Current trends in sensitizing immune checkpoint inhibitors for cancer treatment

Abstract

Immune checkpoint inhibitors (ICIs) have dramatically transformed the treatment landscape for various malignancies, achieving notable clinical outcomes across a wide range of indications. Despite these advances, resistance to immune checkpoint blockade (ICB) remains a critical clinical challenge, characterized by variable response rates and non-durable benefits. However, growing research into the complex intrinsic and extrinsic characteristics of tumors has advanced our understanding of the mechanisms behind ICI resistance, potentially improving treatment outcomes. Additionally, robust predictive biomarkers are crucial for optimizing patient selection and maximizing the efficacy of ICBs. Recent studies have emphasized that multiple rational combination strategies can overcome immune checkpoint resistance and enhance susceptibility to ICIs. These findings not only deepen our understanding of tumor biology but also reveal the unique mechanisms of action of sensitizing agents, extending clinical benefits in cancer immunotherapy. In this review, we will explore the underlying biology of ICIs, discuss the significance of the tumor immune microenvironment (TIME) and clinical predictive biomarkers, analyze the current mechanisms of resistance, and outline alternative combination strategies to enhance the effectiveness of ICIs, including personalized strategies for sensitizing tumors to ICIs.

Introduction

Immune checkpoint inhibitors (ICIs) block regulatory pathways that suppress T cell activity and immune responses, leading to robust activation of the immune system [1,2,3,4]. Mechanistically, cancer cells can exploit these immune checkpoints to evade host immune surveillance, offering a potential strategy for cancer immunotherapy [5,6,7,8,9,10]. The successful clinical development of ICIs, such as CTLA-4 and PD-1/PD-L1 inhibitors, has revolutionized cancer treatment by delivering unprecedented anti-tumor activity. This progress has facilitated the clinical use of ICIs as monotherapies or in combination with other therapies [11,12,13,14,15,16,17,18]. Since the approval of ipilimumab, a CTLA-4-targeting monoclonal antibody, in 2011 for advanced metastatic melanoma, ICIs have become a standard treatment for several solid tumors, including those with microsatellite instability-high (MSI-H) tumors [19,20,21,22,23,24,25]. Numerous immune checkpoints are currently being investigated for their translational potential, from preclinical studies to clinical trials, based on promising preclinical results [26,27,28,29,30,31,32]. For example, relatlimab, a recently FDA-approved drug targeting the lymphocyte-activation gene 3 (LAG-3) protein, is used in combination with nivolumab for treating unresectable or metastatic melanoma [33,34,35]. Ongoing research is focused on exploring novel immune checkpoints and developing rational therapeutic combinations with synergistic anti-tumor mechanisms to expand the indications and clinical benefits of ICIs.

Despite the significant clinical benefits observed in some patients, with notable tumor regression in response to ICIs, a substantial proportion of patients either do not respond to these treatments or exhibit low response rates [36,37,38,39]. Some patients experience innate resistance or develop acquired resistance over time, which diminishes the effectiveness of ICIs [40,41,42]. Currently, numerous clinical trials are investigating various combination therapies with ICIs as the core component to enhance treatment sensitivity [15, 43,44,45]. However, few strategies have yielded encouraging clinical results so far [46,47,48]. Understanding the mechanisms underlying both resistance and response to ICIs is crucial for predicting immune response outcomes and potentially converting non-responders into responders. Furthermore, it is essential to incorporate robust biological principles and prospective biomarkers when evaluating different drug combinations for patient screening [43, 49,50,51,52,53,54,55]. This review explores the mechanisms and factors involved in immune checkpoint response and resistance, assesses clinically integrated immunotherapy response biomarkers, and highlights recent advances in immunotherapy combination strategies aimed at expanding the clinical applications of ICIs.

Immune checkpoint inhibitors (ICIs) in cancer immunotherapy

Over the past decade, advances in tumor immunology have significantly improved our understanding of the immune system’s role in cancer treatment. This has led to the development of immunoregulatory monoclonal antibodies targeting T-cell immune checkpoints such as CTLA-4 and PD-1, which have revitalized anti-tumor immunotherapy and cancer precision medicine. Additionally, antibodies against other immune checkpoint molecules like LAG3, TIM3, TIGIT, ICOS, GITR, and 4-1BB are actively being explored in clinical trials. This section focuses on the mechanisms of CTLA-4 and PD-1 in T-cell-mediated anti-tumor immunity and reviews key clinical trials related to these treatments.

Programmed cell death protein 1 (PD-1)

Programmed death 1 (PD-1, CD279) was identified in 1992 as a key regulator of peripheral tolerance. It is expressed on activated/ exhausted T cells, as well as on B cells, natural killer (NK) cells, macrophages, dendritic cells (DCs), and immature Langerhans’ cells [56, 57]. PD-1 functions through its ligands, PD-L1 and PD-L2, by inhibiting T-cell receptor (TCR) signaling. This inhibition occurs through the phosphorylation of immunoreceptor tyrosine-based switch motifs (ITSM) and the recruitment of Src homology region 2 domain-containing phosphatase-1 (SHP-1) and - 2 (SHP-2), which dephosphorylate ZAP70 and CD3ζ, impairing TCR activation [30, 58,59,60,61,62]. Additionally, PD-1 signaling downregulates the PI3K-Akt-mTOR and RAS-MEK-ERK pathways. Specifically, PTEN phosphorylation, regulated by CK2, suppresses PI3K signaling, while inhibition of Ras and PLC-γ1 affects the RAS-MEK-ERK pathway [63,64,65,66,67,68,69]. PD-1/PD-L1 interactions also inhibit B-cell responses by affecting the phosphorylation of signaling molecules such as PI3K and Syk [70, 71]. Mechanistically, CTLA-4 acts early in T cell activation to block CD28-B7 interactions, downregulating antigen-presenting cells (APCs)-induced T cell responses and primarily affecting CD4⁺ T cell amplification and trafficking. In contrast, PD-1 is expressed later in T cell activation and regulates the effective stage of T cell response by restraining TCR signaling, with anti-PD-1/PD-L1 therapy primarily enhancing the antitumor immunity of exhausted CD8⁺ T cells. Thus, the distinct mechanisms of CTLA-4 and PD-1 highlight their nonoverlapping roles in T cell functional inhibition, supporting the potential benefits of combinatorial targeted strategies [72,73,74,75,76,77,78].

Over the past few years, several pivotal trials have reshaped the treatment landscape. In a phase III trial comparing pembrolizumab to ipilimumab in advanced melanoma, pembrolizumab demonstrated superior progression-free and 12-month overall survival without high-grade toxicity (KEYNOTE-006, NCT01866319) [79]. This finding was reinforced by subsequent analyses, leading to pembrolizumab’s first approval for the treatment of advanced melanoma, particularly after initial treatment with ipilimumab and BRAF inhibitors [80,81,82]. Nivolumab, another PD-1 inhibitor, first entered clinical trials in 2006 and showed promising results in a 2012 phase I trial for refractory solid tumors. In phase III trials, nivolumab exhibited durable objective responses and lower toxicity compared to other chemotherapy regimens in advanced melanoma patients who had progressed after previous treatments. A separate phase III trial also showed nivolumab’s superior overall survival (OS) compared to dacarbazine in treatment-naive advanced melanoma patients, with survival benefits linked to PD-L1 expression [83,84,85]. Combining CTLA-4 and PD-1 inhibitors has shown promise in metastatic melanoma, with a phase III trial revealing that combination therapy significantly improved progression-free survival (PFS) compared to either nivolumab or ipilimumab monotherapy alone, even in PD-L1 negative tumors patients [84]. This led to the FDA’s 2014 approval of nivolumab for melanoma treatment. Advances in the field of immune checkpoint blocking therapy for melanoma have facilitated the exploration of ICI in other tumor types. For example, in non-small cell lung cancer (NSCLC), pembrolizumab outperformed traditional chemotherapy in improving PFS and OS, leading to its FDA approval as a first-line treatment for NSCLC with high PD-L1 expression [86]. Currently, several anti-PD-1/PD-L1 therapies have proven beneficial in NSCLC, small cell lung cancer (SCLC), gastric cancer (GC), hepatocellular carcinoma (HCC), colorectal cancer (CRC), and bladder cancer, and many more are currently under investigation [87,88,89,90,91].

