Decoding the tumor immune microenvironment in lung squamous cell carcinoma: characteristics, regulatory mechanisms, and future directions in immunotherapy

Introduction

Lung cancer is the leading cause of cancer-related death worldwide (1). Lung squamous cell carcinoma (LUSC) constitutes a major subtype of non-small cell lung cancer (NSCLC), representing approximately 20–30% (2). Unlike lung adenocarcinoma (LUAD), LUSC lacks suitable targeted therapies due to the scarcity of driver mutations (3). Recently, immunotherapy has become the principal therapeutic approach for patients with advanced or metastatic LUSC, regardless of programmed death-ligand 1 (PD-L1) expression levels, significantly altering the treatment landscape (4,5). However, the median overall survival (mOS) in patients with LUSC has only modestly increased by 5.5 months compared to the 11.4-month increase observed in patients with non-squamous NSCLC receiving a combination of immunotherapy and chemotherapy versus chemotherapy alone (5,6). Furthermore, over 50% of patients develop resistance to immune checkpoint inhibitors (ICIs) within 1 year (7). These challenges underscore the critical need to explore the underlying mechanisms driving immunotherapy resistance and the limited clinical benefits in LUSC. Thus, it is imperative to gain an in-depth understanding of the features and regulatory mechanisms of LUSC’s tumor immune microenvironment (TIME), as the TIME plays a pivotal role in shaping immune responses and therapeutic outcomes.

The TIME constitutes a sophisticated ecosystem encompassing tumor cells, immune components, and stroma elements in modulating tumor growth, invasion, and metastasis (8-10). A previous study has noted differences in molecular subtypes, tumor mutation burden (TMB), immunogenicity, and immune cell infiltration between LUSC and LUAD (11). Recently, multi-omics techniques such as single-cell RNA sequencing (scRNA-seq) and spatial omics revealed that the TIME of LUSC has more pronounced immune heterogeneity, especially regarding the activities of T cells and the dynamics of cell-cell interactions (12-14). These differences may indicate variations in the immunological efficacy between LUSC and LUAD, advancing our understanding of LUSC’s unique immune epitopes. Therefore, accurate classification and comprehensive generalization of the TIME can facilitate precise selection of immune populations and treatment strategies.

In this review, we propose novel immunotypes to LUSC and provide a comprehensive overview of the features of LUSC’s TIME based on current knowledge. From the perspectives of metabolomics, epigenetics, and genomics, we analyze the regulatory mechanisms of TIME. Finally, we propose significant therapeutic strategies that offer novel insights and hold promise for future advancements in immunotherapy.

Immunotypes of LUSC

Although there is no precise classification of the LUSC’TIME, previous molecular expression profiles of LUSC showed the correlation with different patterns of immune infiltration, suggesting the heterogeneity in the TIME of LUSC. In light of existing evidence, we propose novel immunotypes for LUSC.

In 2010, Wilkerson et al. classified LUSC into four mRNA-based subtypes—primitive (16%), classical (37%), basal (26%), and secretory (21%)—each with distinct gene expression profiles, clinical outcomes, and patient characteristics (15). Faruki et al. demonstrated distinct TIME across the four molecular subtypes: the secretory subtype (high immune infiltration, immunotherapy-sensitive), the classical subtype (immunosuppressive TIME), the primitive subtype (low immune infiltration), and the basal subtype (moderate immune responses) (11). As the advancement of proteomics, Stewart et al. further categorized LUSC into three distinct molecular subtypes: inflamed type (40%), redox type (47%), and mixed type (13%), linking these to the aforementioned four subtypes. The inflamed type comprises two subtypes: A (neutrophil infiltration, immunosuppressive TIME, 43% concordance with basal subtype) and B (enhanced antigen presentation, elevated programmed death-1 (PD-1) expression, 75% concordance with secretory subtype). The redox type (84% concordance with classical subtype) comprises A (NFE2L2/KEAP1 mutations, blood metabolic pathway activation) and B (pan-metabolic features). The mixed type exhibits WNT/stromal pathway activation, involving basal, secretory, and primitive subtypes but excluding the classical subtype (16). Satpathy et al. refined the classification by subdividing the basal subtype into basal-inclusive (B-I) and epithelial-mesenchymal transition-enriched (EMT-E), and further stratified LUSC into “hot”, “warm”, and “cold” tumors (17).

