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Introduction

The immune system serves a pivotal role in both the initiation and progression of cancer (1,2). In 1909, Paul Ehrlich hypothesized that the immune system regulates tumor development (3). In 1957, Burnet (4) introduced the cancer immunosurveillance theory, which suggests that lymphocytes serve a role as ‘guardians’ of the body by identifying, eliminating and killing mutated cells, thereby preventing tumor formation. However, tumors evade immune detection through immune escape mechanisms, leading to cancer progression.

Over the past decades, immunotherapy (therapeutic strategies aimed at targeting and modulating the immune system) has notably changed cancer treatment (5,6). The United States Food and Drug Administration (FDA) has approved numerous types of cancer immunotherapy, including immune checkpoint inhibitors, cancer vaccines and adoptive immune cell therapy (7–13). However, despite these advancements, immunotherapy remains largely effective only against tumors that are intrinsically sensitive to immune responses. A challenge is presented by the immunosuppressive tumor microenvironment (TME), which is characterized by regulatory immune cells [such as regulatory T cells (Tregs) and myeloid-derived suppressor cells (MDSCs)] and immunosuppressive cytokines (14). The TME impedes T cell infiltration and function, thereby presenting a barrier to effective immunotherapy. Therefore, strategies aimed at reversing immune suppression within the TME are key for expanding the applicability of cancer immunotherapy.

Flavonoids, which are abundant in fruits, vegetables, tea and other plant-based foods, exhibit anti-cancer properties, including antioxidant and anti-inflammatory activity, induction of apoptosis, inhibition of angiogenesis and modulation of the immune system (15–19). Flavonoids regulate immune cells, cytokines and antigen presentation, thereby effectively reversing the immunosuppressive TME (19,20). These properties position flavonoids as promising adjuvants in immunotherapy. Although the majority of studies remain at the preclinical stage, clinical trials involving flavonoid compounds in combination with immunotherapy have already been approved by the FDA (Table SI). Furthermore, the application of nanotechnology has promise in enhancing the bioavailability and targeting of flavonoid compounds, thereby improving their efficacy in immunotherapy (21).

The present review aimed to explore the role of flavonoids in cancer immunotherapy, emphasizing their ability to modulate the immune system and to reverse the immunosuppressive TME, as well as their potential to enhance efficacy and expand application of existing immunotherapies.

Mechanisms of tumor-induced immune suppression

Cytotoxic lymphocytes (CTLs) serve a key role in the immune system via identifying and eliminating cancer cells. However, the TME exerts inhibitory effects on their function. Key contributors to immune suppression within the TME include immune inhibitory factors, immunosuppressive cells and immune checkpoint pathways (22).

Immunosuppressive cells and factors

The TME exhibits immunosuppressive properties through a variety of mechanisms; these include the infiltration of immunomodulatory cell populations, such as MDSCs, tumor-associated macrophages (TAMs), cancer-associated fibroblasts (CAFs), Tregs and fetal-like immune and stromal cells (23,24). Moreover, the TME is characterized by the expression of immunosuppressive cytokines, including TGF-β, IL-10, IL-35, chemokine ligand (CCL) 5 and C-X-C motif chemokine ligand 12 (also known as stromal cell-derived factor 1) (25). These factors create an immunosuppressive milieu that impairs cytotoxic T cell activity and inhibits effective anti-tumor immune responses.

Tumor cells secrete chemokines, such as CCL22, to recruit macrophages into the TME, which induces their polarization into the immunosuppressive M2 phenotype via factors such as vascular endothelial growth factor (VEGF), galectin-1, gangliosides, TGF-β, prostaglandin E2 (PGE2) and IL-10 (26–28). Additionally, IL-10, TGF-β and VEGF inhibit antigen presentation mediated by dendritic cells (DCs), thereby facilitating recruitment of Tregs into the TME and suppressing CTL activity (29,30).

Immunological checkpoints

Tumors induce T cell exhaustion and decrease anti-tumor activity by upregulating immune checkpoint molecules, including programmed cell death protein 1 (PD-1) and CTL-associated protein 4 (CTLA-4) (31–33). For example, PD-1 binds to PD-ligand (PD-L) 1/2, which are expressed on tumor or stromal cells, thereby inhibiting T cell proliferation and cytokine production (34). Similarly, CTLA-4 competes with CD28 for binding to CD80/CD86, thereby decreasing T cell activation (35,36). In addition to PD-1 and CTLA-4, other immune checkpoints also serve roles in immune evasion and T cell dysfunction. For example, lymphocyte activation gene 3 (LAG-3) binds major histocompatibility complex (MHC) class II molecules, thereby impairing antigen presentation and decreasing T cell activation, while also enhancing the suppressive activity of Tregs, which exacerbates immune suppression (37). The immune receptor T cell immunoglobulin and immunoreceptor Tyrosine-based Inhibitory Motifdomain, expressed on T cells and natural killer (NK) cells, binds to CD155 on antigen-presenting cells (APCs), inhibiting T cell receptor (TCR) signaling and reducing NK cell cytotoxicity, which thereby promotes tumor immune escape (38,39). In addition, T cell immunoglobulin and mucin domain 3 interacts with galectin-9 and phosphatidylserine, which induces T cell exhaustion and decreases cytokine production; this protein is often co-expressed with PD-1 to produce a synergistic inhibitory effect (40,41). The effects of these immune checkpoints are not isolated; their functions are amplified by the presence of immunosuppressive cells, including MDSCs and Tregs. These cells secrete cytokines (TGF-β and IL-10) and express ligands for immune checkpoint molecules, thereby intensifying immune suppression (42). For example, MDSCs upregulate PD-L1 expression, enhancing PD-1-mediated T cell suppression (43), whereas Tregs exacerbate immune inhibition by highly expressing CTLA-4 and LAG-3 (44,45).