Cytotoxic T lymphocyte-associated protein 4 (CTLA-4)

Cytotoxic T lymphocyte antigen-4 (CTLA-4) is a type I transmembrane protein primarily expressed on T cells and upregulated upon T cell activation. It plays a crucial role in regulating T cell responses early in activation, as independently demonstrated in the mid-1990s [92,93,94,95]. CTLA-4 suppresses T cell activation through both intrinsic and extrinsic mechanisms. CTLA-4 binds with high affinity to B7 molecules, competing with CD28 for binding to B7 family members on APCs, thereby blocking the CD28-B7 signaling pathway and preventing CD28-mediated T cell activation [96]. Additionally, CTLA-4 exerts negative regulation by inhibiting IL-2 production and IL-2 receptor expression, suppressing T cell proliferation and activation by blocking the transition from G1 to S phase, and disrupting TCR signaling through interactions with PP2A and SHP-2 [97,98,99,100,101]. CTLA-4 is constitutively expressed on regulatory T cells (Tregs) that maintain self-tolerance, CTLA-4-knockout Tregs fail to regulate autoimmunity, leading to severe immune hyperactivation and premature death in mice [102,103,104]. These mechanisms collectively enable CTLA-4 to play a critical role in immune tolerance.

Initially, two fully humanized CTLA-4-directed monoclonal antibodies, ipilimumab (MDX-010) and tremelimumab, were developed and tested in clinical trials for advanced melanoma starting in 2000 [11, 105]. Ipilimumab was first evaluated in single-dose trials for patients with metastatic melanoma, ovarian cancer (OC), and prostate cancer [106,107,108,109,110,111,112]. Subsequent multi-dose studies showed that administering ipilimumab at 3 mg/kg every 3 weeks, combined with peptide vaccines, significantly improved OS and tumor regression compared to peptide vaccines alone [113]. This established the importance of CTLA-4 in cancer immunotherapy and led to a surge in immuno-oncology research. In 2010, a pivotal phase III trial demonstrated that ipilimumab provided a 3.5-month median OS benefit over the gp100 peptide vaccine alone, leading to its FDA approval in 2011 for late-stage melanoma [106, 114]. However, tremelimumab, another anti-CTLA-4 mAb, did not show a significant survival advantage in several late-phase trials. For instance, a phase III trial comparing tremelimumab to standard chemotherapy for metastatic melanoma failed to show significant improvement [115]. These setbacks led to the exploration of combination therapies [116, 117]. Phase III HIMALAYA trail combining tremelimumab with durvalumab showed promising results, including a potent immune response and enhanced anti-tumor activity in HCC (NCT03298451) [118]. Based on these findings, the combination was approved by the FDA in October 2022 for unresectable hepatocellular carcinoma (uHCC) and in November 2022 for metastatic non-small cell lung cancer (mNSCLC) [105, 116, 119]. Despite the progress, many combination trials still fail to meet primary OS endpoints [120].

Other immune checkpoints

Apart from CTLA-4 and PD-1, several positive and negative immune checkpoint molecules have been identified and investigated over the past decade as potential immunotherapeutic targets [121,122,123].

LAG-3 is highly expressed on activated T cells and various other immune cells, where it binds to MHC class II and other ligands, delivering inhibitory signals that suppress T cell activity and enhance Treg-mediated immunosuppression [124]. Several studies have demonstrated that LAG-3 and PD-1 synergize on CD8 ⁺ T cells to drive T cell exhaustion and impede autocrine IFN-γ-dependent anti-tumor immunity [125]. This synergy provides insight into how combinatorial targeting of LAG-3 and PD-1 can enhance therapeutic efficacy. Subsequent clinical studies have confirmed these synergistic effects. For instance, the RELATIVITY-047 trial, a global, double-blind, randomized Phase 2/3 study, showed improved PFS in patients with metastatic or unresectable melanoma when treated with a combination of relatlimab and nivolumab, compared to PD-1 inhibition alone [33]. Other clinical studies, including those on head and neck squamous cell carcinoma (HNSCC), GC and lung cancer, are ongoing (NCT04811027, CTR20221603) [126].

TIM-3 is a co-inhibitory receptor expressed on a range of immune cells, including IFN-γ-secreting CD4 ⁺ and CD8 ⁺ T cells, NK cells, myeloid cells, mast cells, Tregs, and B cells. It interacts with ligands such as galectin-9, phosphatidylserine, high-mobility group protein B1, and carcinoembryonic antigen cell adhesion molecule-1 to suppress anti-tumor immunity via inducing terminal dysfunction and death of CD8⁺ T cells [127,128,129,130]. A range of anti-TIM-3 agents have been developed based on preclinical data and effects observed in advanced cancer patients, with sabatolimab and cobolimab being the most advanced. Several clinical trials are ongoing for anti-TIM-3 therapies across hematologic malignancies and solid tumors [131,132,133,134,135]. Notably, sabatolimab in combination with hypomethylating agents has shown durable responses, earning Fast-Track designation from the FDA and orphan drug status from the European Commission for MDS [136]. In a Phase I study, cobolimab combined with dostarlimab (PD-1 inhibitor) achieved a 42.9% objective response rate (ORR) in NSCLC patients without severe adverse events (AEs). Cobolimab is now being tested in combination with chemotherapy (docetaxel) and/or anti-PD-1 in various cancers [137].

TIGIT is a newly identified co-inhibitory receptor that suppresses T cell proliferation and cytokine production by interacting with its ligands CD155 and CD112. Clinical trials investigating anti-TIGIT monoclonal antibodies, both alone and in combination with anti-PD-1 and anti-PD-L1 therapies, have shown promising early results [138,139,140,141]. Notably, the CITYSCAPE-02 trial demonstrated that tiragolumab (TIGIT inhibitor) plus atezolizumab improved overall response rates in PD-L1-positive, recurrent or metastatic NSCLC, earning FDA breakthrough therapy designation [139]. In addition, the MORPHEUS-liver study showed that tiragolumab combined with atzolizumab and bevacizumab improved ORR and FPS in first-line liver cancer patients [142]. Despite these advancements, the phase III SKYSCRAPER-02 trial found no clinical benefit for TIGIT targeting in SCLC [143]. Overall, while anti-TIGIT therapies have demonstrated notable progress in specific indications, the results across different studies have been mixed [144].

Co-stimulatory checkpoint molecules are being explored for cancer immunotherapy in both preclinical and clinical studies. These molecules, found not only on T cells but also on other immune cells, enhance anti-tumor immune responses. Immune co-stimulator (ICOS), expressed on T cells, is induced by T cell activation and facilitates T cell differentiation and expansion. Several clinical trials are currently investigating the effects of targeting ICOS alone or in combination with ICIs (NCT05695898, NCT02520791, NCT04319224) [145, 146].

Checkpoint molecules of the tumor necrosis factor receptor (TNFR) superfamily, including 4-1BB, OX40, and glucocorticoid-induced TNFR-related gene (GITR), are being investigated as therapeutic targets [147,148,149,150]. 4-1BB, also known as CD137 or TNFRSF9, is a transmembrane protein upregulated upon T cell activation by mitogens or antigens. Engagement of 4-1BB with its exclusive ligand 4-1BBL forms a hexameric complex, which enhances the transcription of anti-apoptotic genes in T cells, thereby promoting long-lasting memory responses of cytotoxic CD8⁺ T cells. The overexpression of 4-1BBL and administration of agonistic monoclonal antibodies targeting 4-1BB have been shown to improve CD8⁺ T cell-mediated anti-tumor responses and induce tumor rejection in preclinical models. This underscores the potential of incorporating 4-1BB signaling domains into CAR-T cell constructs to enhance activation and therapeutic efficacy [151]. OX40, expressed transiently on T cells after activation, enhances T cell expansion and memory formation upon interaction with its ligand OX40L. It also modulates Tregs by inhibiting their function while promoting their production, thereby playing a complex role in anti-tumor immunity [152]. Several agonist antibodies targeting OX40 and GITR are currently being explored in preclinical and clinical trials [153,154,155,156,157,158].