Based on the above evidence, we propose that the TIME of LUSC can be classified into three distinct types: immuno-response type, immuno-suppression type, and immuno-desert type. The immuno-response type characterizes by enhanced antigen-presenting capacity, the cytotoxicity of T cells, and elevated PD-1/PD-L1 expression, indicating a “hot tumor” phenotype with a favorable response to immunotherapy, which is aligned with the inflamed B type and the secretory subtype. The immuno-suppression type featured by increased neutrophil infiltration and enrichment of stroma components indicating a “warm tumor” phenotype and an immunosuppressive status. The immuno-desert type is potentially associated with KEAP1/NFE2L2 gene mutations, aberrant WNT signaling pathways, and other dysregulated metabolic pathways suggesting a “cold tumor” phenotype with minimal immune infiltration, which may potentially respond to a variety of metabolic regulation treatments. Targeting and eliminating immunosuppressive components may reshape the TIME of immuno-suppression type to enhance immunotherapy efficacy. The application of anti-tumor immune cells combined with the modulation of specific regulatory pathways may reprogram the immuno-desert type’s TIME. Therefore, the selection of TIME for specific populations is crucial for advancing future precision diagnosis and treatment (Figure 1).

Figure 1 Immunotypes of LUSC. The illustration categorizes LUSC into distinct immunotypes based on features of the TIME. The immuno-response type exhibits enhanced antigen-presenting capacity, abundant cytotoxic T cells, and elevated PD-1/PD-L1 expression. The immuno-suppression type shows increased neutrophil infiltration and stromal enrichment. The immuno-desert type is associated with mutant KEAP1/NFE2L2 genes, aberrant WNT/stroma signaling, and dysregulated metabolic pathways. These immunotypes highlight the heterogeneity of LUSC. CAF, cancer-associated fibroblast; DC, dendritic cell; LUSC, lung squamous cell carcinoma; MDSC, myeloid-derived suppressor cell; PD-1, programmed death-1; PD-L1, programmed death-ligand 1; TAM, tumor-associated macrophage; TAN, tumor-associated neutrophil; TCR, T-cell receptor; TIME, tumor immune microenvironment; Treg, regulatory T cell.

Features of LUSC’s TIME

As the data of omics continues to be updated, we delve deeper into the intricate properties of the heterogeneous TIME in LUSC. CD8⁺ T cells represent pivotal cellular subsets mediating the response to immunotherapy and their substantial differences between LUAD and LUSC may underlie the differential efficacy of immunotherapy. The upregulation of immune checkpoint molecules in LUSC compared to LUAD implies distinct CD8⁺ T cell functional profiles (18-20). The spatial separation between CD8⁺ T cells and tumor cells reveals distinct spatial localization of CD8⁺ T cells in LUSC relative to LUAD (21). And the enhanced infiltration of immunosuppressive cells in LUSC compared to LUAD implies fundamental differences in their TIME landscapes (22,23). Thus, we comprehensively examine the features of TIME in LUSC, with a focus on the functional state of CD8⁺ T cells, their spatial location, and cellular crosstalk.

CD8⁺ T cells functional subsets and spatial distribution in LUSC

A study has demonstrated that elevated levels of CD8⁺ T cells correlate with their increased infiltration (24). However, the functional activity and spatial distribution of these infiltrating cells with the TIME are considered more critical to clinical outcomes than their mere abundance (25). Thus, precisely characterizing the functional state and spatial organization of CD8⁺ T cells is essential.

The phenotypic conversion of CD8⁺ T cells

CD8⁺ T cell subsets, including effector, memory, and exhausted populations, exhibit distinct functional and phenotypic characteristics. The use of scRNA-seq has enabled a more refined categorization of CD8⁺ T cells.