Immune modulation of flavonoids to immune cells

The TME maintains a balance between tumor-promoting and tumor-antagonistic immune cells. Tumor-promoting cells, such as Tregs, MDSCs and M2-polarized macrophages, foster cancer progression by inhibiting anti-tumor immunity and creating an immunosuppressive environment (46). By contrast, tumor-antagonistic cells, including CTLs (CD8+ T cells), NK cells, DCs and M1-polarized macrophages, recognize and destroy cancer cells. Flavonoids modulate these immune cells, thereby shifting the TME from an immunosuppressive to an immune-supportive state (47).

Effect of flavonoids on monocytes and macrophages

Monocytes and macrophages serve a key role in the immune system via the detection of pathogen-associated molecular patterns, mediating inflammatory responses, promoting immune killing and facilitating antigen presentation (48–50). Macrophages recruited to the TME are known as TAMs. TAMs are primarily classified into two types: M1 and M2. M1 macrophages are typically pro-inflammatory and kill tumor cells, whereas M2 macrophages are associated with tissue repair and tumor progression (51–53). In numerous types of tumor, such as breast, lung, colorectal and liver cancer, and glioblastoma, TAMs adopt an M2-like phenotype, which promotes tumor growth, invasion and metastasis. M2-like TAMs stimulate cancer cell proliferation, angiogenesis and migration, thereby contributing to tumor expansion and spread. Moreover, TAMs engage in positive cross-talk with other immunosuppressive cells, such as Tregs, MDSCs and CAFs, further enhancing tumor growth and the release of growth factors (54,55).

Flavonoids reverse immune suppression through inhibiting macrophage recruitment. Luteolin and catechin, for example, inhibit the cytokine CCL2, which is secreted by TAMs, thereby suppressing the recruitment of macrophages and monocytes to the TME and inhibiting tumor progression (56,57). Furthermore, a combination of resveratrol, curcumin and quercetin inhibits macrophage recruitment, prevents the polarization of TAMs into the M2 phenotype and alleviates immune suppression within the TME (58).

Flavonoids regulate TAM polarization by modulating key signaling pathways that are involved in macrophage polarization, including the STAT3 and NF-κB signaling pathways. For example, a previous study on total flavonoids from Glycyrrhiza Radix et Rhizoma revealed that these compounds decrease STAT6 phosphorylation and enhance the expression of microRNA-155, which inhibits M2 macrophage polarization and the expression of the M2 marker arginase-1 (Arg-1) (59). Isoliquiritigenin suppresses M2 polarization by inhibiting the PGE2/peroxisome proliferator-activated receptor-δ and IL-6/STAT3 signaling pathways (60). Furthermore, baicalein notably decreases the expression of immunosuppressive factors, including IL-10 and TGF-β, by inhibiting the NF-κB signaling pathway, thereby suppressing M2 polarization and promoting M1 polarization (61). Additionally, baicalin induces TAM polarization towards the M1-like phenotype, potentially through activating autophagy and driving transcriptional activation via the RelB/p52 pathway (62). TriCurin, a formulation combining curcumin with other polyphenols, shifts TAMs from an M2 to an M1 phenotype, thereby promoting the IL-12-dependent recruitment of NK cells and CTLs to the tumor site. This facilitates tumor cell elimination via apoptosis (63). Furthermore, xanthohumol, a natural product found in the female inflorescences of Humulus lupulus, when encapsulated in poly (lactic-co-glycolic acid) (PLGA) nanoparticles, stimulates M1 polarization in macrophages (64).

Flavonoids have also been demonstrated to enhance macrophage phagocytic activity: Hesperidin-loaded gold nanoparticles increase macrophage phagocytic capacity, decrease the secretion of pro-inflammatory cytokines and notably inhibit the proliferation of the human MDA-MB-231 breast cancer cell line (65). Furthermore, epicatechins inhibit macrophage migration inhibitory factor, thereby enhancing both the anti-inflammatory properties of macrophages and phagocytic activity (66). Finally, isorhamnetin (3′-O-methylquercetin) has been identified as a compound that enhances lysosomal degradation in macrophages, thereby increasing phagocytic capacity (67).

Effect of flavonoids on MDSCs

MDSCs are composed of immature myeloid cells (68). Tumor cells and the TME secrete chemokines that recruit MDSCs to the tumor site (69,70). Following their arrival at the tumor site, MDSCs interact with various immune and stromal cells, thereby establishing an immunosuppressive niche. MDSCs inhibit the activity of cytotoxic T and NK cells by producing Arg-1, inducible nitric oxide synthase (iNOS) and reactive oxygen species (ROS) (71–73). Additionally, MDSCs promote the expansion and activation of Tregs within the TME, while suppressing effector T cell function (42,71,74).

Apigenin inhibits the TNF-α-mediated release of CCL2 and other chemokines in breast cancer cells, thereby suppressing the recruitment of immune-suppressive cells such as MDSCs and decreasing MDSC-mediated immune suppression in the TME (75). In a murine breast tumor model, epigallocatechin-3-gallate (EGCG) decreases the immunosuppressive effects of MDSCs by downregulating the canonical signaling pathway that includes Arg-1, iNOS, NADPH oxidase 2, NF-κB and STAT3 (76). Silymarin (milk thistle extract) attenuates the immunosuppressive function of MDSCs by reducing the mRNA expression levels of iNOS2 and Arg-1 and enhancing the infiltration and efficacy of CD8+ T cells, thereby reversing the inhibitory TME (77). Neobavaisoflavone (Neo), a natural isoflavone first isolated from the seeds of Psoralea corylifolia, effectively inhibits the expansion of MDSCs and suppresses their immunosuppressive function by targeting STAT3 signaling (78). In addition, Neo directly inhibits the growth of tumors derived from the 4T1 and Lewis lung carcinoma (LLC) cell lines in vivo (78). Icariin and its derivative, 3,5,7-Trihydroxy-4′-methoxy-8-(3-hydroxy-3-methylbutyl)-flavone, have been revealed to inhibit the JAK2/STAT3 pathway, downregulate S100A8/A9 proteins and promote the differentiation of MDSCs into immune-stimulatory macrophages and DCs (79). In a mouse model, the downregulation of immunosuppressive factors, including IL-10, IL-6 and TNF-α, is observed following treatment with icariin and its derivative (79). Moreover, Chrysin (Chr), a natural flavonoid found in honey, propolis and numerous plants, inhibits the function of MDSCs by targeting the PI3K/AKT pathway, thereby reversing the immunosuppressive TME (80).