The complex tumor immune microenvironment is a highly promising biomarkers for tumor immunotherapy

Immune checkpoint inhibitors response and TIME

The discovery of immune checkpoints has revolutionized cancer treatment, significantly improving clinical outcomes and overall survival. Following the FDA approval of ipilimumab in 2011, interest in immunotherapy surged. Over the past decade, several monoclonal antibodies targeting PD-1 and PD-L1 have also received FDA approval for various cancers, including melanoma, NSCLS, renal cell carcinoma (RCC), HNSCC, GC, HCC, and more [19, 159, 160]. Despite these advancements, resistance to immune checkpoint therapy (ICT) remains common, categorized into primary resistance (no initial response) and acquired resistance (initial response followed by relapse). Some cancers, like pancreatic cancer and glioblastoma, show inherent resistance, while others, such as melanoma and bladder cancer, may develop resistance after initial success [161,162,163,164]. Understanding the mechanisms of resistance is crucial for predicting patient responses and improving treatment strategies. Though robust biomarkers are lacking, analyzing the tumor microenvironment (TME) and immune cell composition pre- and post-therapy may reveal predictive markers for effective ICT. The cancer-immunity cycle highlights the role of T cells in anti-tumor immunity, from activation in lymph nodes to tumor cell elimination [165]. However, tumors often exploit immune checkpoints like PD-1 to evade detection, leading to T-cell exhaustion. Immunotherapy aims to reverse this exhaustion and enhance immune responses.

T cells play a central role in anti-tumor immunity, guided by the classical cancer-immunity cycle. This cycle outlines how APCs, such as DCs, process tumor antigens and present them as major histocompatibility complex (MHC)-peptide complexes to T cell receptors (TCRs). This interaction triggers T cell priming and activation, regulated by co-stimulatory and inhibitory checkpoints in lymph nodes, leading to T cell proliferation, enhanced effector functions, and memory formation [166]. Once activated, T cells migrate to the TME to target and destroy tumor cells. However, tumors often exploit inhibitory checkpoint molecules like PD-1 to evade T cell-mediated destruction. This results in T-cell exhaustion, a state of diminished immune response due to the immunosuppressive TME [167, 168]. Immunotherapy aims to counteract this exhaustion by reactivating T cells and improving their ability to combat tumors. Effective therapies often focus on reversing T cell exhaustion and enhancing anti-tumor immune responses to limit tumor progression and promote tumor elimination.

The complex interactions between the host’s immune system and cancer cells within the TME significantly influence immune cell functions [169]. Understanding these interactions is crucial for deciphering how tumors evade immune detection. The concept of “immune contexture” refers to the diverse immune variables, including cell types, densities, functional states, and distributions within the TME, which can indicate survival outcomes and predict treatment responses [170, 171]. The TME is classified into three categories (hot, altered and cold) based on immune activity and tumor escape mechanisms. Hot Tumors have high lymphocyte infiltration and PD-1 expression, often showing good responses to ICIs. They are characterized by high levels of tumor-infiltrating lymphocytes (TILs), PD-L1 expression on immune cells, and high genomic instability. In contrast, cold tumors show low levels of lymphocyte infiltration and minimal PD-L1 expression. They typically have low antigen presentation capabilities, such as reduced major histocompatibility complex class I (MHC I) expression, and low mutational burden, making them less responsive to ICT. This classification provides a framework to assess tumor immune profiles and predict responses to immunotherapy, potentially guiding more effective treatment strategies [1, 172, 173].

Clinically integrated biomarkers of ICI response

Efforts to predict responses to cancer immunotherapy and guide personalized treatment have intensified. Identifying and standardizing early predictive biomarkers related to immunotherapy sensitivity and resistance are crucial for optimizing ICB therapies. Proposed and tested biomarkers include immune cell infiltration, Immunoscore, tumor mutational burden (TMB), neoantigen load, PD-L1 expression, mismatch repair deficiency (dMMR), microsatellite instability (MSI), and inflammatory gene profiling within the TME [49, 174]. These biomarkers aim to identify the most effective therapeutic interventions for individual patients. Here, immunotherapy biomarker applied for the oncology clinic will be delineated.

PD-L1 expression

PD-L1 expression in total nucleated cells (both malignant and immune cells in the TME), as detected by immunohistochemistry (IHC), is the most commonly used predictive biomarker for selecting patients for ICIs therapy. High PD-L1 expression often correlates with better responses to PD-1/PD-L1 axis blockade, making it a valuable tool for identifying patients who might achieve longer survival [175, 176]. In clinical practice, PD-L1 expression is evaluated using four FDA-approved IHC assays (22C3, 28-8, SP263, and SP142), each validated through rigorous staining processes. Pre-treatment PD-L1 expression is assessed using scoring systems such as the combined positive score (CPS), tumor proportion score (TPS), and immune cell score (IC). Scoring methods and cut-off values for predicting immunotherapy response vary across clinical trials and tumor types (e.g., gastric or lung cancer). Currently, PD-L1 status has associated with regulatory approvals for several cancers, including NSCLC, GC/GEJC, bladder cancer, and cervical cancer [177, 178]. However, PD-L1 status is only predictive in about 30% of cases across these cancer types, with its clinical utility varying by cancer type and ICI agent [178]. Some patients with PD-L1-negative tumors also show responses, and phase III trials have not always shown a clear correlation between PD-L1 expression and survival outcomes. This variability underscores the challenge of using PD-L1 as a standalone biomarker [179]. Furthermore, differences in tissue handling techniques, detecting antibody, immune cell staining heterogeneity, and assay sensitivity complicate the standardization of PD-L1 testing. Therefore, improving standardization and integrating PD-L1 with other biomarkers could enhance predictive accuracy.

Tumor mutation burden (TMB) and MSI

Tumor mutation burden (TMB), defined as the number of non-synonymous mutations per megabase of coding sequence, is being investigated as a predictive biomarker for ICI therapy. High TMB can generate tumor neoantigens (mutation-associated neoantigens, or MANAs) that may be recognized by the immune system, potentially triggering an effective antitumor response. Advances in next-generation sequencing and molecular characterization have enhanced our understanding of cancer biology and facilitated precision medicine. Whole exome sequencing (WES) is considered the gold standard for measuring TMB in tumor biopsies [180, 181]. Clinical studies have shown that higher TMB is associated with better outcomes in ICI therapy, leading to the FDA’s approval of TMB as a companion diagnostic for pembrolizumab in treating pediatric and adult patients with unresectable or metastatic solid tumors with high TMB (≥ 10 mutations/megabase) [182]. Notably, not all mutations are detectable by the immune system, and a high TMB does not always ensure a positive response to ICT, nor does a low TMB rule out an effective immune response. This makes TMB an imperfect standalone biomarker across different tumor types [183]. Additionally, technical and biological limitations, such as the lack of standardization and a universal threshold for defining high TMB, complicate its use. The commonly used threshold of 10 mutations per megabase is constrained by variability across cancer types, making it difficult to apply universally. Future research should focus on validating TMB thresholds and exploring the immunogenicity of various mutation patterns [184]. The analysis of WES data from both germline and tumor DNA is complex, and limited availability of WES data in diagnostic biopsies further hinders its widespread use. In heterogeneous tumors with many subclonal mutations, detecting mutations becomes less effective as tumor purity decreases. These challenges may be addressed through advancements in bioinformatics, targeted next-generation sequencing, and machine learning-based TMB detection [185, 186].

In addition, genome damage due to deficiencies in the DNA mismatch repair system is closely linked to immunogenicity because it results in a high mutational load. DNA damage repair (DDR) defects can lead to increased TMB and more neoantigen formation [187, 188]. For example, tumors with high microsatellite instability (MSI-H) or dMMR show greater sensitivity to ICIs than those with proficient mismatch repair (pMMR). Initial studies found that CRC patients with dMMR had significantly better responses to pembrolizumab compared to those with pMMR status [189]. Subsequent clinical trials confirmed the utility of MSI as a predictive biomarker for immunotherapy, leading to the FDA’s approval of pembrolizumab for MSI-H/dMMR solid tumors and nivolumab for MSI-H CRC.

