Notch1 signaling plays a critical role in tumor-induced immunosuppression. On one hand, Notch1 contributes to tumor immunosuppression through promotion of MDSCs. Notch-activated macrophages increased the liberation of C-C motif chemokine ligand 2 (CCL2) and interleukin-10 (IL-10) to induce MDSC recruitment in glioma (55). The MDSC-rich environment educated T cells to be immunosuppressive and attenuated the efficacy of oncolytic herpes simplex virus-1 (oHSV-1)-based virotherapy. Conversely, pharmacologic blockade of Notch reversed the immunosuppressive brakes and potentiated CD8⁺ T cell-driven antitumor immune responses, thereby improving oncolytic virus-induced therapeutic benefit. Deregulation of Notch signaling in immature T cells induced the expansion of CD11b⁺Gr-1⁺ MDSCs in T-cell acute lymphoblastic leukemia (T-ALL) through IL-6 (56). Depletion of MDSCs inhibited T-ALL cell proliferation and expansion. Notch3 increased the expression of various cytokines such as CCL2, colony-stimulating factor-1 (CSF-1) and C-X-C motif chemokine ligand 12 (CXCL12), which promoted recruitment of macrophages and MDSCs to the TME of colorectal cancer (CRC) (57). Therefore, Notch3 accelerated CRC development by favoring immunosuppressive TME. MDSCs were able to stimulate signal transducer and activator of transcription 3 (STAT3) and Notch in breast cancer cells through IL-6 and nitric oxide (NO), respectively (58). Notch activation supported sustained STAT3 activation via IL-6. The crosstalk between Notch and STAT3 signaling pathways mediated MDSC-induced breast cancer stemness. Notch signaling was strongly inhibited in MDSCs derived from colon carcinoma- and melanoma-bearing mice (59). Notch signaling suppressed the production of polymorphonuclear (PMN)-MDSCs but increased the generation of mononuclear MDSCs. Depletion of Notch signaling reduced the inhibitory effect of MDSCs on T cell function. Manipulation of Notch signaling cascade may represent a potential therapeutic option for cancer patients. PMN-MDSC-derived reactive oxygen species (ROS) activated Notch1 in circulating tumor cells (CTCs) isolated from patients with breast cancer or melanoma, which enabled CTCs to respond to Jag1-mediated Notch activation (60). The crosstalk between PMN-MDSCs and CTCs enhanced the dissemination and metastatic potentials of CTCs. On the other hand, Notch signaling counteracts MDSC-mediated immunosuppression. Activation of Notch/RBPJ signaling pathway blocked monocarboxylate transporter 2 (MCT2)-mediated lactate import in myeloid cells through its downstream molecule HES1 (61). This resulted in the suppression of MDSC differentiation and promotion of M1-like TAM maturation, thereby restricting lung cancer progression. MDSCs decreased the expression of Notch1 and Notch2 in T cells in a NO-dependent manner (62). Notch1 increased the cytotoxic effects of CD8⁺ T cells and induced their conversion into a memory phenotype. Restorement of Notch1 signaling in CD8⁺ T cells overcame MDSC-induced T cell suppression and improved the beneficial effects of T cell-based immunotherapy in lung cancer-bearing mice.

TAMs play dual roles in remodeling of tumor immunosuppressive microenvironment. Notch induced the expression of IL-1β and CCL2, which then recruited TAMs to the tumor, contributing to breast cancer progression (63). High expression of Jag1/Notch1 were positively linked with infiltration levels of M2-like TAMs, contributing to poor prognosis in lung adenocarcinoma (LUAD) (64). Mechanistically, Notch signaling recruited macrophages into LUAD tissues through induction the secretion of CCL2 and IL-1β and promoted their polarization into M2 type. Jag1-mediated Notch signaling increased the secretion of IL-4 and IL-6 in breast cancer cells, leading to M2 polarization of TAMs and breast cancer development (65). Epithelial ovarian cancer (EOC) cells induced the activation of Notch pathway in endothelial cells to upregulate CD44 in TAMs, propelling their education by EOC cells (66). This resulted in formation of immunosuppressive microenvironment and impairment of T cell-mediated immunity, hence accelerating EOC progression. Notch signaling was activated in TAMs in the TME of pancreatic ductal adenocarcinoma (PDAC) (67). Notch activation favored TAM polarization into an immunosuppressive M2-like phenotype characterized by high levels of immunosuppressive cytokines (IL-10, transforming growth factor-β1 (TGF-β1) and tumor necrosis factor-α (TNF-α)), arginase 1 and immune checkpoint molecules. Notch inhibition cooperated with programmed cell death protein-1 (PD-1) blockade to induce CD8⁺ T cell-mediated antitumor immunity in PDAC. The combined treatment achieved a stronger anticancer effect than single treatment. Activation of Notch in triple negative breast cancer (TNBC) induced the expression of IL-1β and CCL2 to attract TAMs into the tumor, culminating in cancer development and immune evasion (68). Notch signaling in PDAC cells fostered the recruitment and activation of macrophages to a M2-like phenotype through upregulation of IL-8, CCL2, IL-1α and urokinase-type plasminogen activator (uPA) (69). In turn, activated TAMs then secreted IL-6 to enhance PDAC cell metastasis. Interruption of Notch-dependent circuit holds potential as a therapeutic option for PDAC treatment. Intriguingly, Notch signaling also limits the immunosuppressive function of TAMs. Myeloid-specific Notch activation remarkably reduced the infiltration of pro-tumorigenic M2 macrophages into PDAC tissues, which led to upregulated antigen presentation and enhanced cytotoxic T effector function (70). Notch signaling in myeloid cells suppressed the expansion of Kupffer cell-like TAMs (kclTAMs), thus inhibiting hepatocellular carcinoma (HCC) progression (71).