Effect of flavonoids on Tregs

Tregs primarily influence tumors through immunosuppressive mechanisms. Tregs suppress the activity of cytotoxic and helper T cells, inhibit anti-tumor immune responses and induce immune tolerance within the TME (29,81,82). Tregs limit the activation of effector T cells and DCs by releasing immunosuppressive cytokines, including IL-10 and TGF-β (83–85). Additionally, Tregs indirectly promote tumor growth through fostering angiogenesis and regulating inflammation (86,87). Moreover, the presence of Tregs within tumors is often associated with poorer prognoses (88).

Flavonoids exert immunomodulatory effects on Tregs (89,90). Wogonin, a flavonoid compound found in the roots of the Scutellaria baicalensis plant, inhibits Treg activity, which results in the reversal of the suppressive TME (91). The underlying mechanism may involve inhibition of the Smad-3, GSK-3β and ERK1/2 signaling pathways, along with the enhancement of p38 MAPK phosphorylation (91). In addition, naringenin, a flavonoid found in fruits, decreases reduce the abundance of Tregs by downregulating TGF-β1, thereby reversing the immunosuppressive microenvironment in the lung (92). Flavonoids from Radix tetrastigmae have been revealed to lower the levels of serum immunosuppressive molecules, including TGF-β, PGE2 and cyclooxygenase-2, in tumor-bearing mice, thereby inhibiting the development and function of Tregs (93). Scutellarin, a Chinese herbal medicine of flavone glycoside origin, disrupts the interaction between TNF-α and TNF receptor 2 (TNFR2), preventing TNFR2 activation in Tregs, which decreases Treg activation and proliferation (94). This notably enhances the efficacy of tumor immunotherapy in a mouse model of CT26 colon cancer (94).

Effects of flavonoids on DCs

DCs serve a key role in the TME through capturing, processing and presenting tumor-associated antigens to T cells, thereby initiating an adaptive immune response against cancer (95–97). They serve as a key link between innate and adaptive immunity, activating cytotoxic T and T helper (Th) cells. Moreover, specialized killer DCs have been demonstrated to express various TNF family members, including Fas ligand (FasL), TNF-related apoptosis-inducing ligand and TNF-α, which enable them to promote tumor cell apoptosis (98,99). Th1 lymphocytes enhance DC-mediated tumor-killing activity via an IFN-γ-dependent pathway (100); by contrast, immunosuppressive cytokines (TGF-β and IL-10) and immunosuppressive cells (MDSCs and Tregs) inhibit DC maturation and antigen presentation (101).

Wogonin promotes the migration and infiltration of DCs into the TME (102). Furthermore, wogonin enhances the immunogenicity of dying tumor cells through stimulating the release of calreticulin and high-mobility group box protein 1 (HMGB-1). This triggers DCs and facilitates their efficient uptake of tumor antigens (102,103). The administration of kaempferol and quercetin increases the secretion of granulocyte-macrophage colony-stimulating factor by PC-3 prostate cancer cells, thereby promoting the recruitment of DCs to the tumor site (104). Naringenin, a flavonoid derived from grapefruit, increases antigen cross-presentation in murine DCs, and enhances the activation of CTLs (105). Sea buckthorn flavones enhance the expression of co-stimulatory and pro-maturation molecules of DCs, as well as regulate the expression of immunity-associated genes (106). Water-soluble astragalin-galactoside (Ast-Gal) activates DCs by binding specific receptors, thereby triggering intracellular signaling pathways. The activation of these pathways leads to increased expression of maturation markers (CD80, CD86 and MHC II) on DCs and the secretion of immune-stimulating cytokines, especially IL-12. Ast-Gal-treated DCs preferentially drive the differentiation of naive CD4+ T into Th1 cells, thereby promoting the secretion of IFN-γ (107).

Effect of flavonoids on NK cells

NK cells recognize abnormal cells due to their decreased MHC-I expression or via interaction with stress-induced ligands and are activated by receptors such as killer inhibitory and natural cytotoxicity receptors (108–111). Upon activation, NK cells form an immune synapse with target tumor cells, releasing cytotoxic granules containing perforin and granzymes (111). NK cells induce apoptosis via death receptor pathways, such as the pathway involving the interaction of Fas with FasL (112). Furthermore, NK cells secrete cytokines such as IFN-γ and TNF-α (113,114), which exert an anti-tumor role and stimulate other immune cells to participate in the immune response.

The combined action of naringenin and asiatic acid rebalances the TGF-β1/Smad3 signaling pathway in NK cells, thereby promoting their differentiation, maturation and cytotoxicity against cancer cells (115). The administration of apigenin, a plant-derived flavonoid, enhances NK cell proliferation by increasing the expression of Bcl-2 and decreasing Bax expression (116). Additionally, apigenin activates the JNK and ERK signaling pathways in NK cells, leading to an upregulation of the expression of perforin, granzyme B and the receptor NK group 2, member D (NKG2D), thereby boosting NK cell cytotoxicity against cancer cells (116). Apigenin promotes the upregulation of NK cell-activating receptors (NKG2D, NKp30 and NKp44), which enhances the expression of CD95L on NK cell surfaces, resulting in the induction of apoptosis in hepatocellular carcinoma (HCC) cells (117).

