Temporal dynamics in cancer immunotherapy: the interplay between circadian rhythms, tumor microenvironment, and immune checkpoint blockade

Abstract

The tumor microenvironment (TME) is a highly intricate and dynamic system composed of various cellular and non-cellular components, which plays a crucial role in regulating key biological processes such as tumor initiation, progression, and therapeutic response. Recent studies have uncovered a complex regulatory network between the circadian rhythm system and TME, which significantly impacts the clinical efficacy of immune checkpoint inhibitors (ICIs). This review systematically delineates the multi-level regulatory mechanisms by which circadian rhythms influence key components within the TME, including tumor cell biological characteristics, immune cell functional states, angiogenesis processes, and cancer-associated fibroblast activity. It deeply analyzes the systemic impact of the TME on ICI treatment efficacy via immunosuppressive networks, immune checkpoint molecule expression, T cell functional states, and drug bioavailability. The review underscores the central role and clinical significance of circadian rhythms in regulating immune checkpoint molecule expression, immune cell function, ICI drug pharmacokinetics, and TME homeostasis. Based on these research advancements, this paper systematically presents ICI treatment optimization strategies grounded in circadian rhythm dynamics and TME characteristics, encompassing time-dependent dosing regimens, combination therapies targeting the TME, circadian rhythm modulation methods, and precision personalized treatment approaches. Additionally, it offers a systematic outlook on the research frontiers and clinical translation prospects in this field, encompassing multi-omics integrated analysis of circadian regulatory networks, artificial intelligence (AI)-based precision chronomedicine strategies, novel chronotherapeutic approaches, and standardized clinical translation systems. This review establishes a crucial theoretical foundation for understanding the circadian rhythm-TME-ICI interaction network and optimizing cancer immunotherapy strategies.

1. Introduction

The tumor microenvironment (TME) is a highly complex and dynamic network system co-constructed by tumor cells and their surrounding intricate microecosystem. Its main components encompass tumor cells, infiltrating immune cells, cancer-associated fibroblasts (CAFs), vascular and lymphatic networks, and nervous systems, as well as non-cellular components including extracellular matrix, basement membrane, growth factors, and metabolites.1, 2, 3, 4, 5 The TME plays a pivotal role in orchestrating key biological processes, including tumor initiation, progression, metastasis, and treatment response, through the establishment of intricate intercellular signaling networks.^4,6,7 Immune checkpoint inhibitors (ICIs) are a class of therapeutic humanized monoclonal antibodies designed to target key immune inhibitory molecules, primarily programmed cell death protein 1 (PD-1), its ligand PD-L1, and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4).8, 9, 10, 11, 12, 13 ICIs effectively remodel the immune microenvironment and elicit anti-tumor immune responses by specifically recognizing and blocking immunosuppressive signaling pathways (particularly CTLA-4 and PD-1), demonstrating significant efficacy in the clinical treatment of various solid tumors, including breast, renal, and lung cancers.^8,14, 15, 16, 17, 18, 19, 20 The circadian rhythm is a fundamental endogenous time-regulating system within organisms, which exerts temporal control over crucial physiological processes such as sleep-wake cycles, immune system function, metabolic activities, and endocrine networks through an intricate network of transcription-translation feedback loops. Circadian rhythm regulation is primarily governed by the function of the biological clock. At the organ level, the biological clock is composed of two major components: the central and peripheral biological clock systems. The core of the central biological clock is the suprachiasmatic nucleus residing in the hypothalamus, which receives photic signals from the eyes and influences rhythmic activities through the controlled secretion of hormones such as melatonin and cortisol. The peripheral biological clocks, through multilevel regulation, enable many genes to exhibit distinct circadian oscillations across various tissues and organs. Furthermore, at the molecular level, the biological clock comprises a specific network of genes and proteins, with core components including Clock, Bmal1 (Arntl), Period (Per), and Cryptochrome (Cry). These clock genes and proteins undergo periodic changes throughout the day through precise negative feedback loops: Clock and Bmal1 genes activate the transcription of Per and Cry genes, while Per and Cry proteins gradually accumulate within cells and bind to the Clock-Bmal1 complex, thereby inhibiting its transcription; as Per and Cry proteins degrade, Clock and Bmal1 expression increases again, thereby initiating a new circadian cycle. Additionally, the nuclear receptors REV-ERB and retinoid‐related orphan receptor (ROR) provide additional regulatory mechanisms by respectively inhibiting and activating BMAL1 transcription.²¹ Accumulating evidence indicates that persistent disruption of circadian rhythms can significantly increase the risk and accelerate the progression of various diseases, particularly malignant tumors.^21,22 A growing body of research has confirmed that circadian rhythms play a crucial regulatory role in various components of the TME. Circadian rhythms orchestrate tumor metabolism, immune cell activity and function, cytokine expression, and other key biological processes within the TME.22, 23, 24 In recent years, researchers have extensively investigated the mechanisms by which the TME influences the efficacy of ICIs, focusing on key factors such as the composition of immune cell subpopulations, cytokine networks, and angiogenic states within the TME.25, 26, 27, 28 Circadian rhythms modulate the therapeutic effects of ICIs by dynamically regulating multiple aspects of the TME,^29,30 thereby revealing a complex regulatory network that offers new intervention strategies and potential targets for tumor immunotherapy.

Despite significant advancements in the fields of circadian rhythms, TME, and ICIs, there remains a notable paucity of integrative research. Existing studies primarily focus on individual domains of circadian rhythms, TME, or ICIs, with a conspicuous absence of systematic integrated research. This limitation impedes our comprehensive understanding of the complex interactive network among these three elements, particularly regarding the regulatory mechanisms by which circadian rhythms influence the efficacy of ICIs. Current research on ICIs predominantly focuses on clinical efficacy and adverse reactions, while in-depth exploration of their mechanisms of action remains relatively insufficient. Notably, the molecular mechanisms underlying the impact of circadian rhythms on the efficacy of ICIs warrant further investigation. Elucidating the interregulatory mechanisms among circadian rhythms, TME, and ICIs is of paramount theoretical and clinical significance. Delineating the regulatory network and molecular mechanisms among these three elements will furnish novel theoretical foundations and identify potential intervention targets for optimizing tumor immunotherapy strategies. Furthermore, elucidating the associations between patients' circadian rhythm patterns, TME characteristics, and responses to ICI therapy will establish a robust scientific foundation for developing personalized precision treatment strategies. In light of the current research landscape, the following scientific questions in the field of ICI therapy require urgent attention: 1) The regulatory mechanisms and key molecular players through which circadian rhythms influence the TME; 2) The tumor microenvironment-mediated pathways that modulate the efficacy of ICIs; 3) The molecular mechanisms underlying the regulatory axis connecting circadian rhythms, TME, and ICI efficacy; and 4) Chronobiology-based strategies for optimizing ICI therapy.

This review comprehensively elucidates the regulatory mechanisms of circadian rhythms in the TME, critical factors influencing the efficacy of ICIs within the TME, and the pivotal regulatory mechanisms of circadian rhythms in the interaction network between the TME and ICIs. Based on current research evidence, this article explores optimization strategies for the synergistic regulation of ICI treatment effects by circadian rhythms and the TME, and provides a comprehensive outlook on the research frontiers and clinical translation prospects in this field. This article focuses on analyzing the multi-level regulatory network of circadian rhythms in the TME, comprehensively expounding their regulatory mechanisms on tumor cell biological characteristics, immune cell functional states, tumor angiogenesis processes, and the activity of tumor-associated fibroblasts. Furthermore, this article provides a comprehensive analysis of the multidimensional impact of the TME on the efficacy of ICIs, including the regulation of immune responses by immunosuppressive cell populations and inhibitory factor networks, as well as the extensive effects of the TME on the expression and regulation of immune checkpoint molecules such as PD-L1, alterations in T cell functional states, and the bioavailability of ICI drugs. Special emphasis is placed on the pivotal role and clinical significance of circadian rhythms in regulating immune checkpoint molecule expression, immune cell function, pharmacokinetic characteristics of ICI drugs, and the overall dynamics of the TME.

2. The influence of circadian rhythms on the TME

TME is composed of various components, including tumor cells, immune cells, tumor vasculature, and tumor-associated fibroblasts.⁵ A growing body of evidence suggests that circadian rhythms modulate tumor cell proliferation, apoptosis, and metabolic processes in the TME, as well as their stemness and metastatic potential. Moreover, circadian rhythms can modulate immune cell quantity, function, migration, and infiltration, as well as influence tumor angiogenesis and the activity of tumor-associated fibroblasts, thus exerting multifaceted effects on tumor initiation and progression.

2.1. The influence of circadian rhythms on tumor cells

Circadian rhythms, mediated by their transcription-translation feedback loops, modulate various aspects of tumor cell activities and characteristics, exerting intricate and specific regulatory effects on tumor cell proliferation, apoptosis, and metabolic processes, as well as their stemness, metastatic potential, and immunogenicity (Table 1).

Table 1.

Key circadian clock genes and their regulatory mechanisms in the tumor microenvironment.