Notch1 overexpression favored melanoma growth in vivo (72). In terms of mechanism, Notch1 suppressed the intratumoral infiltration of CD8⁺ cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells while increased the population of Tregs and MDSCs via upregulation of TGF-β1. It also inhibited T cell proliferation and activation and increased PD-1 expression on CD4⁺ and CD8⁺ T cells. High expression of Dll3 correlated with poor prognosis in patients with invasive breast cancer (73). Patients with high Dll3 expression had high proportions of T follicular helper (Tfh) cells and Tregs and low proportions of M2 macrophages and resting mast cells. The accumulation of tumor-promoting immune cells is likely to facilitate breast cancer pathogenesis. The infiltration of Tregs and T helper 17 (Th17) cells in the TME was increased with the development of gastric cancer, leading to an imbalance between Tregs and Th17 (74). CD4⁺CD25⁺CD127^dim/- Tregs expressing high levels of forkhead box protein P3 (FoxP3) function to maintain peripheral tolerance in cancer by inhibiting the activation of tumor-specific effector T cells (75). Retinoic acid-related orphan receptor γt (RORγt), a key transcription factor of IL-17, is expressed in Th17 cells and correlates with IL-17-driven inflammation (76). Notch signaling pathway was activated in gastric cancer (77). Inhibition of Notch signaling reduced the expression of transcription factors FoxP3/RORγt without affecting Treg/Th17 balance. Notch inhibition blunted the suppressive function of CD4⁺CD25⁺CD127^dim/- Tregs and IL-17 production by Th17 in gastric cancer. Altogether, Notch signaling played a critical role in regulating the activity of Tregs and Th17 cells during GC progression. Another study indicated that high Notch3 expression was associated with decreased infiltration of activated CD8⁺ T cells and emergence of immunosuppressive cells (e.g., Tregs and M2 macrophages) in the TME of gastric cancer (78). High Notch3 expression also contributed to upregulation of immune checkpoint genes (e.g., CD200, CD274, CD276, CTLA4 and HAVCR2), leading to impaired immune responses. Altogether, Notch3 was involved in immune tolerance of gastric cancer, highlighting its potential as a therapeutic target for the treatment of gastric cancer.

Jag1/Notch2 signaling-mediated cell-to-cell contact between CAFs and lung cancer cells facilitated vascular mimicry (VM) generation and permeability (79). The loose junctions of VM networks fostered cancer intravasation and neutrophil penetration, leading to enhanced N2 neutrophil infiltration in lung cancer tissues. Blockade of Jag1/Notch2 interaction inhibited VM formation and lung cancer growth in a murine tumor model. Notch1 activation in TNBC cells promoted contact-dependent induction of CXCL8 in inflammation-induced TNBC-stromal interaction network, leading to TNBC metastasis and progression (80). CAF-derived stanniocalcin 1 (STC1) increased the stemness of HCC cells (81). Mechanistically, STC1 directly combined with Notch1 receptors to motivate the Notch signaling pathway. Moreover, Notch1 was capable of regulating STC1 at the transcriptional level, constituting a tumor-stromal amplifying STC1-Notch1 feedforward signal. Radiation induced the expansion of Dll1-overexpressing breast cancer CSCs, which impelled IL-6-dependent recruitment of CAFs within the TME (82). After that, Dll1-mediated Notch signaling in CAFs triggered Wnt signaling in Dll1-overexpressing CSCs to increase radioresistance in breast cancer. Moreover, blocking Dll1 and IL-6 sensitized Dll1-ovexpressing breast cancer cells to radiotherapy.