Effect of flavonoids on effector T cells

Effector T cells include CD8+ CTLs and effector CD4+ T cells. Activated CTLs undergo differentiation into effector CTLs, leading to the release of cytotoxic molecules such as perforin and granzymes, which leads to the induction of apoptosis in tumor cells (118–120). CTLs also activate the Fas/FasL pathway and stimulate the secretion of cytokines such as IFN-γ to further enhance the anti-tumor immune response (121,122). Effector CD4+ T cells, including the Th1 and Th17 subsets, are activated upon recognizing tumor-specific antigens that are presented by APCs via the TCR (123). Activated effector CD4+ T cells release pro-inflammatory cytokines, such as IFN-γ and TNF-α, which promote the activation and expansion of CTLs, thereby promoting antitumor cell activity (124–126).

Baicalein and baicalin restore the sensitivity of T cells to tumor cells through inhibiting STAT3 activity and suppressing IFN-γ-induced expression of PD-L1 (127). Furthermore, naringenin activates CD169+ macrophages in lymph nodes, thereby upregulating the expression of immune-associated genes such as CD169, IL-12 and CXCL10, leading to the recruitment of CTLs to the tumor site, whereby their activation is promoted, leading to an enhancement of the anti-tumor immune response (128,129). Betulin, a natural triterpene obtained from birch bark, enhances T cell cytotoxicity against tumors by inducing the secretion of IL-2 and IFN-γ from white blood cells (130). Both Chr and hesperetin notably enhance the activity of CTLs (131,132). Hesperidin and linarin specifically stimulate Vδ1+ T cells, thereby enhancing their functional activity (133). Vδ1 T cells have antitumor functions, and their presence is associated with improved patient outcomes in metastatic colorectal cancer and lung cancer (134,135). Moreover, the flavonoid polyphenol melafolone enhances the proliferation and effector function of CD8+ T cells via downregulation of the immunosuppressive factors TGF-β and PD-L1 (136). In a study by Tian et al (137), luteolin was revealed to activate the PI3K/AKT pathway in APCs, which allows activated APCs to efficiently present tumor antigens to CTLs. This activation further stimulates CTLs, thereby strengthening the antitumor immune response. Xanthohumol enhances the cytotoxic immune response by increasing the secretion of perforin and granzyme B and promoting a higher ratio of CD8+ cytotoxic T cells to CD25+ Tregs (CD8+/CD25+); furthermore, xanthohumol shifts the immune response towards Th1 polarization by upregulating the expression of Th1 cytokines (138).

The aforementioned studies demonstrate that flavonoids regulate immune signaling pathways and immune cell functions by targeting multiple pathways, including inhibition of the NF-κB, JAK-STAT and PI3K/AKT pathways, as well as activation of IFN-γ and TNF-α. By suppressing pro-tumor signals and enhancing anti-tumor responses, flavonoids decrease the risk of resistance, thereby demonstrating their notable anti-tumor potential and offering a promising strategy to overcome the limitations of single-target immunotherapy.

Flavonoids enhance the therapeutic effect of immune checkpoint inhibitors

Immune checkpoint inhibitors restore immune responses against cancer by targeting the PD-1/PD-L1 pathway; however, immunosuppressive TMEs often hinder their efficacy. A number of studies have demonstrated that flavonoids modulate the expression of PD-1 and PD-L1, thereby reversing immune suppression within the TME (127,139–152).

Flavonoids inhibit the expression of PD-L1 and reverse immunosuppression of the TME

PD-L1 expression is regulated by two primary signaling pathways, namely the JAK/STAT and NF-κB pathways. In the JAK/STAT pathway, external stimuli activate cell surface receptors, triggering JAK activation and subsequent phosphorylation of the STAT proteins. Phosphorylated STAT proteins are translocated to the nucleus, where they enhance PD-L1 gene transcription, thereby increasing PD-L1 expression on the cell surface (153–155). In the NF-κB signaling pathway, extracellular signals activate NF-κB by promoting the degradation of inhibitory IκBs, which releases NF-κB transcription factors. These factors enter the nucleus, where they bind specific DNA sequences and promote PD-L1 gene transcription, resulting in increased PD-L1 expression (156,157).

The JAK/STAT signaling pathway has a key role in regulating PD-L1 mRNA expression. Activation of this pathway by cytokines or growth factors leads to transcription of the PD-L1 gene, resulting in PD-L1 protein expression on the cell surface (155,158–160). Agents that block or inhibit the JAK/STAT pathway disrupt this cascade, preventing expression of PD-L1. Flavonoids have potential as natural inhibitors of PD-1 and PD-L1 expression, resulting in an enhancement of the immune response against numerous types of cancer (Fig. 1). In SMMC-7721 and HepG2 liver cancer cell lines, baicalein dose-dependently inhibits IFN-γ-induced expression of PD-L1. Flow cytometric and western blot analyses and revealed that treatment with baicalein (10 µM) and baicalin (40 µM) considerably reduced the expression levels of PD-L1 on the membrane surface (127). Apigenin, combined with curcumin, inhibits the IFN-γ-induced upregulation of PD-L1 in melanoma cells, with apigenin demonstrating a more marked inhibitory effect. At the concentration of 30 µM, apigenin decreases expression of PD-L1, and this effect is associated with a decrease in STAT1 phosphorylation (139,140). In KRAS-mutant lung cancer, luteolin and apigenin inhibit STAT3 phosphorylation and downregulate IFN-γ-induced PD-L1 expression levels, thereby exhibiting anticancer properties (141). Pentamethylquercetin, a methylated quercetin derivative, inhibits expression of PD-L1 in HCC cells by modulating IFN-γ, especially in the context of obesity, via the IFN-γ/JAK-STAT signaling pathway (142). In addition, nobiletin, a natural flavonoid isolated from citrus peel, inhibits PD-L1 expression levels in non-small cell lung cancer cells via the EGFR/JAK2/STAT3 signaling pathway (143). Myricetin, a flavonoid compound found in numerous types of plant, fruit, vegetable and tea, interferes with the JAK/STAT/IFN regulatory factor 1 signaling pathway activated by IFN-γ, thereby inhibiting the transcription of PD-L1 in tumor cells (144). Galangin, a flavonoid that is abundant in galangal and propolis, inhibits expression of PD-L1 by blocking STAT3 activation via the JAK1/JAK2/Src pathway and suppressing the activation of Myc via the Ras/RAF/MEK/ERK pathway (145).

