti-tumor efficacy but also induce responses outside the radiation field. In a low immunogenicity mouse model of TNBC, radiotherapy upregulated the expression of genes containing immunogenic mutations. Vaccination with new epitopes encoded by these genes induced CD8 + and CD4 + T cells, thereby enhancing the immune response [84]. A study compared radiotherapy, anti-CTLA-4 antibody, and combined treatment with both in low- immunogenicity metastatic mouse breast cancer 4T1 cells; the findings suggested that combined treatment with ICIs and chemotherapy enhanced T cell infiltration, delayed growth of the primary irradiated tumor, improved survival rates, and inhibited lung metastasis [85].

In the TONIC trial, which had an adaptive design, mTNBC patients received nivolumab for 2 weeks and were then administered low-dose radiotherapy, the final ORR was 10%. The results indicate that CTLA-4 inhibitor therapy before low-dose radiotherapy offers better outcomes than other regimens, as the consumption of immunosuppressive Treg cells limits the function of CD8 effector cells and anti-tumor immunity. In this context, a multi-center phase 2 study evaluated the effectiveness and safety of combined radiotherapy and pembrolizumab in mTNBC patients. The study found that in the intention-to-treat cohort, the ORR of the unselected PD-L1 population was 17.6%; this was higher than that of mTNBC patients who had previously received ICI monotherapy [86]. However, the combination of immunotherapy and radiotherapy may be a double-edged sword. Owing to ICI-induced alterations in the balance between immune resistance and tolerance, increased tumor recognition is accompanied by higher immune response rates in normal tissues. On administration of radiotherapy, both tumor-specific and non-tumor-specific antigens may be released into the TME; this may activate autoreactive T cells, and possibly attack and damage normal tissues. Therefore, the combination of the two may increase the severity of adverse effects. Future large-scale clinical trials are therefore needed to further explore the best combination strategy for radiotherapy and immunotherapy.

There has been some progress in the administration of gene therapy with ICIs in TNBC, and studies are evaluating its immunotherapeutic effect (whether similar or superior). In the early stages, gene therapy utilized the oncolytic effect of Ad.sT, a protein created by fusing an adenovirus and a TGF β receptor II IgG Fc fragment (sTGFβ RIIFc). The Ad.sT interfered with TGF β-1 binding and inhibited TGF-β-dependent transcription when it was infected in MDA-MB-231 cells. More than 85% of the tumors of MDA-MB-231 xenografts in nude mice showed a reduction by the administration of Ad.sT [87]. Ad.sT could increase the amounts of CD4 + and CD8 + T lymphocytes in peripheral blood, thereby improving the immunotherapeutic effect of ICIs. Notably, a combination of PD-1 inhibitors and CTLA-4 inhibitors increases the efficacy of Ad.sT in inhibiting the metastasis and proliferation of tumor [88]. In this context, the LyP-1 receptor is highly expressed in breast cancer and three oncolytic viruses, namely, Ad.sT, AdLyp.sT, and mHAdLyp.sT; the latter two produce higher levels of the sTGFβRIIFc protein compared to viruses without LyP-1 modification. Intravenous injection of AdLyp.sT and mHAdLyp.sT has been found to induce a strong anti-tumor response in a bone metastasis model of TNBC, the delivery of AdLyp.sT in a 4T1 mouse model with normal immune function also enhanced the efficacy of anti- CTLA-4 and anti-PD-1treatment [89]. These findings suggest that it may be possible to develop more potential targeted immunotherapeutic drugs for TNBC.

In some studies, adenovirus-mediated herpes simplex virus(HSV)thymidine kinase was injected into mTNBC tumors; the injection site was then irradiated using stereotactic body radiotherapy (SBRT) and the patients subsequently received pembrolizumab. The median duration of treatment and OS in this study were 9.6 and 14.7 months, respectively. Notably, the median OS had more than doubled in patients who experienced clinical benefits; this indicated that the use of SBRT in conjunction with HSV thymidine kinase and gene therapy prior to pembrolizumab can improve the therapeutic efficacy of the latter in patients with mTNBC. This combined treatment regimen has also shown improved anti-tumor efficacy in a study on patients with liver metastases who had a low or negative PD-L1 status [90]. However, the patients in this study were not randomized, and the response rate to oncolytic viruses (alone or in combination with ICIs) did not exceed 12%. In addition, the combination of ICIs and SBRT only improved the response in preconditioned patients without liver metastasis (and demonstrated a shorter duration of response) [86]. Larger future randomized studies are therefore needed to evaluate the effect of each modality and its impact on the tumor microenvironment.

Nanoscale materials have small particle sizes, stable properties, and high bioavailability. As a new area of interest in the field of cancer research, nano delivery systems are applied for drug delivery and early detection. Certain studies have shown that the combination of nanotechnology with immunotherapy ensures targeted delivery and stability of loaded drugs (via the application of nanocarriers) and enhances drug uptake and biocompatibility in breast cancer.