Clock gene	Primary function	TME target	Regulatory mechanism	References
BMAL1	Core positive regulator	Tumor cells, immune cells, metabolites, and ECM	Cell cycle checkpoint, chemokine signal, glucose metabolism, and TGF-β signal regulation	39, 103, 127, 132, 153, 155
CLOCK	Transcriptional activator	Immune cells, vessels	Migration factor and OLFML3-HIF1α pathway regulation	135, 141, 147, 218
PER1/2	Negative feedback regulators	Tumor cells, soluble factors	AKT/mTOR pathway, cell cycle checkpoint, EMT, and VEGF transcription regulation	24, 43, 49, 74
CRY1/2	Transcriptional repressors	Endothelial cells	ICAM-1, VCAM-1, and E-selectin regulation	137
REV-ERBα	Secondary repressor	Immune cells, ECM	CCL2 and TGF-β signal regulation	131, 174
RORα	Secondary activator	Tumor cells	Cell cycle checkpoint, metabolism, and TMB regulation	37, 47, 55

Abbreviations: CCL2, C—C motif chemokine ligand 2; CRY1/2, cryptochrome 1/2; ECM, extracellular matrix; EMT, epithelial-mesenchymal transition; HIF1α, hypoxia-inducible factor 1α; ICAM-1, intercellular adhesion molecule-1; OLFML3, Olfactomedin-like 3; PER1/2, period 1/2; RORα, retinoid‐related orphan receptor α; TGF-β, transforming growth factor-β; TMB, tumor mutational burden; VCAM-1, vascular cell adhesion molecule-1; VEGF, vascular endothelial growth factor.

2.1.1. Regulation of tumor cell proliferation

Extensive research has demonstrated that circadian rhythm disruption significantly affects tumor cell proliferation processes.³¹ Elevated circadian rhythm disruption (CRD) scores are significantly associated with reduced DNA repair capacity and increased expression of G2/M checkpoint, MYC target pathways, and cell cycle proteins.³¹ Sleep pattern disturbances result in significantly elevated levels of reactive oxygen species, which subsequently induce DNA damage through oxidative stress cascade reactions.^32,33 Research has shown that chronic sleep deprivation significantly increases the expression levels of Ki-67, a key marker of cell proliferation, in tumor tissues.³⁴ The circadian molecular clock systematically controls DNA replication, DNA repair, and cell cycle transition processes, thus precisely regulating tumor cell proliferation. Key circadian factors Timeless and Cry2 form functional complexes with cell cycle checkpoint protein Chk1 and the ATR-ATRIP complex, thereby playing a central regulatory role in the DNA damage checkpoint response.³⁵ Per2, a key regulatory factor, exerts its tumor suppression function by modulating DNA damage response pathways and cell cycle progression.³⁶ RORa4/RORγ and REV-ERBα/β respectively activate and inhibit the expression of the cell cycle regulatory gene p21, thereby regulating the G1 progression phase of cell proliferation.³⁷ The core clock gene Bmal1 promotes G2-M phase arrest by activating the ATM signaling pathway, consequently inhibiting tumor cell proliferation.³⁸ Research has demonstrated that overexpression of Bmal1 and Clock can inhibit the G1/S phase transition in cancer cells, and the molecular mechanism is closely related to the downregulation of Cyclin D1 expression.³⁹ Moreover, the circadian system participates in the regulation of tumor cell proliferation by modulating the activation states of multiple signaling pathways.40, 41, 42 ARNTL2 exhibits a significant pro-tumor proliferation effect, and its expression level positively correlates with key oncogenic signaling pathways, including hypoxia, KRAS, P53, and MYC.⁴⁰ BMAL1 inhibits mTOR1, while overexpression of Per1 and Per2 can suppress cancer cell proliferation by promoting autophagy through the inhibition of the PI3K/Akt/mTOR signaling pathway.43, 44, 45 Per1 accelerates the malignant progression of oral squamous cell carcinoma by inhibiting autophagy-dependent cell death and activating the AKT/mTOR signaling pathway, thereby promoting cell proliferation.⁴³ Studies have revealed that NPAS2 can promote the malignant proliferation of liver cancer cells by transcriptionally activating CDC25A.⁴¹ NFIL3 enhances the activation of the NF-κB signaling pathway, thereby promoting the proliferation of triple-negative breast cancer cells.⁴² Furthermore, circadian rhythm-related genes can regulate tumor cell proliferation processes by influencing lipid and iron metabolism in tumor cells.46, 47, 48

2.1.2. Regulation of tumor cell apoptosis

Accumulating evidence has demonstrated that circadian rhythms play a crucial role in regulating tumor cell apoptosis. Single-cell transcriptome analysis has revealed a negative correlation between CRD scores and cellular apoptosis levels.³¹ The circadian gene Per1 forms functional complexes with checkpoint proteins ATM and Chk2, and its overexpression significantly enhances the sensitivity of tumor cells to DNA damage-induced apoptosis.⁴⁹ The transcription factor NFIL3 exerts anti-apoptotic effects by transcriptionally suppressing the expression of apoptosis-related genes, including TRAIL, FGF7, FAS, and GADD45a, thereby promoting tumor progression.⁵⁰

2.1.3. Regulation of tumor cell metabolism

A substantial body of research has confirmed that circadian rhythm disruption can facilitate tumor growth by interfering with normal metabolism,⁵¹ while the disruption of central clock components contributes to metabolic dysregulation.⁵² Circadian rhythms play a crucial regulatory role in various key metabolic processes, particularly glycolysis and fatty acid metabolism pathways. Single-cell transcriptomic analysis reveals that CRD significantly upregulates the expression of key metabolic enzymes involved in fatty acid metabolism and glycolysis under hypoxic conditions, including G6PD, HK2, and ANXA2. Notably, HK2 and ANXA2 influence tumor growth and drug tolerance through metabolic reprogramming.³¹ The functional loss of BMAL1 can enhance glycolytic activity by upregulating the expression of hexokinase HKDC1 or driving APC heterozygous loss and activating the Wnt signaling pathway.^53,54 The expression levels of TIMELESS and RORα show significant correlations with multiple key lipid metabolism molecules in non-small cell lung cancer.⁵⁵ Research has found that TIMELESS promotes tumor cell proliferation by regulating sphingolipid metabolism through the Sp1/ACER2/S1P signaling axis.⁵⁶ RORα regulates lipid metabolism by suppressing the expression of lipogenesis-related genes, thereby inhibiting colorectal cancer cell proliferation.⁴⁷ Clock can promote tumor cell proliferation by regulating iron metabolism through time-dependent transcriptional promotion of iron regulatory protein 2 (IRP2).⁴⁸ Melatonin may inhibit tumor progression by suppressing the uptake and metabolism of linoleic acid.⁵⁷ Furthermore, circadian rhythms can regulate energy metabolism by modulating mitochondrial fission-fusion dynamics, where the circadian expression regulation of dynamin-related protein 1 (DRP1) can influence mitochondrial morphology and respiratory activity.⁵⁸ Notably, in triple-negative breast cancer, BMAL1 can inhibit the plasticity of mitochondrial substrate utilization, and its downregulation enhances cellular oxidative capacity for glutamine, fatty acids, and pyruvate.²³

2.1.4. Impact on cancer cell stemness

Cancer stem cells (CSCs), a distinct subpopulation of tumor cells, possess stem cell-like properties, including self-renewal capacity and multi-lineage differentiation potential, thereby playing a crucial role in tumor initiation, progression, and metastasis. High aldehyde dehydrogenase (ALDH) activity is widely recognized as a key marker for identifying and isolating cancer stem cells. Circadian rhythms have been shown to influence the stemness of cancer cells. Studies in mouse breast tumor models have demonstrated that the number of ALDH-high cells exhibiting stem cell-like properties exhibits significant circadian oscillations, which are primarily regulated by the rhythmic expression of Wnt 10a produced by ALDH-negative cells in the tumor microenvironment.⁵⁹ Core circadian clock genes, including CLOCK and Per2, are significantly suppressed in ALDH-high cells. Research has demonstrated that CLOCK negatively regulates ALDH activity and the mRNA expression levels of stemness-related transcription factors Klf4, Myc, and Nanog, and its upregulation leads to diminished cancer cell stemness characteristics.⁶⁰ Studies have demonstrated that overexpression of the clock gene PER3 in colorectal CSCs is negatively correlated with the expression levels of stemness markers LGR5, CD44, CD133, and SOX2, indicating the critical regulatory role of PER3 in modulating CSC stemness.⁶¹ The circadian clock gene Bmal1 contributes to tumor progression by modulating the expression of stem cell-related genes, and disruption of its expression leads to a gradual accumulation of cancer stem cells.⁶² Subsequent studies have demonstrated that the depletion of Bmal1 can facilitate the development of intestinal tumors by upregulating the Hippo signaling pathway.⁶³ Accumulating evidence has established that CRD can augment the stemness characteristics of primary tumor cells. In a breast cancer model subjected to chronic jet lag (JL) conditions, researchers detected a significant increase in the fraction of cancer stem cells, concomitant with elevated expression levels of stem cell markers.⁶⁴ The core circadian clock genes BMAL1 and CLOCK exert critical functions in modulating the energy metabolism of glioblastoma stem cells, particularly in maintaining the homeostasis of cellular oxidative phosphorylation and the tricarboxylic acid (TCA) cycle. Attenuation of these genes' expression diminishes the expression level of succinate dehydrogenase (SDH), resulting in the downregulation of key stemness regulatory factors MYC, OLIG2, and SOX2.⁶⁵