Figure 1. Flavonoids suppress PD-1 and PD-L1 expression through multiple signaling pathways, primarily targeting the JAK-STAT and NF-κB pathways. PD-1, programmed cell death protein 1; PD-L1, programmed cell death ligand 1; JAK, Janus kinase; STAT, signal transducer and activator of transcription; NF-κB, nuclear factor kappa B.

Similarly, the NF-κB signaling pathway markedly regulates expression of PD-L1. When the NF-κB pathway is mutated or hyperactivated, PD-L1 expression is increased (156,161). Inhibiting the NF-κB signaling pathway decreases PD-L1 expression in numerous types of cancer (162). One study revealed that hesperidin inhibits breast cancer cell proliferation via the downregulation of PD-L1 expression via inhibition of the AKT and NF-κB signaling pathways (146). Moreover, Chr notably downregulates PD-L1 expression levels in HCC cells by blocking the STAT3 and NF-κB pathways (147). Additionally, Chr increases the concentration of IL-2, stimulating T cell proliferation (147). Icaritin, an active ingredient of the Chinese herb Epimedium, binds specific amino acids in IκB kinase-α (IKK-α), namely Cys-46 and Cys-178, preventing the formation and activation of the IKK complex. This inhibition disrupts the activation of the NF-κB signaling pathway, thereby hindering NF-κB nuclear translocation and reducing PD-L1 expression levels (148). Treatment with icaritin decreases PD-L1 expression levels on MDSCs and neutrophils (149). When icaritin is combined with immune checkpoint therapies, such as anti-PD-1/CTLA-4, it notably increases antitumor efficacy (149,150).

In addition, licochalcone A has been demonstrated to inhibit the phosphorylation of eukaryotic translation initiation factor 4E-binding protein 1, to activate the Protein Kinase R-like endoplasmic reticulum (ER) kinase/eukaryotic initiation factor 2α pathway and induce the generation of ROS, thereby suppressing expression of IFN-γ-induced PD-L1 in cancer cells (151). Finally, isorhamnetin directly targets the cell membrane receptor EGFR, thereby inhibiting the EGFR/STAT3/PD-L1 signaling pathway, with subsequent downregulation of PD-L1 expression levels in tumor cells (152).

Taken together, the aforementioned studies demonstrate that flavonoids inhibit the PD-1/PD-L1 pathway by regulating the associated signaling pathways, thereby reshaping the immune-suppressive TME.

Combining flavonoids with anti-PD-1/PD-L1 therapy

Flavonoids, in addition to lowering PD-1 and PD-L1 expression, offer substantial therapeutic benefits when combined with anti-PD-1/PD-L1 therapy. This synergistic approach enhances the immune response against cancer by decreasing inhibitory PD-1/PD-L1 interactions through amplifying T cell activation and cytotoxicity and favorably modulating the TME. The combination of cryptotanshinone with low-dose anti-PD-L1 therapy exerts a synergistic effect, effectively controlling tumor growth and inducing long-term specific immunity against LLC in mice (163). Wu et al (164) observed that, in an HCC mouse model, 100 mg/kg/day quercetin with the anti-PD-1 antibody remodeled the HCC TME, thereby enhancing the efficacy of the anti-PD-1 antibody. Furthermore, Neo increases the effectiveness of anti-PD-1 treatment in a breast cancer 4T1 tumor model, which is initially insensitive to immunotherapy, by inhibiting MDSCs and modulating the TME (78). In the 4T1 model, the combination of Chr and a PD-1 inhibitor decreases the immunosuppressive function of MDSCs, enhances T cell activity and alleviates T cell exhaustion, outperforming single therapies in terms of the ability to inhibit tumor growth (80).

Flavonoids broaden indications and improve the efficiency of cancer vaccines

Cancer vaccines represent a notable advancement against cancer, serving as a cornerstone of cancer-specific active immunotherapy. These vaccines are classified into two primary categories: Cancer-preventive and therapeutic vaccines. Cancer-preventive vaccines are designed to prevent the development of cancer by targeting specific viral infections that increase cancer risk (165,166). Human papillomavirus vaccines, such as GARDASIL® and CERVARIX®, are cancer-preventive vaccines (167,168). On the other hand, therapeutic vaccines are designed to treat cancer that has already developed. Unlike conventional treatments, such as chemotherapy and radiation, therapeutic vaccines exert their effects by activating the immune system to target and destroy cancer cells (169,170). For example, PROVENGE® induces an immune response against prostate cancer cells by stimulating CD8+ CTLs to attack the tumor (171–173).