During the initial phases of tumor development, effector and memory CD8⁺ T cells serve as primary functional populations driving the anti-tumor response. As tumor evolution continues, these cells progressively transition into an exhausted state (26). Precursor exhausted CD8⁺ T (Texp) cells act as an intermediate population linking effector and exhausted CD8⁺ T cells. These cells can be reactivated by ICIs to target tumors, marked by the simultaneous expression of cytotoxic and immune inhibitory markers like granzyme K (GZMK) and PD-1 (27-29). Recent researches provide a further breakdown of the functional transition of exhausted CD8⁺ T cells which undergo a stepwise functional transition from progenitor 1 to progenitor 2 state to an intermediate state, and then reaching an irreversible terminal state (30,31). In LUSC, two types of progenitor exhausted subsets and one intermediate subset can be reinvigorated by ICIs leading to durable clinical benefits, but they only account for a minority. The majority are terminally exhausted CD8⁺ T cells characterized by elevated levels of co-inhibitory molecular expression do not respond to immunotherapy in LUSC (28,29,32,33). Parra et al. demonstrated that the expression levels of immune checkpoints such as PD-1, T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3), and lymphocyte-activation gene 3 (LAG-3) were significantly higher in LUSC compared to LUAD (18,34). The co-expression of TIM-3 and LAG-3 with PD-1 is associated with poorer clinical responses to immunotherapy (35-40). Additionally, CD39 and CD101 are inhibitory molecules linked to irreversible terminal exhaustion and disease progression in LUSC (34,35). Therefore, the elevated expression levels of co-inhibitory molecules may explain the differences in immunotherapy efficacy between LUSC and LUAD.

The spatial distribution of CD8⁺ T cells

Multiplex immunofluorescence analysis reveals that reduced spatial distance between CD8⁺ T cells and tumor cells correlates with improved clinical outcomes. However, LUSC exhibits greater spatial separation and limited interactions among CD8⁺ T cells, whereas PD-L1⁺ cells, CD133⁺ cancer stem cells (CSCs), and macrophages are more closely positioned relative to their arrangement in LUAD (21,41).

Parra et al. revealed that T cells expressing immune checkpoints exhibit closer proximity to tumor cells in LUSC compared to LUAD (18), suggesting that exhausted CD8⁺ T cells may cluster around malignant cells in LUSC. Additionally, adenosine, a product catalyzed by CD39 and ATP-degrading enzymes such as CD73, accumulates in the stroma, forming a barrier that hinders immune cell infiltration into the tumor region (34,42). Elevated levels of CD73⁺CD90⁺ cells in the stroma hinder CD8⁺ T cell cytotoxicity and are strongly linked to epithelial-mesenchymal transition (EMT), negatively impacting survival outcomes in LUSC patients relative to LUAD (43). These factors may compromise the functionality of CD8⁺ T cells in LUSC, resulting in their partial infiltration into the tumor area. Recent research has highlighted CD73 as a promising biomarker (42).

Collectively, CD8⁺ T cells are highly heterogeneous in LUSC and undergo significant phenotypic changes. The distinct spatial arrangement of CD8⁺ T cells directly hinders anti-tumor immunity. While some CD8⁺ T cells remain functional and infiltrate the tumor area, tumor cells can also escape immune destruction through diverse interactions with other TIME components.

The impact of other cells on CD8⁺ T cells

Beyond the characteristics of CD8⁺ T cells, the spatial organization of other cell types deepens our insight into the complex interactions between these cells and tumor cells (18,21). The TIME comprises diverse cell populations including natural killer (NK) cells, tumor-associated neutrophils (TANs), tumor-associated macrophages (TAMs), CD4⁺ T cells, cancer-associated fibroblasts (CAFs), and myeloid-derived suppressor cells (MDSCs), all exerting direct and indirect effects on CD8⁺ T cells in LUSC (13,44-47).

NK cells as the first line of defense of innate immunity directly exert cytotoxic effects on tumor cells (48). The proportion of CD56⁺ NK cells is positively correlated with the efficacy of ICIs (49). However, the spatial omics profiles of Desharnais et al. indicated that the proportion of NK cells within the TIME was extremely low in LUSC (50). Moreover, the function of NK cells undergoes changes with the increased expression of inhibitory receptors such as NKG2A, PD-1, TIM-3 and LAG-3 similar to CD8⁺ T cells with the occurrence of immune resistance (49). In addition, due to the loss of major histocompatibility complex class I (MHC-I) molecules, the ability of NK cells to produce interferon-γ (IFN-γ) together with CD8⁺ T cells is greatly weakened in LUSC (51). Therapeutic strategies targeting NK cell checkpoints or adoptive NK cell transfer may thus synergize with PD-1/PD-L1 blockade to reinvigorate exhausted CD8⁺ T cells and overcome immunosuppression in LUSC.