2.1.5. Impact of circadian rhythms on tumor cell metastatic potential

A growing body of evidence indicates that circadian rhythms play a pivotal role in regulating the metastatic potential of tumor cells. Research has consistently demonstrated that CTCs generated during the rest phase exhibit heightened metastatic potential compared to those produced during the active phase, a phenomenon largely attributed to the specific upregulation of mitosis-related genes during the rest period. The production kinetics of CTCs are intricately regulated by various circadian rhythm-associated hormones, including melatonin, testosterone, and glucocorticoids.⁶⁶ The circadian molecular clock system exerts a profound regulatory influence on tumor cell metastasis. Overexpression of NFIL3 has been demonstrated to promote epithelial-mesenchymal transition (EMT) in cancer cells,^67,68 while CLOCK has been found to exhibit tissue-specific regulatory effects on EMT in colorectal and breast cancer cells.⁶⁰ Recent studies have demonstrated that ectopic expression of Bmal1 promotes the transition of melanoma cells towards a mesenchymal phenotype,⁶⁹ while its deficiency results in a shift in the epithelial-mesenchymal balance towards epithelial characteristics.⁷⁰ Furthermore, BMAL1 promotes the metastatic process of colorectal cancer by modulating the expression levels of Rab27a, a key mediator in exosome secretion.⁷¹ CLOCK and BMAL1 enhance the migration and invasion capabilities of cancer cells via the RHOA-ROCK-CFL signaling pathway.⁷² Loss of TIMELESS expression induces the EMT process by upregulating ZEB1 expression.⁷³ PER2 inhibits the expression of EMT-related genes by recruiting a protein complex comprising EZH2, SUZ12, and HDAC2.⁷⁴ Decreased PER2 expression may activate the Snail/Slug axis by suppressing p53 activity, thereby promoting EMT pathway activation.⁷⁵ Deficiency in PRRX1, an effective EMT inducer, may promote the transition towards mesenchymal-epithelial transition through temporal inhibition of the TGFβ signaling pathway.⁷⁶ The circadian rhythm-related gene TP53I11 inhibits the EMT process in breast cancer cells through suppression of HIF1α expression.⁷⁷ Melatonin inhibits EMT by downregulating RSK2 expression and attenuates the invasive ability of breast cancer cells by suppressing the p38 MAPK signaling pathway.^78,79

2.1.6. Impact of circadian rhythms on tumor cell immunogenicity

Accumulation of genetic alterations in tumor cells leads to enhanced antigenicity, a key factor in tumor immunogenicity. Deficiencies in DNA mismatch repair (MMR) mechanisms result in substantial accumulation of microsatellite instability (MSI) and elevated tumor mutational burden (TMB) levels, both of which are crucial indicators of tumor immunogenicity.^16,33 Multiple studies have demonstrated that elevated TMB and MSI levels are positively associated with significantly improved clinical outcomes in patients receiving ICI therapy.^80,81 In several cancer types, including head and neck squamous cell carcinoma, kidney renal clear cell carcinoma, acute myeloid leukemia, sarcoma, and skin cutaneous melanoma, the expression of specific circadian rhythm genes exhibits a significant positive correlation with TMB. Conversely, other cancer types demonstrate a significant negative correlation.⁸² For example, in non-small cell lung cancer, the expression of the circadian rhythm gene TIMELESS demonstrates a significant positive correlation with TMB, whereas RORA expression shows a significant negative correlation.⁵⁵ Analysis of diverse human cancer samples suggests that high expression indices of core circadian clock genes (BMAL1, PER1/2/3, CLOCK) are negatively correlated with the downregulation of MMR pathway activity. This correlation may indicate a decreased frequency of mismatch repair events, although this hypothesis necessitates further validation.⁸³ It is important to emphasize that the precise molecular mechanisms through which circadian rhythm hormones regulate MMR, MSI, and TMB processes remain to be elucidated and warrant further in-depth investigation.

2.2. Impact of circadian rhythms on immune cell function and dynamics

Accumulating evidence indicates that circadian rhythms profoundly influence the activities of diverse immune cells within the TME, modulating multiple aspects including their quantity, function, migration, and infiltration (Fig. 1 and Table 2). Consequently, these rhythms play a pivotal role in critical processes such as immune defense and immunosurveillance.

Fig. 1.

Circadian rhythm effects on immune cell function. Circadian rhythms regulate the abundance and functional activity of immune cells, including neutrophils, DCs, CD8⁺ and CD4⁺ T cells, macrophages, NK cells, and B cells. These rhythms also govern immune cell migration via CXCL/CXCR signaling pathways, CCL chemokines, and adhesion molecules within the tumor microenvironment. Circadian hormones such as glucocorticoids and adrenaline further modulate immune cell trafficking by influencing chemokine and receptor expression. CCL, C-C motif chemokine ligand; CRD, circadian rhythm disruption; CRY, cryptochrome; CXCL, C-X-C motif chemokine ligand; CXCR, C-X-C motif chemokine receptor; CTLA-4, cytotoxic T-lymphocyte-associated protein 4; DCs, dendritic cells; GSC, glioma stem cell; ICAM-1, intercellular adhesion molecule-1; IFN-γ, interferon γ; IL, interleukin; LEC, lymphatic endothelial cell; MDSC, myeloid-derived suppressor cell; NK cells, natural killer cells; PD-L1, programmed death-ligand 1; PER, period; ROR, retinoid‐related orphan receptor; TNF-α, tumor necrosis factor α; VCAM-1, vascular cell adhesion molecule-1; VEC, vascular endothelial cell; WBC, white blood cell.

Table 2.

Circadian characteristics of key immune cell populations in the TME.

Immune cell type	Circadian feature	Key regulators	Functional impact	References
CD8+ T cells	Cytotoxic activity and infiltration rhythm	BMAL1, RORα, Cortisol	Enhanced tumor killing capacity	30, 91, 93, 94, 105, 138
CD4+ T cells	Th1/Th2/Th17/ Tregs balance oscillation	NFIL3, Per1, RORγ, circadian hormone	Immune response polarization	85, 86, 108, 111, 112, 113, 114, 115
NK cells	Cytotoxicity rhythm	Circadian gene expression	Natural tumor surveillance	116, 117, 118
Macrophages	Phagocytic activity, cytokine secretion, and metabolic rhythm	BMAL1, Per2, CRY, REV-ERBα	Anti-tumor capacity regulation	95, 96, 97, 98, 99, 100, 101, 102, 103
Dendritic cells	Migration and activation rhythm	REV-ERBα, REV-ERBβ	Antigen presentation efficiency	121, 122
Neutrophils	Circadian function and aging	BMAL1	Inflammatory response timing	29, 119, 120
B cells	Differentiation and function rhythm	BMAL1, CLOCK	Humoral immunity	104, 123, 124, 125

Abbreviations: CRY, cryptochrome; NK cells, natural killer cells; Per1, period 1; RORα, retinoid‐related orphan receptor α; TME, tumor microenvironment; Tregs, regulatory T cells.

2.2.1. Regulation of immune cell quantities

The circadian system plays a pivotal role in regulating the maintenance of immune cell quantities within the TME. Circadian rhythm disruption can substantially impact the quantitative balance of various immune cells in the body. Alterations in circadian rhythms can result in a reduction in the number of certain immune cells within the TME. For instance, sleep deprivation can induce a significant decrease in the number of natural killer cells (NK cells),³⁴ monocytes, and various lymphocyte subsets.⁸⁴ Multiple circadian genes play crucial roles in the dynamic regulation of immune cell quantities. For example, the rhythm gene Arntl2 inhibits Th17 cell proliferation through suppression of the interleukin (IL)-21 signaling pathway,⁸⁵ while NFIL3 can reduce the number of regulatory T cells (Tregs).⁸⁶ However, alterations in circadian rhythms can also positively modulate the quantities of certain immune cells. Studies have demonstrated that in chronic JL mouse models, there is a significant increase in the number of senescence-associated immune cells in the spleen and mesenteric lymph nodes, including PD-1⁺CD44⁺CD4⁺ T cells, PD-1⁺CD44⁺CD153⁺CD4⁺ T cells, follicular helper T cells, and regulatory T cells.⁸⁷ CRD has been demonstrated to induce an increase in the number of M2-type macrophages, regulatory T cells, and myeloid-derived suppressor cells (MDSCs).^88,89 Deficiencies in clock genes Cry1 and Cry2 have been shown to lead to an increase in the number of activated CD3⁺CD69⁺ T cells.⁹⁰ Studies have demonstrated that the generation of tumor antigen-specific CD8⁺ T cells in draining lymph nodes exhibits significant circadian rhythmicity.⁹¹ Similarly, the number of regulatory T cells in arthritis models also exhibits notable circadian fluctuations.⁹² Research has revealed that dendritic cell-ovalbumin immunization at midday induces a stronger T cell proliferative response; however, this time-dependent response is completely abolished in Bmal1-deficient DCs or CD8 T cell-specific Bmal1 knockout mice.⁹³ The circadian rhythmicity of T cell proliferation is likely closely related to the regulation of proximal T cell receptor (TCR) signaling pathways by the biological clock.⁹⁴

2.2.2. Effects on immune cell functions

Monocytes and macrophages

Extensive research has established that circadian rhythms modulate the biological functions of monocytes and macrophages by regulating several key biological processes, including phagocytic activity, cytokine secretion, TLR signaling pathways, and immune metabolic reprogramming.95, 96, 97 Investigations have demonstrated that the phagocytic activity of ex vivo macrophages displays significant circadian oscillations.⁹⁸ Further studies have elucidated that BMAL1 influences macrophage phagocytic function by regulating RhoA-dependent macrophage cytoskeleton remodeling.⁹⁵ However, the rhythmic production of cytokines by macrophages is governed by distinct circadian mechanisms and does not depend on the circadian oscillations of phagocytic activity.⁹⁸ Experimental data reveal that sleep deprivation and the absence of clock genes Per2 and CRY lead to a significant increase in tumor necrosis factor (TNF)-α expression levels in monocytes and macrophages.99, 100, 101 Additionally, BMAL1 knockout results in increased IL-1β secretion in macrophages.⁹⁶ Furthermore, REV-ERBα exerts an inhibitory effect on IL-6 production in macrophages.¹⁰² Notably, macrophage Toll-like receptor (TLR) signaling pathways are subject to precise regulation by the biological clock.¹⁰⁰ In-depth research has uncovered that Per2 functional deficiency regulates the production of TLR9-dependent cytokines in macrophages by altering TLR9 expression levels.¹⁰⁰ Systematic analysis of macrophage transcriptomes and proteomes has unveiled extensive post-transcriptional regulatory mechanisms through which circadian rhythms influence macrophage immunometabolic states.⁹⁷ Under lipopolysaccharide stimulation, Bmal1 deficiency results in impaired macrophage mitochondrial function, augmenting Hif-1α-dependent metabolic reprogramming and inflammatory responses, consequently promoting the formation of an immunosuppressive TME.¹⁰³