Flavonoids increase tumor immunogenicity by promoting immunogenic cell death (ICD)

ICD is a process in which stressed or dying cells release damage-associated molecular patterns (DAMPs) into the extracellular space (174–176). DAMPs include HMGB1, ATP and calreticulin (CRT). These DAMPs have a key role in activating immune responses by modulating immune cell functions via specific molecular pathways. For example, HMGB1 and ATP bind pattern recognition receptors on DCs, such as toll-like receptor (TLR)2 and 4 and the receptor for advanced glycation end-products (RAGE), triggering DC activation (177,178). This induces DC maturation and migration to lymph nodes, where mature DCs process antigens from dying tumor cells and present them to T cells via MHC class I and II molecules, thereby stimulating anti-tumor immunity (179,180). Additionally, activated DCs upregulate co-stimulatory molecules such as CD80, CD86 and CD40, enhancing T cell activation (96,179). ATP also binds the purinergic receptor P2X7 on NK cells, activating them to enhance cytotoxic activity against tumor cells via the secretion of perforin and granzymes (181–183). Furthermore, in monocytes and macrophages, DAMPs bind TLRs and RAGE, promoting their recruitment to the tumor site (184). Activated macrophages secrete pro-inflammatory cytokines such as TNF-α, IL-1β and IL-6 via the M1 phenotype, thereby enhancing anti-tumor immunity (185). DAMPs also activate effector T cells, especially CTLs, inducing the release of pro-inflammatory cytokines such as IFN-γ and upregulating co-stimulatory molecules on tumor cells, thereby enhancing T cell cytotoxicity and activation (186). Flavonoids enhance tumor immunogenicity by inducing immunogenic cell death (ICD) (Fig. 2), converting cancer cells into ‘therapeutic vaccines’ that activate anti-tumor immunity without the need for adjuvants (187).

Figure 2. Flavonoid-induced immunogenic cell death (ICD) enhances antitumor immunity by triggering the release of DAMPs (calreticulin, HSP70, HMGB-1) from tumor cells. These signals activate dendritic cells (DCs), promote antigen presentation, and prime CTLs), further fostering a pro-inflammatory tumor microenvironment and boosting immune responses. ICD, immunogenic cell death; CRT, calreticulin; HSP70, heat shock protein 70; HMGB-1, high-mobility group box protein 1; DAMP, damage-associated molecular pattern; DC, dendritic cell; CTL, cytotoxic T lymphocyte.

Silymarin induces ICD in CT26 colon cancer and B16F10 melanoma cells, as evidenced by the release of DAMPs, including CRT, HSP70 and HMGB-1. When combined with doxorubicin, silymarin markedly enhances this ICD response, and promotes Th1-type immune responses by increasing the secretion of IL-12 (188). Afzelin, a flavonol glycoside, induces ICD in lung cancer cells by activating ER stress and promoting the release of ICD-associated molecules, including ATP, HMGB1 and CRT (189). Moreover, LW-213, a synthesized flavonoid, induces ICD in tumor cells by activating ER stress and releasing DAMPs (181). These DAMPs activate APCs, leading to DC maturation and the infiltration of CD8+ T cells into the TME (190). Wogonin triggers the production of ROS within tumor cells, resulting in ER stress. ER stress induces the PI3K/AKT pathway, causing the translocation of CRT and annexin A1 to the cell membrane (102). This allows immune cells to recognize tumor cells. Scutellarin, a Chinese herbal medicine of flavone glycoside origin, induces ICD in HCC, leading to a notable increase in the levels of CRT, ATP and HMGB-1 in the extracellular space (191). PLGA@Icaritin nanoparticles (PGLA nanoparticles loaded with icaritin) induce the generation of ROS, leading to subsequent mitochondrial dysfunction, including the loss of mitochondrial membrane potential and oxidative damage to mitochondrial DNA. This triggers the release of DAMPs from the impaired mitochondria. DAMPs activate the immune system, resulting in ICD within the tumor cells (192). A study of microsatellite-stable colorectal cancer revealed that combination of quercetin and alantolactone induces CRT translocation and HMGB1 release, thereby inducing ICD in cancer cells (193). Furthermore, in a mouse colon cancer model, the combination of 8 procyanidins and 2 mg/kg mitoxantrone induces a higher level of immunogenic cell death in CT26 tumor cells, resulting in the release of HMGB-1 and CRT. This promotes DC maturation and enhanced T cell infiltration within the TME, improving the efficacy of immunotherapy (194).

Flavonoids improve the therapeutic effect of cancer vaccines as adjuvants

Flavonoids serve as adjuvants in tumor vaccines, thereby enhancing their efficacy. Flavonoids notably enhance antigen presentation, improving the efficacy of tumor vaccines. Hesperetin enhances the ability of APCs to process and present tumor antigens by activating the PI3K/AKT signaling pathway, thereby strengthening the immune response (195). In a study of inactivated B16F10 melanoma cells, hesperetin served as an adjuvant, improving the immune response and extending the survival of tumor-bearing mice (195). Moreover, in a melanoma mouse model, the flavonoid compound Chr serves as an adjuvant for tumor vaccines by activating APCs, enhancing the function of Th1 cells and promoting CTL-mediated antitumor responses (196). Transplantation of CD8+ T cells isolated from immunized mice into tumor-bearing mice notably prolongs survival of recipient mice (196).

Flavonoids also serve a key role in enhancing the responses of CTLs when used as vaccine adjuvants. Luteolin serves as an adjuvant for malignant melanoma vaccines. In a mouse model, intramuscular injection 5×106 inactivated B16F10 cells and 10 mg luteolin enhances the responsiveness of CTLs and suppresses the immunosuppressive function of Tregs, thereby inhibiting tumor growth and prolonging the survival of tumor-bearing mice (137). Procyanidin enhances T cell-mediated immune responses and anti-tumor activity when used as a vaccine adjuvant by promoting CD8+ T cell activation and cytokine secretion, which inhibits tumor growth and prolongs survival in tumor-bearing mice (197). In another study, the combination of EGCG with DNA vaccination notably enhanced tumor-specific T cell responses and improved antitumor efficacy, exceeding the effects of either immunotherapy or EGCG alone (198). Finally, in a mouse TC-1 tumor model, intraperitoneal injection of 25 mg/kg apigenin combined with the E7-HSP70 DNA vaccine increases the production of E7-specific CD8+ T cells, thereby enhancing the immune response (199).