Adagloxad simolenin is an active anti-cancer immunogen, which targets the Globo-H antigen which is highly prevalent on the surface of various cancers including TNBC [119]. It can be used as a target to identify cancer cells (which may stimulate the human immune response) and eliminate malignant tumors. On combined with the saponin adjuvant, OBI-821, adagloxad simolenin can induce an immune response to challenge tumor cells. An ongoing international multicenter phase III study (NCT03562637) is evaluating the anti-Globo-H vaccine in high-risk early Globo H-positive TNBC [120].

The α-lactalbumin is being considered a potential target for developing a vaccine to treat and prevent TNBC. Although this protein does not exist in normal and aging tissues after lactation, it exists in most TNBCs. Activating the immune system against this redundant protein may provide pre-emptive immune protection against α- lactalbumin protein expression. In addition, the vaccine contains an adjuvant that can stimulate the innate immune response, thereby promoting the production of a specific immune response against tumors and preventing their further progression. In a study performed in a mouse model, α- lactalbumin engineered vaccine boosts antitumor immunity in breast cancer which could prevent breast cancer recurrence [121]. An open-label phase I clinical trial (NCT04674306)is ongoing to determine the safety as well as the optimal dose of α- lactalbumin vaccine to treat TNBC patients with a high risk of recurrence.

DR5, also referred to as TNF-related apoptosis-inducing ligand receptor 2, has the ability to attach to TNF-related apoptosis-inducing ligands and facilitate cellular apoptosis. In a study using the BALB/c mouse model, a DR5 DNA vaccine induced the expression of proapoptotic antibodies, thereby triggering tumor cell apoptosis. In addition, the vaccine-induced DR5-specific T cells to secrete IFN-γ. The investigators concluded that DR5 can be used as an immunogenic target for a TNBC vaccine [122].

A study evaluated a DC fusion vaccine in TNBC. Compared with the control group, the group that was administered a co-culture of DC, T cells, and TNBC cells (created by electrofusion technology) demonstrated significant stimulation of T cell expansion. The increased levels of IL-12 and IFN-γ demonstrated the specific cytotoxic effect of the DC fusion vaccine on tumor cells [123].

Research has indicated the efficacy of personalized peptide vaccination (PPV) in managing TNBC [124]. During a phase II study involving patients with metastatic and recurrent TNBC, 1/18 patients each developed a complete and partial immune response after PPV administration (NCT02427581). PPV appeared to be safe, as it did not lead to any serious adverse effects in these patients. These results reveal that PPV has a potential effect in treating patients with TNBC [125].

Chimeric antigen receptor T cell (CAR-T) therapy has shown excellent therapeutic effects or potential in treating diverse forms of cancer. Although breakthroughs have been achieved in hematological tumors, their use in the field of solid tumor therapy remains in the explorative stage. Studies in patients with TNBC have identified an antigen target related to breast CSCs (disialoganglioside GD2), which is widely found in highly invasive breast cancer subtypes; GD2-CAR-T cells were therefore created. In a study on a mouse model, GD2-CAR-T cells demonstrated a significantly higher inhibitory effect on tumor cell growth compared to CD19-CAR-T cells. Notably, no metastatic cancer cells were observed in the lungs of mice treated with GD2-CAR-T cells, all mice treated with CD19-CAR-T cells exhibited metastatic cancer cells in the lung [126]. Investigators have used TAB004 (a highly specific monoclonal antibody against the tumor-associated form of MUC1) to engineer the MUC28z (a chimeric antigen receptor) fusion molecule and generate CAR T cells; they also performed phenotypic and functional analyses. The results showed that MUC28z CAR-T cells can efficiently suppress the proliferation of TNBC tumors, under both in-vivo and in-vitro conditions. The CAR-T cells exhibited significant therapeutic promise in combating MUC1-positive TNBC tumors [127]. In a study on a TNBC mouse model, a fibrin gel containing CAR-T cells was applied to the surgical wound after partial tumor resection. The CAR-T cells significantly eliminated residual tumor cells, allowing the mice to survive. The results indicate that CAR-T cells could potentially act as a supplementary approach to enhance the efficiency and success rate of surgery in TNBC [128].

The regulation of NK cell activities, such as degranulation, cytokine secretion, and cytotoxicity, is determined by the combination of signals received from activating and inhibitory receptors. NK cells possess the ability to identify and eliminate tumor cells that exhibit diminished or suppressed expression of MHC-I, a phenomenon referred to as the “missing self” recognition mechanism [129]. NK cells possess the ability to not only secrete cytotoxic particles, such as perforin and granase, via exocytosis but also directly induce the lysis of target cells and initiate cell apoptosis by activating the expressionof FASL/TRAIL in NK cells. Furthermore, other immune cells can be recruited to generate secondary immune responses by synthesizing and secreting IFN-γ, TNF-α, and chemokines [130, 131]. Additionally, another significant mechanism employed by NK cells in tumor eradication is antibody-dependent cell-mediated cytotoxicity (ADCC), which triggers the degranulation response of NK cells to eliminate target cells that are coated with antibodies. Presently, the repertoire of NK cell approaches employed in tumor mmunotherapy encompasses the following: in vitro administration of activated autologous or allogeneic NK cells; the induction of antibody-specific cytotoxicity through the amalgamation of NK cells and monoclonal agents (e.g., ICIs); and the implementation of CAR-NK cell immunotherapy. Notably, NK cells derived from cancer patients exhibit comparable expansion potential to those derived from healthy donors, exhibit cytotoxicity against TNBC cell lines and xenograft tumor models, and can be harmoniously integrated with existing.