T cells

Circadian rhythms are intricately linked to diverse T cell functions. Research has demonstrated that CD8⁺ T cells possess an endogenous cell-autonomous circadian clock system,⁹³ enabling their immune responses to antigen presentation in vivo to display significant circadian variations. Conversely, certain studies have indicated that the cell-intrinsic molecular clock does not exert a decisive influence on T cell development and immune responses. Specifically, the absence of the core clock gene BMAL1 neither affects T cell differentiation and function nor can be compensated for by CLOCK expression. Rather, the functional regulation of T cells appears to primarily depend on the modulatory effects of antigen-presenting cells and the influence of extrinsic circadian signals (including cytokines and hormones).¹⁰⁴ The periodic activation process of CD8⁺ T cells is orchestrated by the rhythmic migration of dendritic cells (DCs) and the cyclic production of their co-stimulatory molecule CD80. Subsequent studies have revealed that CD80 expression is regulated by the transcriptional control of the core clock gene BMAL1.⁹¹ The nuclear receptor RORα modulates the cholesterol metabolism network of CD8⁺ T cells by inhibiting the transcriptional activity of NF-κB, thus influencing their cytotoxic effector functions.^105,106 Pan-cancer genomic analyses have elucidated that functional dysregulation of circadian genes leads to aberrant activation of immune-related pathways, alterations in immune cell infiltration patterns, and systemic upregulation of immune checkpoint molecules (such as CTLA-4 and PD-L1), phenomena closely associated with T cell exhaustion.¹⁰⁷ Studies have demonstrated that the circadian rhythm system plays a pivotal role in regulating the balance of CD4⁺ T cell subsets, specifically in the dynamic regulation of Th1/Th2 and Th17/Treg ratios.¹⁰⁸ NFIL3, a crucial regulatory factor, not only promotes T cell polarization towards the Th1 phenotype and induces IL-13 secretion but also stimulates Th2 cells to produce cytokines including IL-4 and IL-10.^86,109,110 Furthermore, NFIL3 orchestrates circadian regulation of Th17 subset differentiation through REV-ERBα-mediated transcriptional repression and modulation of RORγ transcriptional activity.¹¹¹ Per1 facilitates the conversion of T helper cell subsets to the Th2 phenotype by negatively regulating the Akt/mTORC1 signaling pathway.¹¹² Research has established that RORγ is an essential transcription factor in regulating the fate determination of CD4⁺ T cells towards Th17 cells.¹¹³ CD4⁺ T cells harbor an endogenous circadian clock system that regulates the periodic expression of interferon(IFN)-γ and CD40L through the NF-κB signaling pathway.¹¹⁴ Moreover, endocrine hormones and sleep play crucial roles in regulating the Th1/Th2 balance. Epinephrine and glucocorticoids can inhibit Th1 cell differentiation by suppressing mTORC1 signaling through modulation of the PI3K/AKT pathway or influencing PER1 expression levels.¹¹² The early stages of sleep promote Th1 responses, while the later stages shift towards a Th2-dominant state, and this dynamic change is likely closely related to the rhythmic release of prolactin and growth hormone.¹¹⁵

NK cells

NK cells demonstrate significant circadian secretion patterns of cytokines and cytolytic factors.¹¹⁶ Chronic long-term circadian rhythm disruption significantly alters the circadian-coordinated expression of IFN-γ, perforin, and granzyme B in NK cells, leading to a substantial reduction in their peak lytic activity.¹¹⁶ Deficiency of the transcription factor NFIL3 results in significant impairment of NK cells' ability to produce IFN-γ and their overall functionality.¹¹⁷ CRD promotes NK cell apoptosis and senescence while simultaneously altering the NK cell receptor expression profile, characterized by decreased expression of functional receptors (Ly49H, Ly49G2, and Ly49D) and increased expression of immature receptors (CD127 and CD117).¹¹⁸

Neutrophils

Circadian clock disruption and Wnt signaling pathway activation synergistically induce the transformation of neutrophils toward an immunosuppressive phenotype.²⁹ The core clock protein BMAL1 specifically binds to the Cxcl2 promoter, thereby controlling neutrophil aging through a CXCR2-dependent signaling pathway.¹¹⁹ Circadian rhythm disruption mimics chronic stress, which can promote glucocorticoid release, subsequently inducing neutrophil extracellular trap formation and accelerating the circadian aging process of neutrophils.¹²⁰

Dendritic cells

Circadian oscillations in mitochondrial calcium (Ca²⁺) levels of DCs significantly modulate their antigen presentation capacity.¹²¹ The nuclear receptor REV-ERB exerts an inhibitory influence on DC development and activation through transcriptional repression. In REV-ERBα and REV-ERBβ gene knockout mice, bone marrow-derived DCs demonstrate significantly elevated expression of pro-inflammatory cytokines and maturation markers, including CD86 and MHCII.¹²²

B cells

Current evidence suggests that B cell differentiation and functional regulation operate independently of the cell-autonomous circadian clock system.¹⁰⁴ The compromised B cell development observed in BMAL1-deficient mice is likely attributed to alterations in microenvironmental regulatory factors.¹²³ Disruption of circadian rhythms results in diminished immunosuppressive capacity of IL-10-secreting B cells and impairs the generation of type 1 regulatory T cells. Mechanistic investigations demonstrate that elevated CLOCK expression in IL-10-secreting B cells enhances CMIP protein degradation, consequently suppressing IL-10 expression.¹²⁴ BMAL1 governs PDL1-positive regulatory B cells through modulation of IL-33 transcription. Research has revealed that dysfunctional PDL1-positive B cells in Bmal1 knockout mice can trigger programmed cell death in activated CD4⁺ T cells.¹²⁵

2.2.3. Influence on immune cell migration and infiltration

Circadian regulatory mechanisms of immune cell migration

Immune cell migration demonstrates pronounced circadian characteristics, predominantly regulated by the periodic expression of chemokines and adhesion molecules. The periodic expression patterns of cell migration-related factors, encompassing chemokines and adhesion molecules, govern the rhythmic migration of leukocytes to target tissues,^126,127 a process contingent upon the circadian function of endothelial cells.¹²⁸ The circadian system orchestrates the migration process of immune cells through precise regulation of the spatiotemporal expression patterns of diverse chemokines within the microenvironment. The rhythmic expression of the C-X-C motif chemokine ligand (CXCL)/ C-X-C motif chemokine receptor (CXCR) signaling pathway modulates immune cell migration. Research has demonstrated that peripheral circulating leukocytes in mice and humans display antithetical circadian fluctuations, a species-specific difference intimately associated with the distinct circadian expression patterns of the chemokine receptor CXCR4. The core clock gene ARNTL1 plays a pivotal role in orchestrating this process.¹²⁶ CRD can modulate the migratory capacity of immunosuppressive cells, such as MDSCs, by perturbing the circadian rhythmicity of the CXCL5-CXCR2 and CXCL12-CXCR4 signaling pathways.⁶⁴ Recent studies have revealed that the expression of chemokine CXCL5 exhibits a distinct rhythmicity, with the occupancy rate of glucocorticoid receptors in its promoter region also demonstrating circadian oscillation patterns. Although the absence of BMAL1 in bronchiolar cells disrupts both oscillation patterns, the persistent presence of rhythmic pulmonary neutrophilia indicates the potential existence of other circadian regulatory mechanisms.^129,130 Furthermore, the biological clock influences immune cell migration through the rhythmic regulation of C-C motif chemokine ligand (CCL) family chemokines. For instance, the periodic expression of chemokine CCL21 and adhesion molecules orchestrates the circadian pattern of DC migration to draining lymph nodes.¹²⁷ The nuclear receptor REV-ERBα cooperates with the clock protein complex CLOCK/BMAL1 to co-regulate the circadian expression of CCL2.^131,132 The clock protein BMAL1 regulates the circadian migration rhythm of Ly6Chi monocytes by inhibiting the transcriptional expression of chemokines CCL2, CCL8, and S100A8, whereby the absence of BMAL1 results in the disappearance of this migration rhythm.¹³² The clock gene BMAL1 in lymphocytes governs the rhythmic tissue homing and circulation migration of lymphocytes by modulating the rhythmic expression of chemokine receptor CCR7 and sphingosine-1-phosphate receptor 1 (S1pr1).¹³³ Accumulating evidence has confirmed that the circadian system regulates the expression patterns of stimulus-responsive chemokines secreted by macrophages.¹⁰⁰

Notably, circadian rhythm molecules can precisely modulate the expression levels of endothelial cell adhesion molecules, consequently influencing immune cell migration. The core clock gene BMAL1 modulates the circadian expression of multiple adhesion molecules in lymphatic endothelial cells, including the lymphatic endothelial hyaluronan receptor LYVE-1 and junctional adhesion molecule JAM-A, thus mediating the periodic migration of DCs.¹²⁷ Studies have demonstrated that the expression of BMAL1 in endothelial cells plays a crucial role in maintaining the circadian expression patterns of intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1).^128,133,134 The clock protein Clock facilitates the adhesive interactions between monocytes and vascular endothelial cells by modulating the expression levels of ICAM-1 in endothelial cells.¹³⁵ The migration of dendritic cells from skin tissue to draining lymph nodes demonstrates significant time dependence, a phenomenon attributed to the circadian expression of TNF augmenting BMAL1-regulated ICAM-1 expression in high endothelial venules.¹³⁶ Research has revealed that sleep deprivation suppresses the expression of the clock gene CRY1 in endothelial cells, consequently upregulating the expression of various cell adhesion molecules, including ICAM-1, VCAM-1, and E-selectin, thereby promoting the adhesion of monocytes to vascular endothelial cells.¹³⁷