ICI sensitization strategies and clinical research progress

Based on the increasing understanding of the mechanisms underlying ICB resistance, research is focused on combining ICBs with various therapeutic strategies to enhance treatment sensitivity and efficacy (Fig. 1). Additionally, the ongoing major clinical trials on sensitizing ICIs for cancer therapy are summarized in Table 1.

Fig. 1

Current trends in sensitizing immune checkpoint inhibitors for cancer treatment

Table 1 The ongoing major clinical trials on sensitizing ICIs for cancer therapy

Immune checkpoint combinations

Several ICIs have been approved by the FDA, showing significant clinical benefits across various cancers. However, the response rate of ICIs varies by cancer type, ranging from 10 to 45%, prompting the investigation of combination therapies to improve efficacy [190]. CTLA-4 inhibitors primarily enhance DCs activity, which is suppressed by Tregs, and activate antigen-specific CD4⁺ T cells in the lymph nodes. This boosts T cell priming, expands T cell diversity, and reduces Tregs in the TME [191]. Conversely, PD-1/PD-L1 inhibitors aim to reverse T cell exhaustion and activate NK cells in tumors and lymph nodes, restoring cytotoxic T lymphocytes (CTLs) function. Combining CTLA-4 and PD-1/PD-L1 inhibitors leverages these distinct mechanisms, showing promising clinical results [192]. Therefore, this dual approach has demonstrated higher ORR and improved OS compared to monotherapy in melanoma, CRC, NSCLC, HCC, and RCC.

For instance, the CheckMate 067 trial revealed that nivolumab combined with ipilimumab significantly improved median OS and PFS in advanced melanoma [193]. Similarly, CheckMate 204 showed durable responses and long-term benefits for patients with melanoma brain metastases when treated with nivolumab plus ipilimumab [194]. The CheckMate 227 trial found that this combination improved 5-year survivorship compared to chemotherapy, regardless of PD-L1 expression [195]. Additionally, the CheckMate 142 study demonstrated that nivolumab plus low-dose ipilimumab was well-tolerated and effective as a first-line treatment for MSI-H/dMMR mCRC [196, 197]. Moreover, in the neoadjuvant setting, this combination achieved an impressive 100% pathological response rate in early-stage dMMR colon cancers [197].

While combination therapies offer enhanced efficacy, not all patients benefit equally. Ongoing research is exploring third-generation ICIs with non-overlapping mechanisms, such as targeting LAG-3, TIGIT, TIM-3, VISTA, and BTLA. Recent trials, such as those with relatlimab plus nivolumab and tiragolumab plus atezolizumab, have shown encouraging results, though some have not yet demonstrated significant improvements in PFS or OS [33, 139, 198]. Multiple trials are currently investigating these and other combination strategies to further improve outcomes (NCT05645692, NCT05785767, NCT04811027, NCT05483400, NCT04305054).

Combination with chemotherapy

Cytotoxic chemotherapy remains a cornerstone in cancer treatment, effective for most malignancies. It rapidly reduces tumor burden and induces immunogenic cell death (ICD), which can enhance immune responses by releasing cancer antigens for potential presentation. This effect can complement ICIs, potentially improving treatment outcomes [199, 200]. Chemotherapy’s benefits include stimulating tumor-specific immunity and synergistically enhancing ICI efficacy. For instance, combining chemotherapy with ICIs has shown success in treating NSCLC, SCLC, GC, biliary tract cancer, and triple-negative breast cancer (TNBC) [201,202,203,204,205,206]. Chemotherapy promotes the release of damage-associated molecular patterns (DAMPs) from tumor cells, which activate DCs and enhance antigen presentation. This process improves T cell activation and reduces the presence of immunosuppressive cells like Tregs, myeloid-derived suppressor cells (MDSCs), and M2 macrophages in the TME [207, 208]. It also encourages the differentiation of tumor-associated macrophages (TAMs) into the M1 phenotype, boosting both innate and adaptive immunity.

Several chemotherapy and ICI combinations have received regulatory approval [209]. The KEYNOTE-189 trial demonstrated that adding pembrolizumab to standard chemotherapy (pemetrexed-platinum) significantly improved response rates and OS in untreated metastatic non-squamous NSCLC, with a 48.3% ORR compared to 19.9% for chemotherapy alone. Long-term results from this study showed a 3-year OS of 71.9% among patients receiving pembrolizumab. Similarly, the CheckMate 816 trial found that neoadjuvant Opdivo plus platinum-based chemotherapy significantly extended median event-free survival and increased the rate of pathological complete response (pCR) in resectable NSCLC [210, 211]. In the KEYNOTE-355 study, pembrolizumab combined with chemotherapy notably improved PFS in metastatic TNBC with high PD-L1 expression (CPS ≥ 10), leading to accelerated FDA approval [212]. Numerous ongoing clinical trials are investigating additional combination regimens and their effects on various cancers (NCT0276357, NCT02578680, NCT02366143, NCT02872116, NCT02746796, NCT03138512, NCT03838159, NCT02488759) [205, 213,214,215,216,217]. Despite these advances, only a subset of patients benefits from combination therapies. Identifying biomarkers to better target these treatments remain crucial for optimizing patient outcomes [51, 218,219,220,221].

Combination with radiotherapy

Radiotherapy (RT) is a cornerstone of cancer treatment, with emerging evidence showing it can have both tumor-promoting and tumor-suppressing immune effects. RT induces significant DNA damage, ICD, and tumor shrinkage, which can enhance immune responses by generating neoantigens and increasing the release of inflammatory cytokines. This promotes the infiltration of effector CD8⁺ T cells and DCs into the TME, potentially leading to an abscopal effect on non-irradiated lesions [222]. However, RT also has immunosuppressive effects, such as inducing anti-immunogenic cytokines and increasing the presence of immunosuppressive cells like Tregs, MDSCs, and M2 macrophages [223]. Recent studies have found that RT can upregulate PD-L1 expression on DCs and myeloid cells, which may facilitate metastasis. Blocking the PD-L1/CXCL10 axis or MDSC infiltration during local irradiation can mitigate these effects [224].

Combining RT with ICIs shows potential synergy. For instance, the Phase III PACIFIC trial demonstrated that durvalumab, an anti-PD-L1 antibody, significantly improved PFS and OS in stage III NSCLC patients after chemoradiotherapy [87, 225,226,227]. Similarly, adding durvalumab to neoadjuvant therapy with stereotactic body radiotherapy (SBRT) increased the MPR in early NSCLC [228]. In metastatic NSCLC, combining SBRT with pembrolizumab improved response rates, though not all studies have met predefined efficacy criteria [229, 230]. Despite mixed results in other cancers, such as HNSCC and Merkel cell carcinoma, ongoing research aims to optimize RT and ICI combinations by adjusting treatment timing, dose, and site. Low-dose radiation strategies are being explored as potential enhancers of ICI response [231,232,233,234,235].

Preclinical studies suggest low-dose RT can enhance immune checkpoint inhibitor effectiveness by promoting macrophage differentiation into the M1 phenotype and increasing T cell recruitment [231, 232, 236]. For example, combining low-dose RT with dual PD-L1 and VEGFA blockers has shown promise in treating HCC by activating exhausted CD8⁺ T cells [237]. Additionally, a recent study indicated that low-dose RT combined with immunotherapy can significantly improve outcomes in SCLC patients, with high ORR and prolonged PFS [235]. Overall, while combining RT with immunotherapy holds promise, further research is needed to identify optimal protocols and patient subgroups to maximize therapeutic benefits.

Combination with targeted therapy

Cancer is a genetic disease driven by multiple mutations. Targeted therapies, focusing on molecules involved in cancer processes such as carcinogenesis, angiogenesis, and metastasis, have emerged as crucial components of precision medicine. These therapies target specific molecules that support tumor progression, offering a clinical basis for mechanism-based treatment of malignancies. Key therapeutic targets include BRAF, EGFR, HER2, MEK, and PARP. Many targeted inhibitors have been approved by the FDA, either as monotherapies or in combination with other drugs, showing promising clinical results [238, 239]. Preclinical studies indicate that targeting tumor signaling pathways can enhance T cell infiltration, restore dendritic cell function, neutralize MDSCs, and improve antigen presentation, providing a rationale for combining ICBs with targeted therapies [240].