TANs are highly plastic and heterogeneous, with significantly higher infiltration observed in LUSC compared to LUAD (22,52). The subtype of TANs undergoes dynamic changes with tumor progression, eventually transforming into a pro-tumor N2-type TANs subset (22). TANs induce tumor angiogenesis by generating or releasing pro-angiogenic factors such as vascular endothelial growth factor A (VEGFA) into the extracellular matrix (53). S100A12 and V-domain immunoglobulin suppressor of T cell activation (VISTA) as inhibitory markers expressed on TANs are associated with the exhaustion of CD8⁺ T cells, promoting tumor growth, invasion, recurrence, and metastasis. The combination of VISTA inhibitors and radiotherapy can potentiate the activity of CD8⁺ T cells and attenuate the accumulation of TANs, thus suppressing tumor growth (13,54). The interaction between chemokine CCL3 positive TANs and secreted phosphoprotein 1 (SPP1) positive TAMs facilitates reciprocal amplification of each other (55). Targeting chemokine receptor CXCR2 can reduce TANs infiltration and enhance CD8⁺ T cells activity (56). In the OAK study, the TANs signature gene set has potential as a prognostic indicator for overall survival (OS) in patients receiving immunotherapy (52). Inhibiting the conversion of other subtypes to N2-type TANs has emerged as a research hotspot, and co-targeting TANs and CD8⁺ T cells may have a synergistic anti-tumor effect for LUSC.

TAMs, characterized by both anti-tumor M1-type and pro-tumor M2-type properties, are more abundant in LUSC compared with LUAD (57). Previous studies have indicated that M2-type TAMs could restrict the activity and migration of CD8⁺ T cells (58-60). MARCO, a collagen structural receptor on M2-type TAMs associated with worse survival only in LUSC not LUAD, can enhance the proliferation of regulatory T cells (Tregs) and inhibit the formation of effector T cells by releasing interleukin 37 (IL-37) (57,61). A study showed that CD163⁺ M2-type TAMs could reduce the cytotoxic function of CD8⁺ T cells and elevate PD-L1 expression (13). In LUSC, the protein S100 calcium-binding protein A7 (S100A7) overexpressed on CD68⁺ M1-type TAMs leads to immunotherapy resistance by suppressing the polarization of M1-type TAMs and decreasing CXCL9 expression, thereby limiting CD8⁺ T cell infiltration (62). A pre-clinical study has shown targeting TAMs could enhance the infiltration of CD8⁺ T cells and the efficacy of anti-PD-1 therapy (63). Hence, reprogramming TAMs towards an anti-tumor status should be the challenge of future therapeutic strategies.

CD4⁺ T cells, a crucial T cell subset, play a supportive role by influencing CD8⁺ T cell activity through diverse regulatory pathways (44,45). In LUSC, the lack of IFN-γ expression on Th1 cells limits their anti-tumor effect (64). Elevated levels of Tregs and Th17 cells within the tumor suppress CD8⁺ T cell migration, facilitating immune evasion by tumor cells. However, it is worth noting that Th17 cells also possess anti-tumor properties, making their role controversial (65,66). The body of research on CD4⁺ T cells in LUSC is currently limited, necessitating further comprehensive investigation in the future.

CAFs, as a crucial component of the stroma, closely interact with MDSC. CAFs recruit CCR2-positive monocytes which generate reactive oxygen species (ROS) by secreting the chemokine CCL2 and induce these monocytes to transform into MDSCs, thereby reducing the proliferation of CD8⁺ T cells and the production of IFN-γ. CCR2 inhibitors block monocyte migration, while indoleamine 2,3-dioxygenase (IDO) inhibitors and NADPH oxidase-2 (NOX2) inhibitors reduce the production of ROS (67). Both mechanisms antagonize the CAFs-MDSCs axis, which is a potential treatment strategy for regulating the immunosuppressive TIME. Therefore, CAFs indirectly affect CD8⁺ T cells through their interactions with MDSCs.