With respect to the endocrine system, hormones exert a crucial regulatory role in the circadian migration of immune cells. Specifically, cortisol and epinephrine demonstrate antagonistic effects on the circadian rhythms of different T cell subsets: naive T cell counts exhibit a negative correlation with cortisol rhythms, while effector CD8⁺ T cell levels display a positive correlation with epinephrine rhythms.¹³⁸ Mechanistic studies have elucidated that cortisol facilitates T cell migration to the bone marrow¹³⁸ and promotes tissue extravasation of naive T cells in the morning¹³⁹ through regulating the expression levels of CXCR4 on T cell surfaces. Further research has uncovered that the glucocorticoid receptor signaling pathway sustains the circadian oscillation of T cell distribution and immune responses by orchestrating the diurnal expression pattern of IL-7 receptors in mouse T cells, which subsequently regulates the expression of the chemokine receptor CXCR4.¹⁴⁰ Moreover, studies have demonstrated that the adrenergic nervous system mediates the circadian recruitment process of leukocytes through regulating the rhythmic expression of chemokines and adhesion molecules.¹³⁴

Circadian regulatory mechanisms of immune cell infiltration

Studies have revealed that leukocyte infiltration in tumor tissues exhibits significant circadian rhythmic fluctuations, with the number of tumor-infiltrating lymphocytes (TILs) reaching its peak at night and the quantity of infiltrating T cells being significantly higher during nighttime compared to daytime levels.³⁰ The time-dependent process of leukocyte infiltration into tissues is primarily governed by the expression of diverse pro-migratory factor subgroups and the modulation of corresponding tissue-specific rhythms.^131,141 Circadian rhythms demonstrate a strong association with immune cell infiltration in the TME. Circadian rhythm scores show a significant inverse correlation with the degree of immune cell infiltration in the TME and patient prognosis. Low CR scores typically suggest the presence of an immunosuppressive microenvironment and are predictive of poor response to immune checkpoint blockade therapy.¹⁴² A predictive model of circadian rhythm-related genes for breast cancer, developed based on The Cancer Genome Atlas data, demonstrates that high-risk patient populations characteristically display significantly reduced infiltration levels of CD8⁺ and CD4⁺ T cells, concomitant with a marked increase in the proportion of immunosuppressive cells, notably M0 and M2 macrophages.^22,143 Recent studies have demonstrated that high infiltration of CD8⁺ T cells in tumor tissues is significantly correlated with low expression levels of circadian rhythm proteins.¹⁴⁴ Different circadian rhythm genes have been found to exhibit significant positive or negative correlations with TIL levels. Specifically, expression levels of TIMELESS, ARNTL2, and ARNTL have been observed to show positive correlations with TIL levels in various cancers, while the expression of period genes PER2, PER3, and PER1 has been found to demonstrate negative correlations.^46,82 The core clock gene CLOCK has been shown to promote microglia tissue infiltration by regulating the expression of the microglia-attracting chemokine OLFML3.¹⁴¹ The core clock protein Rev-erbα has been demonstrated to modulate macrophage tissue infiltration by suppressing the expression level of the chemokine CCL2.¹³¹ Furthermore, it is noteworthy that somatic copy number variations in circadian rhythm genes have been observed to frequently lead to significant inhibition of immune cell infiltration capacity.¹⁴⁵

2.3. Impact of circadian rhythm on tumor angiogenesis

Circadian rhythm, functioning as an intrinsic clock in the human body, exerts a significant influence on tumor angiogenesis. Based on key circadian genes such as NPAS2, NR1D1, PER1, RORC, and TIMELESS, researchers have constructed mRNA expression profiles, revealing that angiogenesis-related pathways are significantly enriched in low-risk groups.¹⁴⁶ Single-cell transcriptome analysis has revealed a significant positive correlation between CRD scores and angiogenic processes.³¹ These findings suggest a close functional connection between circadian regulatory mechanisms and tumor angiogenesis processes. In-depth mechanistic studies have demonstrated that in glioblastoma, the CLOCK-BMAL1 transcription factor complex upregulates the expression of the angiogenic promoter POSTIN via the OLFML3-HIF1α signaling pathway, thereby inducing TBK1-dependent tumor angiogenesis in endothelial cells.¹⁴⁷ BMAL1 promotes angiogenesis by binding to the promoter region of the vascular endothelial growth factor (VEGF) gene for transcriptional regulation, whereas PER2 exerts a negative regulatory effect.²⁴ However, recent studies have shown that although BMAL1 can bind to the VEGFA promoter region, its regulatory effect on VEGFA expression is relatively weak and insufficient to drive the angiogenesis process. Further research has elucidated that BMAL1 primarily regulates endothelial cell cycle activity by modulating the expression of cyclin A1 and cyclin-dependent kinase 1, ultimately influencing angiogenesis.¹⁴⁸

2.4. The influence of circadian rhythms on cancer-associated fibroblasts

Cancer-associated fibroblasts (CAFs), a crucial component of the TME, exhibit a dual role in modulating tumor immune responses and progression. In vitro studies have demonstrated that co-culturing colon cancer cells with CAFs significantly alters the circadian characteristics of tumor cells, accompanied by metabolic reprogramming and enhanced malignant phenotypes.^149,150 The expression level of the clock gene TIMELESS exhibits a positive correlation with CAF infiltration in adrenocortical carcinoma, esophageal cancer, head and neck squamous cell carcinoma, low-grade glioma, mesothelioma, and uveal melanoma. Conversely, it demonstrates a negative correlation in testicular germ cell tumors and stomach adenocarcinoma.¹⁵¹ Expression of the clock gene Per2 enhances CAF infiltration in colon cancer tissues, whereas Per2 deficiency significantly attenuates CAF infiltration levels.¹⁵² Deficiency of the core clock gene Bmal1 suppresses the expression of plasminogen activator inhibitor-1, consequently facilitating the phenotypic transformation of CAFs to myofibroblast-like CAFs via activation of the transforming growth factor β (TGF-β) signaling pathway, ultimately expediting tumor metastasis.¹⁵³

2.5. Non-cellular components in circadian tumor immunomodulation

Non-cellular components play pivotal roles in circadian tumor immunomodulation, primarily encompassing metabolites, soluble factors, and the extracellular matrix. The synthesis, secretion, and function of these components are collectively regulated by circadian clocks, thereby influencing immune dynamics within the TME and promoting tumor progression through circadian mechanisms, including metabolite balance regulation, rhythmic cytokine secretion, and extracellular matrix remodeling.

2.5.1. Metabolites

A close relationship exists between circadian rhythm disruption and tumor immune metabolism, whereby metabolic pathways regulated by the biological clock, such as glycolysis, oxidative phosphorylation, and amino acid metabolism, are crucial for regulating immune cell function within the TME.¹⁵⁴ Studies demonstrate that Bmal1 serves as a metabolic checkpoint in macrophages, maintaining the balance between oxidative and glycolytic metabolism through the Bmal1-Hif-1α axis under inflammatory stimulation, whereas Bmal1 knockout leads to metabolic dysregulation and inflammatory damage, resulting in TME immunosuppression.¹⁰³ Furthermore, Bmal1 mediates the production of the pro-inflammatory factor IL-1β through two metabolic pathways: regulating metabolite succinate generation and modulating the levels and distribution of the glycolytic enzyme PKM2.¹⁵⁵ In prostate cancer, the circadian clock gene NPAS2 is upregulated, promoting cell survival by upregulating HIF-1A expression to enhance glycolysis and inhibit oxidative phosphorylation. Following NPAS2 knockdown, both glucose uptake and lactate production are reduced, which inhibits glucose metabolism while increasing oxygen consumption and pH, thereby suppressing tumor growth.¹⁵⁶ Another study reveals that PD-1-resistant cancer cells upregulate Fabp7, thereby reprogramming lipid metabolism and disrupting clock gene oscillations in CD8⁺ T cells to evade immune cell-mediated ferroptosis.¹⁵⁷

2.5.2. Soluble factors

Soluble factors, including cytokines, chemokines, growth factors, and hormones, exhibit secretion levels that are regulated by circadian rhythms and represent critical components in tumor immune regulation. Circadian rhythmicity can alter the functional networks of key cytokines and chemokines within the tumor microenvironment.⁶⁴ Circadian rhythm disruption significantly increases the levels of TNF-α and IL-10.^158,159 The circadian regulatory factor NFIL3 enhances the production of cytokines such as IL-4, IL-10, and IL-13 in Th2 cells.^109,110 The expression of chemokines CCL2 and CCL8 in monocytes is regulated by the inhibitory action of BMAL1.¹³² Additionally, VEGF gene transcription is regulated by the circadian rhythm genes BMAL1 and PER2, wherein BMAL1 promotes VEGF gene transcription while PER2 exerts negative regulatory effects.²⁴ Furthermore, hormone levels, including glucocorticoids and cortisol, are also subject to circadian rhythm regulation.^120,158 For instance, chronic stress promotes glucocorticoid release, disrupts neutrophil circadian rhythmic activity, and promotes their senescent phenotype, thereby leading to increased neutrophil extracellular trap formation.¹²⁰ Moreover, circadian rhythm disruption can alter plasma cortisol levels.¹⁵⁸