The BRAF oncogene, present in many malignant melanomas and other cancers, is a major focus of targeted drug discovery. BRAF and MEK inhibitors are first-line treatments for BRAFV600 mutation-positive melanoma, and combinations with immunotherapy are being actively studied (NCT02967692; NCT02752074; NCT02625337; NCT03178851; NCT02908672) [241,242,243,244,245]. The Phase III IMspire150 study found that adding atezolizumab to BRAF and MEK inhibitors (vemurafenib and cobimetinib) significantly improved PFS (15.1 months vs. 10.6 months) in BRAFV600 mutation-positive melanoma [246, 247]. This combination also showed activity in patients with CNS metastases [248].

Sequencing immunotherapy and targeted therapy is being explored to optimize combination efficacy and identify biomarkers for predicting long-term benefits [249,250,251,252,253,254]. Additionally, anti-PD-1/PD-L1 therapy has shown comparable efficacy to other targeted therapies such as PARP inhibitors, MERTK inhibitors, PI3K inhibitors, and CDK4/6 inhibitors, which are being tested for improved responses to ICBs [255,256,257,258,259].

Combination with anti-angiogenic therapy

Aberrant tumor angiogenesis, a hallmark of cancer, significantly contributes to tumor immunosuppression and immune evasion. Tumor blood vessel abnormalities can cause intra-tumor hypoxia (ITH) and a low pH TIME, which are linked to decreased T cell infiltration [260,261,262]. Additionally, vascular endothelial growth factor (VEGF) and angiopoietin-2 (ANGPT2) influence the TIME by inhibiting DCs maturation, upregulating PD-L1 on endothelial cells, and promoting the polarization of TAMs into immunosuppressive M2-like phenotypes [263].

Anti-angiogenic therapies, including anti-VEGF agents (bevacizumab), anti-VEGFR agents (ramucirumab), and tyrosine kinase inhibitors (sorafenib, lenvatinib, apatinib, sunitinib, axitinib), work by disrupting neovascularization and limiting tumor nutrient supply. This leads to improved vascular perfusion, increased immune cell infiltration, and reduced presence of MDSCs, Tregs, and M2 macrophages in the TIME, thereby enhancing anti-tumor effects [264]. Emerging preclinical and clinical evidence indicates that combining ICBs with anti-angiogenic therapies during the “vascular normalization” phase can improve clinical outcomes [265]. In various preclinical models, VEGFR inhibitors combined with ICBs have demonstrated synergistic effects by regulating neo-angiogenesis, reversing ITH, and re-sensitizing tumors to ICB therapy.

Clinical trials have confirmed the benefits of this combination approach. The Phase III KEYNOTE-426 study showed that the combination of pembrolizumab and axitinib resulted in significantly longer OS and PFS compared to sunitinib in untreated advanced clear-cell RCC, establishing a new standard of care [266]. Similarly, a Phase II trial is evaluating nivolumab plus axitinib in anti-PD-1 refractory unresectable advanced melanoma [267]. In addition, the Phase III IMbrave150 study led to the FDA approval of atezolizumab plus bevacizumab in 2020 as a first-line treatment for unresectable or metastatic HCC [268]. Numerous ongoing Phase III trials are investigating this combination therapy across various cancers, with initial results showing promising anti-tumor activity (NCT03764293, NCT03468218, NCT04047017, NCT03950154, NCT04547088, NCT03141177) [269,270,271,272,273,274].

Combination with adoptive T cell therapy

Combining ICIs with adoptive T cell therapy is an area of significant interest. Adoptive T cell therapy involves large infusions of tumor-specific T lymphocytes isolated from the TIME. These T cells are specific to tumor antigens and can bypass defects in endogenous antigen delivery, making them a promising strategy for stimulating anti-tumor immune responses.

Chimeric antigen receptor (CAR) T cells are engineered T cells with receptors specific to tumor antigens. The CAR fusion protein, comprising an antibody binding domain and a T cell receptor signaling domain, is introduced into autologous T cells using lentivirus or retrovirus. CAR-T cells can recognize tumor-specific cell surface antigens without MHC restrictions, allowing for precise targeted cytotoxicity and expanding therapeutic potential applications. Preclinical and clinical studies have demonstrated that CAR-T cell therapies can provide durable and meaningful responses, particularly in hematological malignancies [275]. However, the complexity of the TIME in solid tumors and upregulation of inhibitory immune checkpoints can impair CAR-T cell function. Combining CAR-T cell therapy with ICBs may enhance anti-tumor efficacy by overcoming these challenges [276].

Studies have shown that CAR-T cells with endogenous PD-1 checkpoint blockade exhibit improved anti-tumor activity [277,278,279,280]. For instance, Rafiq et al. engineered CAR-T cells to secrete a PD-1 blocking single chain variable fragment (scFv), which enhanced the anti-tumor activity of CAR-T cells in a xenogeneic mouse model [281]. Currently, the mechanism of blocking the PD1/PD-L1 axis to reverse the function of CAR-T cells is also under intensive investigation. Recent studies have also shown that PD-L1 upregulation on M2 macrophages significantly inhibits CAR-T cell activity. Combining PD L1 blockade with CAR-T cell therapy has been shown to promote M1-like macrophages and reduce M2 macrophages, thereby improving CAR-T cell function [282]. Moreover, downregulation of PD-1 expression on CAR-T cells can enhance their anti-tumor efficacy by preserving early cellular memory and reducing T cell depletion [283,284,285,286]. CAR-T cells combined with PD-1/PD-L1 axis blockade have shown promising results in preclinical studies across various cancers, including breast cancer, TNBC, advanced thyroid cancer, CRC, and glioblastoma [287,288,289,290]. Clinical trials are actively exploring these combinations [291, 292]. For example, a Phase Ib study of a novel anti-CD19 CAR expressing PD-1/CD28 chimeric converter receptor demonstrated high remission rates in patients with PD-L1-positive B-cell lymphoma [293]. Similarly, a Phase I study of mesothelin-targeted CAR- T cells combined with PD-1 blockade showed potent anti-tumor activity and long-term CAR-T cell survival in malignant pleural mesothelioma patients [294]. Similarly, a Phase I study of mesothelin-targeted CAR-T cells combined with PD-1 blockade showed potent anti-tumor activity and long-term CAR-T cell survival in malignant pleural mesothelioma patients [295].

In summary, combining CAR-T cell therapy with PD-1/PD-L1 blockade has shown significant potential in preclinical and clinical studies, with ongoing research aimed at optimizing these strategies for improved outcomes in cancer treatment [296,297,298,299,300,301].

Combination with oncolytic viruses

Oncolytic viruses (Ovs) offer a promising therapeutic approach that combines direct tumor cell destruction with the induction of systemic anti-tumor immunity. These viruses selectively replicate within tumor cells, leading to cell lysis and the release of tumor-specific antigens, which in turn stimulates both innate and adaptive immune responses. Additionally, Ovs can be engineered to enhance their immunostimulatory effects, thereby amplifying pro-inflammatory responses in the TIME and improving immune-mediated tumor elimination at both local and distant sites [302]. Given the challenges associated with tumor resistance to immunotherapy, combining Ovs with ICBs is being explored as a potentially synergistic strategy to boost immune responses. This combination approach has been extensively studied in preclinical and clinical trials.

Talimogene Laherparepvec (T-VEC), an attenuated herpes simplex virus type 1 (HSV-1), is the first FDA-approved oncolytic viral therapy. It is used to treat skin and lymph node lesions in patients with unresectable melanoma [303, 304]. T-VEC is genetically engineered to express GM-CSF, which enhances anti-tumor responses by promoting DCs maturation and stimulating tumor antigen-reactive T cells. In a Phase II study, T-VEC combined with ipilimumab showed a significantly higher persistent ORR compared to ipilimumab alone in unresectable stage IIIB/IV melanoma patients, without additional safety concerns. This was the first randomized controlled trial to assess the addition of an oncolytic virus to a checkpoint inhibitor and achieved its primary endpoint [305, 306]. However, a subsequent global Phase III trial found that combining T-VEC with pembrolizumab did not significantly improve PFS or OS compared to pembrolizumab alone, despite promising Phase Ib results [307].