In contrast, Notch-activated CAFs can exert tumor-suppressive actions. Apoptotic lung cancer cells activated Notch1/Wnt-induced signaling protein-1 (WISP-1) signaling pathway in CAFs, thus inhibiting the migration and invasion of lung cancer cells (83). Intracellular Notch1 signaling in CAFs reduced the plasticity and stemness of melanoma stem/initiating cells (MICs), hence affecting cancer heterogeneity and aggressiveness (84). Increased activity of Notch pathway in melanoma-associated fibroblasts strikingly retarded cancer growth and angiogenesis (85). Activating Notch signaling in melanoma-associated fibroblasts may be a promising therapeutic strategy for melanoma treatment.

Previously, Wood et al. (86) reported that neutrophils with activated Notch and TGF-β signaling cascades were enriched in metastatic CRC tissues, suggesting the potential implication of neutrophils in formation of a pre-metastatic niche. Radiation exposure in lung tissues induced accumulation and activation of neutrophils, contributing to a series of tissue perturbations including persistent Notch activation in epithelial cells (87). Such changes facilitated a metastatic niche within irradiated lung tissues, accelerating the growth of arriving breast cancer cells. Notch ligands expressed by cancer cells or tumor-associated neutrophils (TANs) activated Notch1 receptors on endothelial cells (88). Endothelial Notch1 hyperactivation facilitated neutrophil infiltration and metastasis through upregulation of vascular cell adhesion molecule 1 (VCAM1), forming a pre-metastatic niche to drive cancer colonization. These events promoted circulating cancer cell migration, intravasation and homing at distant sites.

Jag2-induced Notch signaling could enhanced NK cell cytotoxicity mediated by DCs (89). This caused increased effector activity of cytotoxic T cells. Thus, overexpression of Jag2 retarded the proliferation of B cell lymphoma cells. Notch2 signaling was implicated in conventional DC (cDC)-mediated antitumor immunity (90). Depletion of Notch2 signaling dampened cDC terminal differentiation and inhibited antigen cross-presentation to CD8⁺ T cells through downregulation of chemokine receptor 7 (CCR7). Consequently, Notch-primed DCs inhibited the development of inflammation-associated colon cancer. Notch signaling in DCs prevents cancer progression by eliciting antitumor immune responses.

Unlike DCs, neutrophils function to impair T cell-mediated antitumor immune responses. Epithelial Notch1 induced TGF-β-dependent neutrophil recruitment into the TME of CRC, contributing to decreased infiltration of CD8⁺ T cells and cancer metastasis (91). EOC cells recruited TANs into the TME and triggered Jag2 expression in TANs via IL-8 release (92). Jag2-activated TANs dampened cytotoxic activity of CD8⁺ T cells. Blockade of Notch signaling inhibited EOC growth and strengthened the killing effect of CD8⁺ T cells.

Notch signaling facilitates adaptive immune evasion in cancer. B cells, CD45⁺ cells, DCs, macrophages, mast cells, neutrophils and T cells were significantly reduced in Notch1-mutant relapsed T1-2N0 laryngeal cancer in comparison with wild-type cancer samples (93). Notch1-mutant recurrent tumors exhibited an immunosuppressive phenotype characterized by decreased B cells score, T cells score and tumor-infiltrating lymphocytes (TILs) score. Notch1 mutation leads to compromised immune response and immunosurveillance escape in recurrent laryngeal cancer. Tumor-derived Jag1 drove tumorigenesis through macrophage recruitment and functional repression of CD8⁺ T cells in breast cancer-bearing mice models (94). Jag1-induced Notch activation fostered the production of multiple cytokines including IL-6 and WISP-1, contributing to the recruitment of macrophages into the TME. Following recruitment, Notch-activated macrophages interacted with tumor-infiltrating T cells to suppress their proliferation and tumor-killing activity by enhancing CD14 and CD93 secretion. Combination of PD-1 blockade and a Notch inhibitor produced significantly greater inhibition of breast cancer growth than either individual agent. Jag1-mediated Notch activation propels adaptive immune evasion of cancer cells and offers novel therapeutic opportunities for cancer treatment. Inhibition of Notch activation reduced the production of proinflammatory cytokines (e.g., CCL2 and IL-1β) in TNBC (95). This triggered a switch to an immune-inflamed tumor phenotype characterized by a reduction in immunosuppressive Tregs and M2-polarized TAMs and increased infiltration of cytotoxic T cells into tumors. Notch signaling can program tumor immune landscape to facilitate TNBC immune evasion. Notch1 signaling decreased the expression of human leukocyte antigen (HLA) class II genes through downregulation of class II transactivator (CIITA) in chronic lymphocytic leukemia (CLL) cells (96). Notch1 also increased the expression of programmed cell death protein-ligand 1 (PD-L1) and promoted the exhaustion of CD8⁺ T cells. Notch1 signaling facilitated CLL escape from immune surveillance by blocking antigen presentation and inhibiting T cell activation.