2.5.3. Extracellular matrix

The extracellular matrix (ECM) within the TME plays a pivotal role in tumor progression by providing structural support, regulating cellular signaling, and forming physical barriers; ECM remodeling can modulate tumor cell proliferation, migration, and invasion, while simultaneously promoting tumor immune evasion and attenuating therapeutic responses.¹⁶⁰ Circadian rhythms govern the expression patterns of ECM-related genes, influence the rhythmic assembly and degradation of collagen, thereby participating in ECM synthesis and remodeling.^{152,153,161,162} Fibroblasts represent key cell types responsible for constructing and maintaining the extracellular matrix, with their protein synthesis and secretory activities being regulated by circadian rhythms.¹⁶¹ Studies have demonstrated that Bmal1 deficiency reduces plasminogen activator inhibitor-1 expression in the TME, subsequently enhancing fibrinolytic activity and activating TGF-β signaling, thereby inducing increased tumor fibrotic components and myoCAF phenotypes.¹⁵³ A subsequent study demonstrated that wild-type mice with colorectal cancer liver metastases exhibited significant ECM remodeling, while Per2-/- mice showed reduced collagen deposition and CAF infiltration compared to wild-type mice, indicating that PER2 plays a crucial role in promoting a tumor-suppressive TME.¹⁵² Furthermore, researchers have identified a circadian-regulated lncRNA called ADIRF-AS1 that exhibits oncogenic properties in clear cell renal carcinoma. ADIRF-AS1 is regulated by the BMAL1-CLOCK complex, and its oncogenic effects are partially ECM-dependent, stemming from its regulation of ECM-related gene NID1 (also known as entactin) expression.¹⁶²

3. Regulatory role of circadian rhythm in the interaction between TME and ICIs

3.1. Regulation of immune checkpoint molecule expression by circadian rhythm

Pan-cancer analyses have revealed that circadian disruption can lead to the systematic upregulation of key immune inhibitory molecules, including CTLA-4 and PD-L1, a phenomenon closely associated with T cell exhaustion.¹⁰⁷ Clinical studies have demonstrated that ovarian cancer patients engaged in night shift work display significantly upregulated expression levels of PD-1 and PD-L1.¹⁶³ Research has indicated that elevated expression levels of clock genes are significantly and positively correlated with the expression levels of T cell dysfunction markers, including PD-1 and CTLA-4.^55,164 The expression level of the clock gene ARNTL2 demonstrates a significant positive correlation with the expression of multiple immune inhibitory checkpoint molecules, including PD-1, PD-L1, LAG3, CTLA4, and TIGIT.⁸⁸ In breast cancer cells, overexpression of the circadian rhythm-related gene NDRG2 has been shown to reduce PD-L1 expression levels through inhibiting the activity of the NF-κB signaling pathway.¹⁶⁵ The clock gene TIMELESS has been demonstrated to enhance the transcriptional activity of the transcription factor c-Myc, consequently upregulating the expression of PD-L1.¹⁶⁶ The nuclear receptor RORA has been shown to significantly inhibit the expression of PD-L1,¹⁶⁷ while in endometrial cancer Ishikawa cells, overexpression of the clock gene PER1 has been found to promote the expression of the PD-1/PD-L1 signaling pathway.¹⁶⁸ Research has demonstrated that deficiency of the clock gene Bmal1 in macrophages results in increased expression of PKM2, which subsequently promotes PD-L1 expression in a STAT1-dependent manner.¹⁶⁹ Importantly, the expression of PD-L1 demonstrates significant time dependency and is regulated directly and precisely by the circadian system.¹⁰⁷ The mRNA levels of PD-1 and Pdcd1 genes in TAMs exhibit circadian rhythmic fluctuations. Likewise, Pdcd1 expression in CD8⁺ T cells displays significant circadian characteristics at both mRNA transcription and cell surface protein levels.^30,144 The expression of PD-1 on the surface of TAMs demonstrates significant circadian rhythmicity. Mechanistic investigations have elucidated that this rhythmic expression is mediated through time-dependent inhibition of p65-mediated transactivation by the clock gene DEC2 (Table 3).¹⁷⁰

Table 3.

Circadian regulation of immune checkpoint molecules.

Checkpoint molecule	Circadian regulator	Regulatory mechanism	Cell type	Therapeutic implication	References
PD-1	ARNTL2, DEC2	Transcriptional regulation	T cells, TAMs	Timing optimization for anti-PD-1 therapy	88, 170
PD-L1	TIMELESS, PER1, RORA, Bmal1, NDRG2	Transcriptional activation/ suppression	Tumor cells, APCs	Chronotherapy scheduling	164, 165, 166, 167, 168, 169
CTLA-4	Circadian disruption	Systematic upregulation	T cells, Tregs	Biomarker for T cell dysfunction	107, 164, 219
LAG-3	ARNTL2	Positive correlation	T cells, Tregs	Biomarker for T cell dysfunction	9, 88, 220
TIGIT	ARNTL2	Positive correlation	T cells, NK cells	Combined checkpoint blockade timing	88, 20

Abbreviations: APCs, antigen-presenting cells; CTLA-4, cytotoxic T lymphocyte-associated antigen-4; NK, natural killer; PD-1, programmed cell death-1; PD-L1, programmed death ligand 1; PER1, period 1; RORA, retinoid‐related orphan receptor α; TAMs, tumor-associated macrophages; TIGIT, T cell immunoreceptor with Ig and ITIM domains; Tregs, regulatory T cells.

3.2. Time-dependent regulation of immune cell functions by circadian rhythms

The biological circadian system orchestrates precise time-dependent regulatory effects on the functional performance of immune cells. Accumulating evidence has demonstrated that the endogenous circadian clock systems in DCs and T lymphocytes govern their circadian antitumor immune functions. The core circadian regulator BMAL1 orchestrates the circadian antitumor immune function of CD8⁺ T cells by regulating the transcriptional expression of the costimulatory molecule CD80 and coordinating the periodic migration of DCs to lymph nodes. This finding implies that synchronizing the timing of cancer immunotherapy administration with the optimal phase of DC function may significantly enhance therapeutic efficacy.⁹¹ The circadian system modulates DC antigen processing and presentation functions, as well as the T cell immune responses they mediate, by regulating the circadian dynamics of mitochondrial Ca²⁺ in dendritic cells.¹²¹ Emerging research has confirmed that the functional state of T cells in the TME exhibits significant circadian variations: CD8⁺ T cells predominantly display an immunosuppressive phenotype in the morning, while demonstrating enhanced antitumor effector functions at night. Specifically, the expression of Pdcd1, the gene encoding PD-1, in CD8⁺ T cells exhibits pronounced circadian oscillations, peaking at zeitgeber time 1 (ZT1) and reaching its nadir at ZT13, elucidating a molecular mechanistic explanation for the time-dependent effects of anti-PD-1 immunotherapy.³⁰ Additionally, the core circadian gene Per2 orchestrates the expression of TLR9, and functional deficiency of the Per2 gene results in the ablation of rhythmic TLR9 expression in macrophages, thereby perturbing the time-dependent characteristics of immune responses.¹⁰⁰

3.3. Circadian regulation of non-immune components and non-cellular TME factors in modulating ICI efficacy

Circadian rhythm disruption modulates non-immune components and non-cellular TME factors through multiple mechanisms, including the alteration of CAF functional states,¹⁵³ modification of cytokine or hormone levels,⁶⁴ and regulation of metabolic reprogramming within the TME,¹⁷¹ thereby inducing an immunosuppressive microenvironment that subsequently impacts ICI therapeutic efficacy. The loss of the core clock gene Bmal1 suppresses the expression of plasminogen activator inhibitor-1, thereby promoting CAF transformation toward a myofibroblast phenotype and ultimately accelerating tumor metastasis.¹⁵³ TGF-β signaling within the tumor microenvironment is closely associated with tumor fibrosis and plays a crucial role in ICI resistance.^172,173 Studies demonstrate that activation of REV-ERBα impedes TGF-β signaling in the liver and potentially slows fibrosis progression.¹⁷⁴ Melatonin is a key circadian rhythm hormone that plays a critical role in anti-tumor immunity. Melatonin downregulates PD-L1 expression on hepatocellular carcinoma cells to inhibit immune escape, with mechanisms potentially involving upstream reduction of HIF-1α expression or activation of JNK and P38 pathways.¹⁷⁵ The loss of the core circadian clock gene Bmal1 causes mitochondrial dysfunction, leading to abnormal activation of hypoxia-inducible factor-1α and metabolic dysregulation, ultimately promoting the formation of an immunosuppressive TME that impacts immunotherapy efficacy.¹⁰³

4. Optimization strategies for ICIs based on circadian rhythms and TME

Despite significant breakthroughs in cancer immunotherapy with ICIs, substantial clinical data demonstrate that the response rate to ICI treatment remains suboptimal for the majority of patients.^26,81,176, 177, 178, 179, 180, 181, 182 Consequently, optimizing ICI treatment strategies has emerged as a paramount scientific challenge in the field of cancer immunotherapy. Recent studies have elucidated that ICI optimization strategies based on biological circadian rhythm regulation and TME remodeling exhibit significant potential for enhancing treatment efficacy. These multidimensional optimization strategies encompass: (1) circadian phase-dependent dosing regimens; (2) combination therapies targeting key components of the TME; (3) biological rhythm regulation interventions; and (4) precision treatment strategies based on individual patient characteristics (Fig. 2). These strategies, through their multifaceted synergistic effects, offer novel research directions and therapeutic approaches for optimizing the clinical application of ICIs and enhancing cancer immunotherapy response rates. However, the clinical translational value and long-term efficacy of these innovative approaches necessitate validation through additional prospective clinical studies.

Fig. 2.