Flavonoids enhance the sensitivity of adoptive cell immunotherapy

Adoptive cell immunotherapy, an approach in cancer treatment, involves the extraction, modification and reinfusion of immune cells, especially T cells, to more effectively target cancer (200,201). However, adoptive cell immunotherapy faces challenges such as immunosuppression, limited T cell efficacy in vitro and high treatment costs (200,202). Flavonoid compounds with immune-modulating properties may provide potential solutions.

In an E.G7 mouse lymphoma model, intraperitoneal injection of 70 mg/kg/day curcumin, combined with adoptive T cell therapy, enhances CD8+ T cell-mediated tumor cytotoxicity (203). This effect is mediated by modulating the TME through the blockade of immunosuppressive factors, including TGF-β, indoleamine 2,3-dioxygenase and Tregs, thereby increasing T cell accumulation and activity (203). Apigenin improves the efficacy of adoptive cell immunotherapy by promoting the activation of CTLs, enhancing antigen presentation and inhibiting Tregs (204). Quercetin, by contrast, enhances the sensitivity of cancer cells to adoptive cell immunotherapy by inducing an imbalance of ROS, mitochondrial dysfunction and apoptosis (205).

Challenges in the clinical application of flavonoids

The optimization of flavonoid dosing is key for clinical application as pharmacological effects are dose-dependent (206). Low doses may be ineffective, whereas high doses may lead to toxicity or side effects. Therefore, personalized dosing regimens should be developed based on cancer type and patient needs. For example, baicalein inhibits expression of PD-L1 at a concentration of 10 µM in vitro, whereas in animal studies, oral doses typically range from 50 to 200 mg/kg (127,207). In clinical trials, quercetin is administered at daily doses of 500–1,000 mg, demonstrating tolerability and immune modulation (208,209). When combined with immune checkpoint inhibitors, the dosing range for synergistic effects should be optimized to avoid increased toxicity.

The low water solubility and rapid metabolism of flavonoids limits their bioavailability (210–213). To enhance therapeutic efficacy, various novel formulations have been developed, including nanoparticle formulations, prodrug designs and sustained-release systems (214–216). Nanoparticles, such as liposomes and polymeric and solid lipid nanoparticles, encapsulate flavonoids to improve stability and targeting (214). Prodrug design involves chemically modifying flavonoids into forms that release active ingredients in specific in vivo environments, thereby enhancing both efficacy and safety (215). Sustained-release systems, such as microspheres or hydrogels, allow prolonged release of flavonoids, decreasing dosing frequency and improving patient compliance (216).

The clinical efficacy of flavonoids is not only influenced by pharmacological properties but is also associated with the route of administration. A rational choice of administration route may notably enhance drug absorption, targeting and therapeutic effects, while minimizing the risk of adverse reactions. Oral administration is the most common route; however, this route is limited by poor solubility, gastrointestinal degradation and first-pass metabolism (213). To overcome these challenges, strategies such as nanoparticle carriers, prodrug design and excipient improvements have been employed to enhance bioavailability (214–216). Intravenous injection is suitable for efficient anti-tumor treatment, with liposomes, polymeric nanoparticles or suspensions used to improve water solubility and plasma stability. Inhalation is ideal for treating respiratory diseases such as lung cancer, where nebulized delivery markedly increases pulmonary drug concentration (217,218). c. The combination of intravenous with local injection, or oral administration with inhalation, produces synergistic effects.

Nano-drug delivery system enhance the therapeutic outcomes of flavonoids in cancer immunotherapies

Novel drug delivery systems, especially nanosystems, improve flavonoid bioavailability and enable targeted tumor delivery (219). Nano-drug delivery systems use biocompatible, surface-modifiable nanocarriers to specifically target tumor sites, enhancing drug concentrations at the target site, while minimizing toxic effects on normal tissue (219–221). This approach has demonstrated promising results in tumor targeting and anti-tumor activity (220,221).

Nano-drug delivery systems markedly improve the bioavailability of drugs. In a microsatellite-stable colorectal cancer mouse model, QA-M, an innovative type of nanotherapy, uses a unique nanodelivery system that synergistically encapsulates quercetin and alantolactone in a 1:4 molar ratio. This system prolongs drug circulation time and increases drug accumulation in tumor tissue, thereby enhancing bioavailability (184). Additionally, it promotes induction of ICD, further boosting the immune response and contributing to more effective tumor control (193).

Nano-drug delivery systems enable the targeted delivery of therapeutics. In a mouse melanoma model, a dual pH-sensitive nanocarrier loaded with curcumin and anti-PD-1 antibodies enhances cancer immunotherapy. This carrier selectively binds to circulating PD-1+ T cells, directing them to the TME. Upon reaching the tumor site, the nanocarrier releases anti-PD-1 antibodies, blocking PD-1 on T cells and enhancing their anti-tumor response (222). Moreover, curcumin inhibits the NF-κB signaling pathway, modulating the expression of immunosuppressive factors and further boosting the anti-tumor immune response (222).

Nano-drug delivery systems encapsulate multiple drugs or therapeutic agents, enabling combination therapy to enhance efficacy and decrease drug resistance. In a mouse melanoma model, Trp2 peptide vaccine combined with curcumin-polyethylene glycol (CUR-PEG) micelles improves the effectiveness of the immunotherapy (223). Lu et al (223) revealed that CUR-PEG effectively reshapes the TME by reducing immunosuppressive factors and increasing proinflammatory signals. This approach strengthens CTL responses and enhances the production of IFN-γ, thereby promoting the transition of immune-suppressive M2 to immune-activating M1 macrophages. By decreasing the populations of immunosuppressive cells such as MDSCs and Tregs, and immunosuppressive molecules such as IL-6, while increasing the levels of proinflammatory cytokines such as TNF-α and IFN-γ, CUR-PEG effectively transforms the inhibitory TME.