Euchromatic histone methyltransferase 2 (EHMT2)-mediated activation of Notch1 signaling increased PD-L1 expression and reduced major histocompatibility complex-I (MHC-I) expression to dampen T cell immune responses, culminating in stemness maintenance of glioma stem cells (GSCs) (97). The binding of Jag1 expressed on tumor epithelial cells to Notch receptors on TAMs in PDAC microenvironment favored the activation of Notch signaling, which induced TAM polarization into an immunosuppressive phenotype (67). TAMs secreted immunosuppressive mediators (e.g., IL-10 and TGF-β) to suppress CD8⁺ T cell function. Furthermore, pharmacological inhibition of Notch signaling combined with PD-1 blockade led to reduced macrophage recruitment and increased CD8⁺ T cell activation, which led to tumor inhibition.

Notch signaling is capable of inducing T cell-mediated immune response ( Figure 2 ). Reportedly, the transcriptional levels of Notch1/2/3 were increased in gastric cancer tissues compared with normal tissues (98). High transcription levels of Notch1/2/3/4 predicted a good prognosis in patients with gastric cancer. Upregulation of Notch family members was positively correlated with infiltration levels of CD4⁺ T cells, DCs, macrophages and neutrophils. Notch expression showed significant association with many immune markers in gastric cancer, including CCL2, CD163, C-X-C motif chemokine receptor 4 (CXCR4), CCR4 and IL-10. Notch1/2/3/4 might represent potential therapeutic targets for precision cancer treatment. Notch signaling pathway increased the expression of MHC-I and interferon-γ (IFN-γ)-dependent cytokines and favored the recruitment of antitumor CD8⁺ T cells in glioma (99). Moreover, Notch signaling promoted the conversion of TAMs into an anti-tumorigenic phenotype. Oppositely, blockade of Notch signaling facilitated tumor immune evasion and increased glioma cell aggressiveness. Collectively, Notch signaling pathway suppressed cancer development by remodeling tumor immune microenvironment. CRC patients with Notch mutation had higher infiltration levels of CD8⁺ T cells, M1 macrophages, neutrophils and NK cells (100). Notch signaling was positively associated with tumor immunogenicity in CRC. Notch mutant status predicted a good prognosis in CRC patients undergoing immune checkpoint inhibitor (ICI) treatment. Notch mutation is anticipated to act as a new therapeutic target and prognostic factor for CRC. Another study indicated that mutated Notch signaling correlated with an enrichment of tumor-specific CD8⁺ T cells and deprivation of Tregs (101). Therefore, Notch signaling pathway mutations augmented antitumor immunity in CRC. The expression levels of Notch1, Notch3 and Notch4 were remarkably decreased in prostate cancer tissues compared with normal tissues (102). The abundance of Notch family genes was connected with intratumoral infiltration of CD4⁺ T cells and CD8⁺ T cells, which influenced clinical outcomes in patients with prostate cancer. Silencing of Notch ligand Dll1 on DCs dampened antitumor immune responses in lung and pancreatic cancers by inhibiting effector CD8⁺ T cell function and T effector memory (Tem) cell differentiation as well as promoting the accumulation of Tregs and MDSCs (103). Consequently, pharmacological inhibition of Dll1 promoted the growth of lung and pancreatic cancers. In addition, enforced expression of Jag1 reduced Treg frequency and enhanced antitumor immunity through downregulation of PD-1 on CD8⁺ Tem cells. Notch ligands are crucial for triggering antitumor T cell immune responses to prevent cancer development.

Immune checkpoints refer to as a class of stimulatory and inhibitory molecules expressed on immune cells and cancer cells (104). They act as accessory molecules that either induce or suppress T cell activation (105). PD-1 is an inhibitory receptor expressed on T cells, while PD-L1 is a ligand of PD-1 (106). The PD-L1/PD-1 signaling cascade constitutes an adaptive immune resistance mechanism in cancer. Notch signaling was responsible for PD-L1 upregulation, contributing to immune evasion in gastric cancer (107). Notch/c-Myc/enhancer of zeste homolog 2 (EZH2) signaling induced PD-L1 expression in CLL, contributing to enhanced resistance to activated autologous T cells in a murine CLL model (108). Likewise, Notch3 was upregulated in PD-L1-overexpressing breast CSCs (109). Notch3 activation increased PD-L1 expression by regulating the activity of mammalian target of rapamycin (mTOR), which was crucial for maintaining the stemness of CSC-like cells. High expression levels of Dll3 were inversely linked with Notch1 but showed significant association with high expression of PD-L1 (110). This resulted in dysfunction of T lymphocytes. However, high Dll3 expression was associated with increased infiltration of immune cells and exerted a protective effect on both progression-free survival (PFS) and overall survival (OS) in PDAC patients.