Circadian-based optimization of ICI therapy. ICI therapy can be optimized by leveraging circadian rhythms and tumor microenvironment characteristics through four main strategies: (1) Optimizing the timing of ICI administration to the early morning or active phases based on circadian patterns; (2) Combining therapies targeting key immune cells, receptors, and cytokines in the tumor microenvironment; (3) Implementing chronobiological interventions including small molecule agonists (RORγ, REV-ERBα/β, CRY), hormone modulation, and non-pharmacological approaches; and (4) Personalizing treatment based on individual circadian profiles. CRY, cryptochrome; ICI, immune checkpoint inhibitor; MDSC, myeloid-derived suppressor cell; RORγ, retinoid‐related orphan receptor γ; TAM, tumor-associated macrophage; TME, tumor microenvironment.

4.1. Impact of circadian rhythms on the pharmacokinetics of ICIs

The circadian rhythm system exerts a profound influence on the pharmacokinetic processes of ICIs, encompassing key aspects such as drug release, absorption, metabolism, clearance, and excretion.¹⁸³ The timing of drug administration plays a crucial role in determining pharmacokinetic parameters, including the area under the curve, clearance rate, peak concentration, time to peak concentration, and half-life.¹⁸⁴ The clearance rate of monoclonal antibodies exhibits temporal variations.^185,186 The distribution of drugs within the body is governed by multiple factors, including blood flow rates in organs or tissues, expression of drug transporters, activity of efflux pumps, and protein binding capacity in plasma and tissues. These physiological processes all demonstrate pronounced circadian oscillations.¹⁸⁷ As the primary site of drug metabolism, the key hepatic enzymes involved in drug biotransformation are intricately regulated by core circadian clock genes. Furthermore, the circadian molecular clock system modulates the renal-mediated drug excretion process, consequently influencing the overall drug metabolism.¹⁸⁸

4.2. Circadian rhythm-based dosing regimens

Clinical evidence suggests that the efficacy of ICI therapy exhibits significant heterogeneity, which is intimately related to chronopharmacological effects. Different ICI administration time points within a 24-hour cycle can lead to variations in patient responses to immunotherapy, underscoring the critical importance of optimizing circadian rhythm-based dosing strategies to enhance patient survival outcomes. Multiple studies have demonstrated that administering ICIs in the early morning or early active phase can significantly prolong patients' progression-free survival and overall survival, resulting in more favorable therapeutic outcomes.^29,189, 190, 191, 192 For instance, studies have revealed that the efficacy of Nivolumab treatment for non-small cell lung cancer patients is significantly higher when administered in the morning compared to the afternoon, demonstrating approximately four-fold greater effectiveness.¹⁸⁹ Similarly, melanoma patients receiving ICI treatment in the morning demonstrated significantly prolonged overall survival (P < 0.001) and progression-free survival (P = 0.009),¹⁹⁰ a finding that was further corroborated by another study on melanoma patients.¹⁹¹ A meta-analysis of seven studies encompassing 1019 stage IV cancer patients revealed that administering ICI in the morning significantly improved overall survival (P < 0.0001) and progression-free survival (P < 0.00001) in patients with advanced cancer.¹⁹² Studies have demonstrated that administering anti-PD-L1 therapy to mice during the early active phase (ZT 16), coinciding with the peak abundance of immunosuppressive MDSCs, yields optimal therapeutic effects.²⁹ Related research has revealed that PD-1 expression on TAMs in melanoma exhibits significant circadian oscillation patterns. Consequently, administering BMS-1 (a small molecule PD-1/PD-L1 inhibitor) during periods of high PD-1 expression on TAMs has been shown to produce stronger antitumor effects.¹⁷⁰ Notably, given the suppressive effect of corticosteroids on ICI efficacy, it is crucial to thoroughly consider the circadian variations of patients' endogenous glucocorticoids when developing dosing regimens, with the goal of achieving optimal therapeutic outcomes.¹⁹³

4.3. Therapeutic strategies targeting key components of the TME

Strategies that combine targeting of TAMs with immune checkpoint blockade therapy have demonstrated a significant enhancement in the clinical response rate of immunotherapy.¹⁴² Studies have demonstrated that B7-H4-positive macrophages exhibit immunosuppressive functions comparable to CD4⁺ regulatory T cells, indicating that targeting B7-H4 molecules or eliminating B7-H4-positive tumor-associated macrophages could potentially provide novel intervention strategies for anti-tumor immunotherapy.¹⁹⁴ Therapeutic strategies targeting TAMs predominantly encompass: 1) promoting their polarization towards an M1 anti-tumor phenotype, 2) inhibiting their chemotaxis and recruitment to tumor sites, 3) blocking the expression of immune checkpoint molecules on their surface, and 4) inducing their functional exhaustion. Combined targeting strategies for MDSCs primarily comprise: 1) inhibiting their proliferation and migration, 2) promoting their differentiation into mature immune cells, 3) modulating their immune metabolism, 4) attenuating their immunosuppressive activity, and 5) eliminating MDSCs from the TME.¹⁹⁵ Various TLR agonists have progressed to clinical trial stages, and their combined application with anti-PD-L1 and anti-CTLA-4 ICIs has demonstrated significant synergistic effects.¹⁹⁶ Inhibitors targeting chemokine receptors CXCR2 or CXCR4 have demonstrated the ability to remodel the TME. Moreover, their combination with immunotherapy exhibits promising results in suppressing tumor metastasis and enhancing treatment efficacy.⁶⁴ Recent studies have demonstrated that within the hypoxic TME, hypoxia-inducible factor-1α (HIF-1α) upregulates the expression of PD-L1 on the surface of MDSCs. This finding suggests that the concurrent application of HIF-1α inhibitors could potentially offer a novel approach to augment the efficacy of immune checkpoint blockade therapy.²⁸ The concomitant administration of bispecific PD-1-IL-2 variants and anti-PD-L1 antibodies has been shown to significantly amplify the clinical benefits of immunotherapy.¹⁰ Pegylated recombinant human interleukin-10 (PEG-IL-10) induces the activation of tumor antigen-specific CD8⁺ T cells and upregulates the expression of IFN-γ and major histocompatibility complex class I and II molecules. Furthermore, its combination with anti-PD-1 antibodies has demonstrated promising clinical responses.¹⁹⁷ The therapeutic strategy combining TGF-β inhibitors and PD-1/PD-L1 pathway blockers has demonstrated a significant improvement in the overall survival rate of prostate cancer patients.¹⁹⁸

4.4. Interventional strategies for circadian rhythm regulation

Recent studies have demonstrated that the coordinated regulation of circadian rhythm mechanisms exhibits significant potential for enhancing the therapeutic efficacy of ICIs. As a result, the development of specific small-molecule compounds targeting key circadian rhythm regulators (such as RORγ, REV-ERBα/β, and CRY) has emerged as a crucial research focus in the field of chronopharmacology. A substantial body of experimental evidence suggests that ROR agonists can concurrently promote immune activation and attenuate immunosuppression, thus playing a pivotal role in anti-tumor immunotherapy. Specifically, RORγ agonists have been shown to enhance the effector function of Th17 cells and improve their survival.¹⁹⁹ Additionally, RORα agonists have been demonstrated to promote CD8⁺ T cell infiltration by inducing the migration of monocyte-derived DCs, while concomitantly regulating cholesterol metabolism to enhance their anti-tumor response.¹⁰⁵ Furthermore, RORγ agonists have been found to attenuate immunosuppression in the TME through multiple mechanisms: inhibiting the generation of Tregs, reducing the expression levels of CD73 and CD39, and downregulating the expression of PD-1 and TIGIT on lymphocyte surfaces.²⁰⁰ Notably, SR9011 and SR9009, which are specific small molecule agonists of REV-ERBα/β, have been shown to suppress BMAL1 transcriptional activity by selectively binding to its gene promoter region.^65,201 Studies have demonstrated that KL001, a small molecule agonist of CRY, exerts its effects through multiple mechanisms, including the inhibition of CRY protein ubiquitination and degradation, reduction in the expression of cancer stem cell markers, suppression of PER1 and PER2 expression, and upregulation of BMAL1 protein levels.²⁰² Recent research has revealed that inhibiting the CLOCK-OLFML3-HIF1α-LGMN—CD162 signaling pathway in combination with anti-PD-1 therapy significantly enhances antitumor effects.²⁰³ Further studies have demonstrated that targeting key circadian rhythm genes ARNTL2 and TIMELESS possesses significant antitumor potential. Research has established that high expression of ARNTL2 is potentially involved in the formation and maintenance of an immunosuppressive TME.⁴⁰ TIMELESS interacts with c-Myc protein to elevate PD-L1 expression levels, consequently promoting tumor progression.¹⁶⁶ This finding offers a theoretical basis for the development of novel drugs targeting TIMELESS to enhance ICI treatment efficacy. Notably, in addition to small molecule agonists targeting circadian rhythms, chronotherapeutic strategies such as endocrine hormone regulation^21,204 and non-pharmacological interventions (e.g., time-restricted feeding protocols)²¹ have demonstrated potential in improving the efficacy of anti-PD-L1 immunotherapy.

4.5. Personalized treatment strategies

Recent studies have revealed significant inter-individual variations in rest-activity rhythms among cancer patients, which are closely associated with the regulation of core body temperature, melatonin and cortisol secretion rhythms, and dynamic changes in peripheral circulating immune cell subpopulations. This heterogeneity in biological rhythms among patients may stem from differences in clock gene expression patterns, which could subsequently influence the therapeutic efficacy of ICIs.^{31,138,139,158} Tailoring chronotherapeutic regimens and personalized dosing strategies based on individual patients' circadian biomarker expression profiles (e.g., Period2 protein)^205,206 holds promise for significantly improving clinical outcomes and overall survival benefits in cancer patients.