RP1 is another HSV-1-based oncolytic virus that encodes a fusogenic protein (GALV-GP-R-) and expresses human GM-CSF. In a Phase 1/2 trial, RP1 combined with nivolumab demonstrated promising anti-tumor activity in patients with cutaneous cancers, including those with anti-PD-1 refractory stage IIIb/IV melanoma. Tumor biopsies from treated patients showed evidence of immune activation, such as increased CD8⁺ T cell infiltration and heightened PD-L1 expression [308]. Initial data from an expanded cohort suggest that RP1 plus nivolumab continues to provide durable and clinically significant anti-tumor activity in patients with progressive melanoma, with an overall ORR of 36.3% [309].

Currently, multiple clinical trials are investigating various Ovs in combination with ICBs (NCT05733611, NCT06311578, NCT05076760, NCT05346484, NCT06067061, NCT05222932, NCT05564897, NCT05271318, NCT04725331), showing promising potential for this combined therapeutic strategy [310,311,312,313,314,315,316,317,318,319].

Combination with vaccine

Cancer immunotherapy has transformed oncology, offering effective treatments across various cancers. Understanding the cancer-immune cycle is crucial for grasping the mechanisms and sensitivities of anti-cancer immune responses. DCs in tumor-draining lymph nodes capture antigens and present them to naive T cells, initiating and sustaining anti-tumor immunity. Therapeutic cancer vaccines aim to stimulate the immune system against tumors by combining antigens with adjuvants to activate DCs and induce a lasting anti-tumor memory [320]. Various vaccine platforms are in clinical trials, including DC-based, DNA-based, RNA-based, peptide-based, and virus-based vaccines [321]. Successful vaccines often target specific antigens, which may be either shared antigens overexpressed in tumors or neoantigens arising from somatic mutations. Current research is focusing on neoantigen-targeted vaccines to improve specificity and minimize off-target effects. However, challenges like low TMB and neoantigen immunogenicity complicate vaccine development [322]. Sipuleucel-T, a DC-based vaccine approved for hormone-refractory prostate cancer, has shown limited survival benefits. To enhance efficacy, combining vaccines with ICBs is being explored to overcome immunosuppressive mechanisms and improve vaccine performance [323, 324].

In recent trials, combining a personalized therapeutic cancer vaccine (PTCV) with pembrolizumab showed promising results in advanced HCC, with an ORR of 30.6% and a complete response (CR) of 8.3%. Neoantigen-specific T cell responses and increased T cell infiltration at the tumor site were observed [18]. Additionally, an ongoing trial involving a vaccine encoding 20 shared neoantigens, utilizing self-amplified mRNA (samRNA) and chimpanzee adenovirus (ChAd68), in combination with ipilimumab and nivolumab, has demonstrated acceptable tolerance and potential efficacy in advanced solid tumors [325]. Future advancements in vaccine platforms and TIME profiling will further enhance the efficacy of these combined therapies [245, 326,327,328,329,330,331].

Combined with FMT

A growing body of preclinical and clinical evidence indicates that the gut microbiome can significantly impact the efficacy of cancer immunotherapy. Advances in high-throughput sequencing technologies have enabled extensive research into how gut microbiota influences the host immune system. Studies have demonstrated that the microbial genome affects hormone secretion, metabolism, immune function, and physiological homeostasis through various mechanisms [332, 333]. For instance, Matson et al. identified a notable correlation between the composition of gut commensal microorganisms and the clinical response of patients with metastatic melanoma by analyzing baseline fecal samples before immunotherapy. They observed that bacterial species such as Bifidobacterium longum, Enterococcus faecalis, and Collinsella globosa were more prevalent in patients who responded to ICIs. Moreover, fecal microbiota transplantation (FMT) from these responders into germ-free mice enhanced T-cell responses and improved the efficacy of anti-PD-L1 therapy [334]. Similarly, Routy et al. found that the abundance of Akkermansia muciniphila was associated with clinical responses to ICIs in cancer patients. Their macro-genomic analysis of fecal samples at diagnosis revealed that higher levels of this bacterium correlated with better treatment outcomes. Furthermore, increased recruitment of CCR9⁺ CXCR3⁺ CD4⁺ T lymphocytes within tumors and the restoration of PD-1 blockade efficacy were observed in an interleukin-12-dependent manner. A meta-analysis of melanoma patients treated with PD-1 inhibitors further supported the complex relationship between gut microbiota and immunotherapy responses [335,336,337]. These findings underscore the potential of gut microbiome biomarker analysis to enhance immune regulation and address tumor immune checkpoint resistance, particularly following antibiotic pretreatment [338, 339].

Molecular mechanisms underlying tumor sensitization to immunotherapy

To expand the clinical benefits of ICIs, it is crucial to understand the mechanisms of drug resistance and response to ICB. While significant progress has been made in understanding ICI resistance, further exploration of sensitization mechanisms is needed. This section reviews the genomic and molecular factors, both intrinsic and extrinsic to tumors, that are associated with resistance and response to tumor immunotherapy (Fig. 2).

Fig. 2

Insights into tumor-intrinsic and -extrinsic mechanisms associated with resistance and sensitivity to ICIs

Tumor intrinsic mechanisms of sensitization to ICI

Antigen-presenting molecules machinery: (MHC, HLA)

Cytotoxic CD8⁺ T lymphocytes initiate effective anti-tumor immunity by recognizing tumor antigens presented by MHC I. However, defects in the neoantigen presentation machinery, mediated by MHC I downregulation due to chromosomal deletions or loss of heterozygosity (LOH), are considered as primary or acquired resistance mechanisms that enable evasion of T cell recognition [340,341,342]. MHC I molecules, consisting of membrane-anchored heavy chains and β2-microglobulin (β2m), are essential for antigen presentation. Mutations in β2m or LOH can reduce MHC I expression on tumor cells, facilitating immune evasion. Additionally, epigenetic alterations, such as chromatin modifications, abnormal DNA methylation, and histone changes, can silence MHC I expression, further promoting immune escape [343,344,345]. Understanding the mechanisms by which tumor cells evade immune elimination is crucial for enhancing the effectiveness of cancer immunotherapy. MHC-I expression is regulated by IFNGR signaling, which involves STAT1-mediated induction of the IRF1 transcription factor that binds to MHC-I gene promoters. In optineurin-deficient CRC cells, impaired IFNγ and MHC-I signaling leads to palmitoylation-dependent lysosomal sorting and degradation of IFNGR1, resulting in decreased MHC-I levels and intrinsic resistance to immunotherapy. This presents a potential pharmacological strategy to sensitize checkpoint therapy in CRC. Additionally, post-transcriptional regulatory mechanisms also impact MHC-I expression. NBR1-mediated macroautophagy can selectively degrade MHC-I molecules, facilitating immune evasion in pancreatic ductal adenocarcinoma (PDAC). This degradation can be reversed by autophagy inhibition with chloroquine, either genetically or pharmacologically [346, 347]. By degrading MHC molecules, tumor cells reduce their visibility to immune cells, particularly cytotoxic T cells, which rely on MHC for recognition and targeting of cancer cells. NK cells can target cells lacking MHC I, but tumors often evade this by reducing MICA/B levels on their surface, presenting a significant challenge for current immunotherapies.

Immunogenic neoantigens: mismatch repair (pMMR/dMMR)

Genomic and chromosomal instability is a hallmark of cancer, altering the genomic landscape and influencing immune responses. Tumors with non-synonymous mutations or genomic rearrangements often produce highly immunogenic neoantigens. Defects in DNA repair pathways, such as the mismatch repair (MMR) system, increase the formation of clonal neoantigens, making tumors more recognizable and sensitive to ICIs [197, 348]. The MMR system, primarily involving the proteins MLH1, MSH2, MSH6, and PMS2, corrects replication errors not fixed by DNA polymerase proofreading. Germline mutations in MMR genes, such as in Lynch syndrome, lead to MSI due to the accumulation of point mutations and defects in repeat sequences. Tumors with MSI-H or dMMR display frequent frameshift mutations and higher TMB, making them more responsive to ICIs [349,350,351]. The increased mutation load stimulates the generation of immunogenic neoantigens, which triggers T cell infiltration and immune activation. However, immune editing can lead to the loss of these neoantigens by selectively removing highly immunogenic tumor cell subpopulations, representing a mechanism of acquired resistance to immune surveillance.