cancer treatments [132]. To enhance the functionality of NK cells in patients with TNBC, it has been observed that the combined approach of radiotherapy (RT) and NK cell therapy can effectively facilitate the infiltration and migration of NK cells into the tumor lesion within the xenograft tumor model of TNBC. Consequently, this approach leads to the reduction of tumor burden and tumor growth. Notably, compared to the NK group, the RT + NK group demonstrated a significantly prolonged presence of NK cells within the primary tumor tissue. Furthermore, the combined therapy of RT + NK cells effectively suppressed the dissemination and distant metastasis of breast cancer cells to the lymph nodes, specifically in the lung and liver [133]. Avelumab, a checkpoint inhibitor targeting PD-L1, demonstrated the ability to elicit cytotoxic effects mediated by NK cells in TNBC patients [134]. Chen et al. developed a nano-system incorporating selenium, which effectively increased NKG2D expression on NK cells and NKG2DLs expression on tumor cells. This augmentation enhanced the recognition and cytotoxicity of NK cells towards tumor cells, thereby bolstering the immune response against tumors facilitated by NK cells [135]. Additionally, studies have shown that Ruthenium complexes and Aptamer-engineered approaches can enhance the sensitivity of TNBC to NK cell therapy and modulate the immune microenvironment [136, 137]. The presence of an immature NK cell population (CD11b CD27 Socs3high) in TNBC has been found to diminish granules-mediated cytolysis and enhance the expression of Wnt16 ligand, which has been implicated in tumor progression and metastasis [138]. The promotion of TNBC progression can be facilitated by the depletion of NK cells, inhibition of Wnt secretion, utilization of immunotherapy involving anti-PD-L1 antibodies, or a combination of chemotherapy and immunotherapy [139].

CAR-NK is an application of genetic engineering cellular therapy that incorporates a chimeric antibody into NK cells, which can significantly improve the specificity of NK cell’ s therapeutic effects. Notably, a study demonstrated that EGFR-CAR NK cells exhibited cytotoxicity and anti-tumor properties against TNBC cell lines with high EGFR expression.

Optimizing the cytotoxic potential of NK cells appears to heavily rely on the tailored design of CAR structures dedicated to NK cells [140]. In addition, it is imperative to optimize the procedures for amplifying and activating harvested NK cells to obtain a large number of memory-like, unexhausted, and homogeneous populations of NK cells in clinical practice. Moreover, the effectiveness of CAR-NK cells in TNBC treatment might necessitate additional modifications to enhance the trafficking capabilities and reduce the sensitivity to immunosuppression in the TME.

Cytokine-Induced Killer (CIK) cells are a diverse group of polyclonal T lymphocytes acquired by expanding lymphocytes in vitro, and exhibit functional and phenotype characteristics of both T cells and NK cells. These cells express two membrane protein molecules, CD3 and CD56, simultaneously, earning them the designation of NK cell-like T lymphocytes. Due to their potent tumor cell recognition abilities, CIK cells are often likened to “cell missiles” that can accurately target tumor cells without harming normal cells. In the case of patients with TNBC, repeated administration of CIK cells and cetuximab to patients with TNBC has been shown to significantly impede the growth, metastasis, and dissemination of TNBC cells and xenotransplanted tumors in lymph nodes and lungs [141]. The use of the treatment approach successfully inhibits the reoccurrence and extends the lifespan of patients with TNBC who have lymph node spread, an advanced TNM stage, and poor histological grade [142].

CIK cells have demonstrated potential benefits in patients with various tumor types, however, their effectiveness varies among individuals. The role of Focal adhesion kinase (FAK).

in controlling cell invasion and migration is vital, which in turn affects the vulnerability of tumor cells to CIK cells. Current evidence suggests a favorable connection between PD-L1 and FAK mRNA expression in PD-L1 positive TNBC, indicating a possible link.

between FAK and the function of immune checkpoints. Furthermore, studies have shown.

that the anti-PD-L1 antibody atezolizumab greatly augments the suppressive impact of FAK inhibitors on cancer cells [143]. The application of FAK inhibitors in the treatment of TNBC cells, along with co-culturing with CIK cells, resulted in a higher incidence of cell death, suggesting that FAK enhances the susceptibility of tumor cells to CIK cells [144]. A study conducted in vivo has substantiated that the combined administration of FAK inhibitors and CIK cells surpasses the efficacy of either treatment in inhibiting tumor growth [145]. Consequently, the integration of CIK cell therapy with PD-L1 inhibitors and FAK inhibitors holds promise as a potentially efficacious and innovative treatment approach for patients with TNBC. The immunotherapy of CAR-T cell, NK cell and CIK cell separately or with others anticancer approaches to treat TNBC is shown in Fig. 5.