5. Future perspectives

Drawing upon current research on circadian rhythms, TME, and ICI therapy, we present a comprehensive outlook on the research frontiers and clinical translation prospects in this field. This includes the application of multi-omics integration to elucidate circadian regulatory networks, the development of artificial intelligence-based precision chronomedicine strategies, the exploration of novel chronotherapeutic approaches, and the establishment of standardized clinical translation systems (Fig. 3).

Fig. 3.

Future research directions in circadian cancer immunotherapy. This framework delineates four critical research directions for circadian-based cancer immunotherapy:integrative multi-omics analyses employing single-cell sequencing, metabolomics, proteomics, and epigenetic studies to characterize circadian regulatory networks within the tumor microenvironment; artificial intelligence-based precision chronotherapy encompassing machine learning algorithms, wearable monitoring, and personalized immune checkpoint inhibitor dosing strategies; novel chronotherapeutic approaches comprising small molecule circadian regulators, nanodelivery systems, and optimized CAR-T cell timing; and standardized clinical translation via prospective randomized controlled trials, multicenter collaborative platforms, and unified assessment protocols for circadian phenotyping and tumor microenvironment analysis. CAR-T, Chimeric Antigen Receptor T-cell; RCT, randomized controlled trial.

5.1. Integrative multi-omics analysis of circadian rhythm regulatory networks

In the context of rapidly evolving cancer research, integrative multi-omics approaches^145,207, 208, 209 play a crucial role in elucidating the complex regulatory networks among circadian rhythms, TME, and ICIs. High-resolution analysis utilizing single-cell sequencing technology facilitates precise characterization of circadian features in various cell subpopulations within the TME,²⁹ thus unveiling the spatiotemporal heterogeneity of circadian-regulated gene expression profiles and signaling pathways.²¹⁰ These findings contribute significantly to a deeper understanding of the temporal dynamics of cells within the TME, thereby establishing a new theoretical foundation for developing personalized precision treatment strategies. Integration of metabolomics and proteomics research methods enables in-depth investigation of the regulatory mechanisms by which circadian rhythms influence metabolite composition and protein expression profiles in the TME.³¹ This multidimensional research strategy facilitates the development of a systematic molecular network model of circadian regulation in the TME, yielding crucial experimental evidence for identifying novel biomarkers and developing innovative therapeutic targets. Epigenetic modifications play a pivotal role in the circadian clock gene-mediated regulation of TME characteristics. For instance, somatic copy number variations in clock genes can significantly affect the degree of tumor immune infiltration.¹⁴⁵ Comprehensive epigenetic studies on circadian rhythms and the TME will elucidate the epigenetic regulatory mechanisms of tumor progression, potentially revealing new intervention targets for cancer therapy.

5.2. Artificial intelligence-based precision chronotherapy strategies

With the advancing understanding of circadian rhythm regulation in the TME and the rapid development of artificial intelligence (AI) technologies,^211,212 AI-based precision chronotherapy strategies are emerging as a critical direction for enhancing the efficacy of ICI treatments. Studies have demonstrated that optimizing the administration timing of ICIs based on individual patients' circadian rhythm characteristics can substantially improve cancer treatment outcomes. To achieve the goal of personalized medicine, there is an imperative need to develop accurate individualized circadian rhythm assessment methods.¹⁹³ Recent studies have demonstrated that machine learning algorithms and mathematical modeling approaches can elucidate circadian parameters and individual circadian patterns by analyzing biological data, particularly gene expression,^31,213 thereby enabling accurate prediction of patient responses to ICI treatments and providing a robust scientific basis for clinical decision-making. Concurrently, wearable sensors integrated with specialized remote monitoring platforms enable real-time detection of circadian and ultradian rhythm characteristics, yielding rich personalized data resources for system modeling and machine learning analysis.^213,214 By integrating patients' circadian rhythm characteristics with the pharmacokinetic properties of ICIs, researchers can develop personalized ICI dosing regimens, encompassing optimization of initial dosing and subsequent dose adjustment strategies. Furthermore, this integration facilitates the design of intelligent drug delivery systems for real-time adjustment of dosing regimens, thus enabling more precise treatment plans for cancer patients and facilitating continuous optimization of therapeutic effects. Beyond circadian rhythm characteristics, the composition and state of the TME exhibit substantial dynamism. Consequently, real-time dynamic monitoring of changes in the TME and treatment responses holds paramount importance for optimizing clinical diagnosis, treatment decisions, and prognostic assessment of tumors. In the realm of chronotherapy, the synergistic application of ICIs and circadian rhythm regulators (including small molecule agonists of molecular clock proteins and rhythm hormones)^{65,199,202,204,215} has exhibited remarkable anti-tumor effects. Elucidating the synergistic mechanisms underlying the interaction of these two approaches in immunotherapy offers substantial potential for further optimizing existing combination treatment strategies.

5.3. Novel strategies for chronotherapy

In recent years, extensive studies on the interaction mechanisms between circadian rhythms and the TME have unveiled new research avenues for developing novel targeted therapeutic strategies to enhance the efficacy of ICIs. In the field of chronopharmacology, research on small molecule agonists targeting key circadian rhythm regulators (such as RORγ, REV-ERBα/β, and CRY) has emerged as a highly promising avenue.^65,199,202 These agents can significantly enhance the therapeutic effects of ICIs by modulating the TME. While ICI therapy faces challenges such as insufficient targeting and limited efficacy, the rapid advancement of nanodelivery technology offers innovative solutions to overcome issues like toxicity and poor targeting associated with conventional drug delivery methods.²¹⁶ Consequently, developing circadian rhythm-dependent nanodelivery systems can not only enhance the targeting and efficacy of ICIs but also facilitate significant breakthroughs in the field of cancer immunotherapy. Recent studies have demonstrated that the efficacy of Chimeric Antigen Receptor T-cell (CAR-T) therapy exhibits significant time dependence, with tumor volume reduction strongly correlated with the tissue homing ability of CAR-T cells at specific time points.^30,217 Optimizing the infusion timing of CAR-T cells to coincide with the optimal window for leukocyte infiltration can significantly augment the therapeutic effect of T cell therapy. This discovery provides novel insights for developing circadian rhythm-based CAR-T cell therapies, potentially further improving their therapeutic efficacy and prolonging their activity in the TME.

5.4. Establishment of a standardized clinical translation system and RCT implementation framework

Advancing the clinical translation of research on circadian rhythm regulation and TME is crucial for optimizing immunotherapy strategies utilizing ICIs. At present, the clinical efficacy of ICI administration regimens based on circadian rhythm patterns lacks sufficient validation, necessitating large-scale, multicenter prospective clinical trials for systematic evaluation. However, our comprehensive literature review reveals a critical limitation in the current evidence base: no high-quality randomized controlled trials (RCTs) specifically examining the circadian timing of ICI administration exist in the published literature or clinical trial registries. This absence represents the most significant barrier to clinical translation of chronotherapy principles in cancer immunotherapy. Future RCT designs must address unique methodological challenges, including individual circadian phenotyping using validated biomarkers, circadian phase-based timing protocols rather than fixed clock-time schedules, and standardized outcome measures for timing-dependent efficacy and safety assessments. A comprehensive analysis of the associations among individual patients' circadian rhythm characteristics, dynamic changes in the TME, and immunotherapy responses through dedicated prospective RCT frameworks is essential for developing precise personalized treatment plans and improving clinical outcomes. The establishment of a standardized multicenter collaborative network platform to systematically collect and integrate patients' clinical characteristics, biomarkers, and prognostic data represents a key initiative in advancing precision medicine research and providing the necessary infrastructure for conducting adequately powered chronotherapy RCTs. Currently, the inadequate standardization of circadian phenotype assessment and TME characterization methods has significantly hindered the optimization and promotion of personalized immunotherapy regimens. The establishment of unified assessment systems for circadian rhythms and standardized analysis protocols for TMEs specifically designed for RCT implementation will provide a reliable scientific basis for the development of precise immunotherapy regimens. This approach will facilitate the standardized application of personalized immunotherapy strategies in clinical practice and enable the systematic clinical validation necessary to advance chronotherapy from preclinical promise to evidence-based therapeutic reality.

6. Conclusions

The systematic convergence of circadian rhythm biology, TME dynamics, and ICI efficacy is increasingly recognized as a key and potentially modifiable factor shaping immunotherapy responses. This review synthesizes robust, multidimensional evidence demonstrating that circadian rhythms regulate the TME at multiple levels. These mechanisms encompass tumor cell dynamics, immune cell rhythms, and vascular and stromal alterations, thereby shaping ICI sensitivity and therapeutic response heterogeneity. As clinical management increasingly emphasizes precision and individualization, integrating circadian rhythm indicators into multidisciplinary approaches for tumor immunotherapy decision-making, drug administration strategies, and immune monitoring systems becomes imperative. Future research urgently requires establishing clinically validated mechanistic frameworks to precisely define individual circadian rhythmic phenotypes and their real-time interactions with TME states, thereby advancing personalized chronotherapy. Additionally, circadian-guided randomized controlled trials are needed to validate and optimize temporal dosing regimens for ICIs, facilitating the transition of immunotherapy from static paradigms to time-precise, biologically synchronized strategies.

Author contributions

Anqi Lin, Xiuhui Fang, Pengpeng Zhang, Bufu Tang, Peng Luo: Writing-Original draft preparation, Investigation. Bufu Tang, Peng Luo: Conceptualization. Anqi Lin, Xiuhui Fang, Pengpeng Zhang, Nan Zhang, Zaoqu Liu, Quan Cheng, Jian Zhang, Bufu Tang, Peng Luo: Writing-Reviewing, editing and revision. Anqi Lin, Xiuhui Fang, Bufu Tang, Peng Luo: Visualization.

Declaration of competing interest

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