Epigenetic reprogramming

Epigenetics involves heritable changes in gene expression that do not alter the DNA sequence. Key mechanisms include DNA methylation, chromatin remodeling, histone modification, and non-coding RNA regulation [352]. These processes ensure precise and lasting gene regulation. Tumorigenesis often features significant genetic and epigenetic alterations. For example, about 20% of cancers exhibit loss-of-function mutations in genes encoding the SWI/SNF chromatin remodeling complex [353]. DNA methylation, primarily regulated by DNA methyltransferase 1 (DNMT1), typically leads to gene silencing and is a stable feature of cells, often disrupted in cancer. Tumor cells exploit epigenetic mechanisms to evade immune surveillance and reduce the efficacy of ICB, often by suppressing endogenous interferon responses. By modifying the epigenetic landscape, tumor cells can either activate or repress human endogenous retroviruses (HERVs), influencing immune responses. Activation of HERVs can trigger an interferon-related innate immune response, enhancing tumor cell detection through viral mimicry. Chromatin-modifying agents, such as DNA methyltransferase inhibitors (DNMTis), can reactivate HERVs, inducing a type I interferon response and promoting apoptosis in OC models. Additionally, DNMTis and histone deacetylase inhibitors (HDACis) can generate HERV-derived immunogenic neoantigens, increasing tumor cell visibility to cytotoxic T cells and boosting immune-mediated tumor destruction [354,355,356,357].

Epigenetic changes can also drive tumor-specific neoantigen formation and reactivation of developmental genes, producing differentiation antigens [358, 359]. Loss of MHC-I expression in tumors may be due to the epigenetic silencing of antigen-presenting genes, such as TAP1, TAP2, and β2m. However, this can be reversed with DNMTis or HDACis. Additionally, global DNA methylation can upregulate PD-L1 and inhibitory cytokines. The cGAS-STING signaling pathway, which detects abnormal DNA and triggers type I interferon responses, is often inhibited by epigenetic silencing in tumors, reducing immunotherapy efficacy. In preclinical models, DNMT1 and EZH2-mediated modifications inhibit T-helper 1 (Th1) chemokines CXCL9 and CXCL10, blocking effector T cell infiltration and correlating with poor clinical outcomes in cancers like OC [360,361,362]. Understanding these epigenetic mechanisms offers potential therapeutic strategies to overcome immunosuppression and enhance immunotherapy effectiveness.

Oncogenic signaling pathways activation

Tumor intrinsic oncogenic signaling pathways drive malignant progression and contribute to the maintenance of an immunosuppressive TME. Mutations in pathways such as IFNγ/JAK/STAT, WNT/β-catenin, PTEN/PI3K, and RAS have been shown to both induce immunogenic responses and mediate immune exclusion, thereby fostering immune resistance. Investigating how these oncogenic pathways influence resistance to ICIs may offer valuable insights for enhancing the effectiveness of immune checkpoint therapies.

IFNγ/JAK/STAT signaling pathway

Type II interferon (IFN-γ), primarily secreted by CD8⁺ CTLs, is crucial for anti-tumor immunity. When IFN-γ binds to its receptors IFNGR1 and IFNGR2, it activates Janus kinases (JAK1 and JAK2) and the STAT signaling pathway. This activation enhances gene transcription and antigen presentation, bolstering anti-tumor effects and increasing cytotoxic T cell infiltration into tumors [363, 364]. However, IFN-γ/JAK/STAT signaling can also contribute to ICI resistance. This occurs when interferon regulatory factor 1 (IRF1) drives the transcriptional upregulation of PD-L1, leading to PD-L1-independent resistance. Mutations in the JAK-STAT pathway, such as loss-of-function mutations in JAK1/JAK2, can impair IFN-γ’s efficacy and serve as potential predictors of ICB resistance [365].

WNT/β-catenin

The Wnt signaling pathway regulates cell homeostasis and stem cell function. Mutation-driven activation of the Wnt-β-catenin pathway is common in cancers, promoting self-renewal and contributing to resistance to ICB therapies [366, 367]. Abnormal Wnt/β-catenin activation induces upregulation of the transcription factor ATF3, leading to immune ignorance in the TME. This pathway inhibits the transcription of chemokine CCL4, reducing the recruitment of CD103⁺ DCs and impeding T cell infiltration. Consequently, this results in CD4⁺ T cell tolerance and CD8⁺ T cell immunosuppression due to ineffective cross-priming with DCs [368,369,370]. Analysis from The Cancer Genome Atlas (TCGA) reveals that Wnt/β-catenin activation correlates with non-T cell inflammatory gene expression across various tumors [371,372,373]. Given its role in immunosuppression, targeting the Wnt/β-catenin pathway is being explored as a strategy to overcome ICB resistance.

PI3K-AKT-mTOR

The PI3K-AKT-mTOR signaling pathway is crucial for cell growth, proliferation, and survival. Mutations activating this pathway are prevalent in various cancers and often lead to increased PD-L1 expression on tumor cells due to PTEN or PIK3CA gene alterations, potentially enabling tumor immune evasion and affecting sensitivity to ICB therapy. PTEN, a frequently inactivated tumor suppressor gene, negatively regulates the PI3K-AKT-mTOR pathway, contributing to anti-tumor activity. PTEN loss is associated with decreased T cell infiltration and reduced expression of oncolytic cytokine genes. This loss is more common in non-T-cell inflammatory tumors, which exhibit less response to PD-1 inhibitors compared to PTEN wild-type tumors [374,375,376]. Conversely, in preclinical models of PTEN-mutated prostate cancer and melanoma, reactivating PTEN through mRNA nanoparticle technology has shown promise in enhancing immune activation and anti-tumor efficacy when combined with anti-PD1 therapy [377]. These findings suggest that targeting the PI3K-AKT-mTOR pathway and PTEN could improve ICB therapy outcomes, supporting the exploration of combination strategies involving PI3K inhibitors.

RAS

RAS genes (KRAS, NRAS, HRAS) are commonly mutated oncogenes that encode small GTP-binding proteins crucial for linking upstream receptors to downstream signaling pathways involved in cell survival, proliferation, migration, and metabolism. Mutations in RAS lock the protein in an active GTP-bound state, leading to continuous activation of downstream pathways such as RAF-MEK-ERK and PI3K-AKT [378, 379]. Clinical trials have shown that KRAS mutations, particularly in NSCLC, are associated with increased PD-L1 expression, which promotes tumor immune escape by stabilizing PD-L1 mRNA. Additionally, RAS pathway alterations can reduce IFNγ-induced MHC expression and affect T cell infiltration in cancers like colorectal and breast cancer. Preclinical studies suggest that combining KRAS G12C inhibitors with anti-PD-1 therapy enhances CD8⁺ T cell infiltration and improves immunotherapy sensitivity [380,381,382,383,384].

Immunological extrinsic mechanisms for sensitizing to ICIs in the tumor microenvironment

Tumor extracellular matrix (ECM) components and cytokines

The TME consists of immune and non-immune components, such as stromal cells, blood vessels, cytokines, and extracellular matrix, which influence tumor characteristics like angiogenesis, invasion, and metastasis. These components interact with tumor and immune cells, significantly affecting tumor progression and resistance to ICB therapies [385]. The tumor-promoting microenvironment is characterized by abundant immunosuppressive myeloid cells, dense desmoplastic stroma that obstructs cytotoxic T-cell infiltration and systemic chemotherapy, and an increase in M2 macrophages. Remodeling the TME with CD40 agonists can polarize M2 macrophages to M1, activate APCs, enhance tumor-specific T-cell responses, degrade fibrotic stroma, and improve chemotherapy sensitivity. In clinical practice, the combination of the CD40 agonist mitazalimab with the mFOLFIRINOX regimen effectively activates immune cells and promotes anti-tumor responses, presenting a promising therapeutic option for metastatic PDAC with poor prognosis [386].