In conclusion, nano-drug delivery systems offer notable advantages in addressing the limitations of flavonoid bioavailability. By enhancing drug stability, prolonging circulation time and enabling precise targeted delivery, these systems increase drug accumulation at tumor sites while minimizing toxicity to healthy tissue. Additionally, the multifunctional design of nanocarriers supports the co-delivery of multiple therapeutic agents, promoting synergistic effects and mitigating drug resistance.

Safety assessment of flavonoids

Flavonoids are generally safe and well-tolerated at standard doses. However, at high doses, they may cause mild gastrointestinal discomfort, headache, skin reactions or slight liver dysfunction. These side effects are typically mild and reversible and readily resolved upon dose adjustment or discontinuation of the treatment. Although flavonoids generally exhibit low toxicity, prolonged or high-dose use may lead to liver and kidney damage, drug interactions or allergic reactions, especially in individuals with pre-existing liver or kidney conditions or a history of allergies (224,225). There is limited evidence on irreversible side effects, but caution is advised in individuals with compromised liver or kidney function to avoid potential long-term damage (224,225).

When combined with immunotherapeutic agents such as PD-1/PD-L1 inhibitors or CTLA-4 inhibitors, flavonoids may induce side effects. Although they enhance the efficacy of immunotherapy by modulating the immune microenvironment and inhibiting immune-suppressive factors (such as TGF-β and IL-10), flavonoids may also result in excessive immune activation, potentially increasing the risk of autoimmune diseases such as rheumatoid arthritis or systemic lupus erythematosus. Additionally, by amplifying the anti-tumor immune response, flavonoids may also trigger a cytokine storm (78,80,152,163).

Although flavonoids exhibit low toxicity in in vitro and animal studies (226,227), their clinical safety requires validation in large-scale clinical trials. Future research should determine the maximum tolerated dose of flavonoids, assessing long-term safety, investigating potential drug interactions and evaluating the risk of adverse effects when combined with immunotherapy.

Conclusion

Flavonoids inhibit the secretion of immunosuppressive factors, promote the release of anti-tumor immune factors, decrease the number and function of immunosuppressive cells and enhance effector T cell activity, contributing to the reversal of the immunosuppressive microenvironment (47). Moreover, flavonoids can considerably enhance the efficacy of cancer immunotherapies, including cancer vaccines, immune checkpoint inhibitors and adoptive cell immunotherapy (228).

Although flavonoids have potential in enhancing the efficacy of immunotherapy, their effective application faces several challenges. First, tumor heterogeneity notably impacts the effectiveness of flavonoids (228). Tumors from different patients typically exhibit notable variation in terms of genetic mutations, immune cell infiltration and the TME, which can lead to differences in the responses to flavonoids across tumor types or individuals (229). Secondly, the role of flavonoids in immune signaling pathways requires further investigation. For example, the JAK/STAT, NF-κB and PI3K/AKT/mTOR signaling pathways are hypothesized to serve key roles in immune-regulatory effects (153–157); however, the underlying mechanisms remain unclear (127,139,146,147). A deeper understanding of how these pathways are modulated by flavonoids may optimize their therapeutic outcomes. To the best of our knowledge, there is currently a lack of clinical data on the use of flavonoids in cancer immunotherapy. Although flavonoids have antitumor effects in animal models, their effective application in clinical settings still requires clinical trial data. Therefore, future studies should focus on validating the use of flavonoids in clinical trials.

In future, the application of high-throughput technologies, such as single-cell sequencing, may enable in-depth analysis of the dynamic changes in immune cells within the TME, thereby revealing the underlying mechanisms of tumor escape and their interactions with flavonoids to support precision medicine (230). Simultaneously, innovative drug delivery systems, including nanotechnology, liposomes and polymeric nanoparticles, may improve the bioavailability, targeted delivery and accumulation of flavonoids within the TME, thereby enhancing anti-tumor efficacy and decreasing toxicity (229–221). Personalized treatment strategies may tailor flavonoid-based therapy according to the genomic features of a patient, TME and immune response differences, to improve efficacy and minimize adverse effects. Furthermore, long-term efficacy and safety assessment of flavonoids should become a research priority, focusing on potential toxicity, drug interactions and effects on normal tissue to ensure the safety and sustainability of clinical applications. Clinical trials evaluating the combination of flavonoids with immune checkpoint inhibitors should also be performed to assess their potential in terms of enhancing immune responses, improving therapeutic outcomes and prolonging survival. Finally, biomarker detection may elucidate the underlying mechanisms and therapeutic prospects, thereby advancing flavonoids as an effective adjunctive therapeutic strategy.

In conclusion, flavonoids have promise in cancer immunotherapy by reshaping the TME and enhancing the capacity of the immune system to combat cancer. Advanced technologies such as metabolomics, single-cell sequencing, innovative drug delivery systems and computer-aided design are needed to develop targeted antitumor immunotherapeutic agents.

Supplementary Material

Supporting Data

Acknowledgements

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Funding

The present was supported by Shandong Provincial Health Commission (grant no. Z-2023064).

Availability of data and materials

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Authors' contributions

CY wrote the manuscript. GW conceived the study and reviewed the manuscript. Both authors have read and approved the final manuscript. Data authentication is not applicable.

Ethics approval and consent to participate

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Patient consent for publication

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Competing interests

The authors declare that they have no competing interests.

Glossary

Abbreviations

Abbreviations:

References