Epithelial-Mesenchymal Transition (EMT) enables epithelial cells to acquire mesenchymal traits, decreasing their susceptibility to immune-mediated lysis. EMT also contributes to immune desensitization through mechanisms such as altered PD-L1/PD-L2 expression, reduced sensitivity to cell death receptor pathways, and immune synaptic defects [387,388,389]. High angiogenesis can lead to abnormal blood vessels and increased interstitial pressure, hindering immune cell infiltration and ICB efficacy.

Cancer-associated fibroblasts (CAFs), influenced by signals like TGF-β, Wnt/β-catenin, PI3K-AKT-mTOR, JAK/STAT, and EGFR, play a complex role in the TME. They promote tumor growth, immune evasion, and resistance to therapy [390,391,392]. TGF-β signaling, which can be activated through canonical Smad pathways or non-Smad pathways (e.g., MAPK, Rho GTPases, PI3K-Akt-mTOR), affects ECM gene expression and immune cell composition, potentially creating an immunologically “hot” TME [393,394,395]. However, CAFs expressing fibroblast-activating protein (FAP) can suppress anti-tumor T cell functions, especially in gastrointestinal tumors. Additionally, TGF-β impairs therapeutic sensitivity of cetuximab by up-regulating CD59 and inhibits T cell proliferation which further reducing ICB therapy susceptibility [396, 397].

Immune cell

Myeloid cells are key components of the TIME and are linked to poor prognosis and reduced efficacy of immunotherapy across various cancers. MDSCs, a crucial myeloid subgroup, are known for their immunosuppressive effects, which contribute to resistance to ICIs. MDSCs release mediators like arginase 1, peroxynitrite, inducible nitric oxide synthase (iNOS), and reactive oxygen species (ROS), creating an immunosuppressive TME [398]. This downregulates T cell and NK cell activity, affects Tregs differentiation, and induces an immunosuppressive macrophage phenotype, reducing immunotherapy effectiveness. Tumor-infiltrating MDSCs also express high levels of PD-L1 and other inhibitory checkpoints. Elevated PI3K-γ expression in myeloid cells can further exacerbate inflammation and tumor progression. In preclinical models, targeting PI3K-γ has been shown to restore ICI sensitivity by reprogramming macrophages and enhancing T cell activity [399].

Tumor-associated macrophages (TAMs), another major myeloid subset, play a significant role in tumor immune regulation. TAMs are categorized into M1 and M2 phenotypes. M1 macrophages promote anti-tumor responses with pro-inflammatory cytokines, whereas M2 macrophages suppress CD8⁺ T cell activation through anti-inflammatory cytokines, recruit Tregs, and facilitate tumor immune escape [400]. M2 TAMs also upregulate inhibitory checkpoint molecules, amplifying their immunosuppressive effects. Inhibiting M2-TAM activity and shifting macrophages toward the M1 phenotype can counteract ICIs resistance [401].

Regulatory T cells (Tregs), characterized by CD25, CD4, and FoxP3, are highly immunosuppressive and crucial for maintaining immune tolerance [402]. Tregs are prevalent in the TIME and bloodstream, where they diminish CD4⁺ and CD8⁺ T cell responses through cytokine secretion (e.g., IL-2, IL-10, IL-35, TGF-β) and checkpoint molecule expression (e.g., CTLA-4, PD-1). Tregs create an immunosuppressive environment by secreting inhibitory cytokines and expressing CTLA-4 and PD-1, which further downregulates co-stimulatory molecules on APCs and increases free PD-L1 [403]. Preclinical studies suggest that depleting tumor-infiltrating Tregs can enhance anti-tumor immunity and ICIs response [404]. However, incomplete depletion may lead to Treg expansion and the upregulation of alternative checkpoints like TIM-3 and LAG-3, contributing to ICB resistance.

Discussion

Immunotherapy has emerged as a crucial pillar in cancer treatment, with significant advances in ICIs marking its progress. Despite notable successes in inducing tumor regression and prolonging disease control across various cancer types, many patients either do not respond to these therapies or develop resistance over time. Understanding the mechanisms behind such resistance and incorporating clinical translational research are essential for improving outcomes and optimizing patient selection through biomarker-based strategies. Recent advancements in genomics, transcriptomics, and other molecular analyses have facilitated the identification of predictive markers for ICI response and resistance. High-throughput sequencing technologies are particularly valuable for dissecting the complex mechanisms underlying treatment resistance. While these technologies offer insights into genomic and functional alterations that contribute to resistance, identifying robust prognostic biomarkers remains a challenge. Broadly exploratory biomarkers have shown promise in guiding clinical trials, yet precise, validated markers are needed for effective patient stratification. Intra- and inter-tumoral heterogeneity complicates the analysis of immune responses, and preclinical models using identical transplantable cell lines often fail to replicate clinical realities. To address this, researchers are developing advanced preclinical tumor models using patient-derived samples that preserve TIME signatures. These models help elucidate both immune and non-immune pathways influencing anti-tumor immunity and provide a better understanding of factors affecting immune response beyond the tumor itself.

Currently, strategies to enhance the efficacy of tumor immunotherapy mainly focus on combining different immune checkpoint inhibitors, targeted therapies (especially anti-angiogenic agents), radiotherapy (including different doses and various radiotherapy modalities), and chemotherapy. New treatment approaches, such as the combination with CAR-T therapy, also play a crucial role in cancer treatment. In recent years, several studies have found high expression of immune-suppressive molecules like LAG3 and IDO in PD-L1⁺ populations, and combined inhibition of LAG-3 and PD-L1 can significantly enhance the activity of effector T cells. Recent research indicates that combination immunotherapy with PD-L1/LAG3 dual-target agents has shown promising clinical response rates in cancers such as esophageal and liver cancer. In addition to LAG3, TIGIT and TIM3 have also been confirmed by multiple studies to co-express with PD-1/PD-L1, and the co-expression of PD-L1/TIGIT and PD-L1/TIM3 is associated with differences in OS. Therefore, shifting from single-target to dual-target approaches might be a strategy to overcome primary resistance to immunotherapy. Currently, the most researched strategy involves using preemptive anti-angiogenic therapy to reverse immune resistance. Regorafenib and apatinib combined with PD-1 monoclonal antibodies have shown good efficacy. In addition, the approach of combining immunotherapy with the blockade of driver genes such as KRAS, MYC, EGFR, and HER2 to reverse resistance is also receiving considerable attention.

While various immune sensitization methods exist, future clinical practice must tailor treatment strategies based on patients’ multi-omics profiles (e.g., genomics, transcriptomics, immunomics, metabolomics) and clinical features (e.g., tumor pathological type, stage, patient age). The goal is to personalize therapies by understanding each patient’s unique clinical and molecular characteristics, optimizing efficacy while minimizing side effects. Despite the potential of personalized immune sensitization, several challenges remain in its clinical application: (1) tumor microenvironment complexity: tumor evolution and intratumor heterogeneity (ITH) drive disease progression and resistance to ICIs. Advances in single-cell analysis and spatial transcriptomics are improving our understanding of TME heterogeneity, enabling novel strategies like cellular immunotherapy. Technologies such as scRNA-seq, scATAC-seq, and whole-genome sequencing provide deeper insights into TME dynamics and resistance pathways; (2) analyzing multi-omics data is resource-intensive and demands advanced technical support and specialized expertise. However, the integration of machine learning and AI models is crucial for advancing the field. Recent advances in combining neural network-based machine learning with multi-omics data may improve predictions of responses to immuno-chemotherapy, helping to address this challenge [405,406,407]; (3) managing side effects in combination therapies: the combination of ICIs with other treatments can lead to increased side effects, making it crucial to balance efficacy with safety. In conclusion, as multi-omics research and AI technologies continue to advance, personalized strategies for sensitizing tumors to ICIs can lead to the development of highly effective treatment plans that maximize immunotherapy outcomes. Personalized immune sensitization is poised to become a cornerstone of cancer treatment.

As the mechanisms of resistance to immune checkpoint therapy are further unraveled, there will be critical advancements in clinical translational research. This progress will drive the development of more effective, personalized immune-oncology strategies, including rational combinations of immune checkpoint blockers with other therapeutic modalities. By integrating these insights, we can enhance the sensitivity of immunotherapy and improve clinical outcomes for individual patients.