Collectively, the presence of the multiple cells mentioned above can directly and indirectly impact the cytotoxicity of CD8⁺ T cells. The immunosuppressive network involving TANs, TAMs, CAFs, and MDSCs contributes to remodeling a specific milieu of the TIME (Figure 2).

Figure 2 Features of LUSC’s TIME. The TIME of LUSC centers on CD8⁺ T cells, emphasizing their functional status, spatial distribution, and interactions with other cellular components. During tumor progression, CD8⁺ T cells transition from cytotoxic effectors to terminally exhausted phenotypes, exhibiting limited tumor infiltration that further restricts immunotherapy efficacy. Moreover, TANs, TAMs, and CAFs, key cellular subsets interacting with CD8⁺ T cells, exacerbate immunosuppression in LUSC. CAF, cancer-associated fibroblast; IFN, interferon; LUSC, lung squamous cell carcinoma; MDSC, myeloid-derived suppressor cell; PD-1, programmed death-1; TAM, tumor-associated macrophage; TAN, tumor-associated neutrophil; TIME, tumor immune microenvironment.

In conclusion, CD8⁺ T cells undergo phenotypic changes across multiple functional states, and their unique spatial distribution excludes them from tumor regions. Epitopes formed via interactions between other cells and CD8⁺ T cells limit anti-tumor effectiveness. However, multi-omics data require fresh or optimally preserved frozen tissue samples, imposing stringent logistical and technical constraints that substantially hinder their clinical translation. Furthermore, these technologies are inherently limited to static snapshots of the TIME, unable to elucidate the temporal dynamics of cellular states and interactions. Compounding these challenges is the substantial financial costs associated with these advanced techniques further narrow their accessibility and utility in routine clinical application. Therefore, it will be crucial to predict the efficacy of immunotherapy by transforming multi-dimensional information of multi-omics data into single-dimensional information such as immunohistochemical (IHC) in the clinical application.

The regulatory mechanism of LUSC TIME

The distinctive characteristics of the TIME in LUSC markedly reduce the efficacy of immunotherapy for most patients. Elucidating the underlying mechanisms that govern the formation of TIME is of critical importance. A comprehensive analysis of metabolomics, epigenetics, and genomics will provide a thorough understanding of their influence on the TIME of LUSC.

The regulation of TIME by metabolomics

LUSC undergoes metabolic reprogramming to fulfill the elevated energy and biosynthetic needs essential for tumor cell proliferation and survival within the TIME (23,68). These contribute to immune evasion by creating an immunosuppressive TIME, characterized by hypoxia and lactate accumulation.

Hypoxia, a main property of tumor proliferation and invasion in LUSC, induces tumor cells to accelerate proliferation and metastasis through glycolysis (69,70). Tumor cells also utilize hypoxic-inducible factors (HIFs) to stimulate the secretion of vascular endothelial growth factor (VEGF), a pivotal regulator in tumor angiogenesis. These aberrant tumor vessels fail to adequately nourish immune cells within the TIME (71). Furthermore, hypoxia activates the PI3K/AKT/mTOR and WNT/β-catenin signaling pathways in various ways, inducing the progression of EMT (72,73). Currently, antiangiogenic therapy such as bevacizumab is not used as the first-line treatment in LUSC due to adverse reactions like pulmonary hemorrhage (74). However, the latest Phase III double-blind, randomized clinical trial, HARMONi-2, demonstrated that ivonescimab, a bispecific antibody targeting PD-1 and VEGF, exhibits significant improvement in prognosis for patients with LUSC compared to pembrolizumab as the first-line treatment. In the LUSC subgroup, the median progression-free survival (mPFS) of patients was 9.7 months, a 3.9-month increase over pembrolizumab-treated group. And the objective response rate (ORR) of patients, marking a 31% improvement compared to pembrolizumab therapy. Notably, the safety of ivonescimab was similar to previous researches (75,76). Therefore, the study marks a new revolution in combining immunotherapy with antiangiogenic therapy as a first-line treatment in LUSC.

The high glucose uptake in LUSC indicates elevated metabolic activity (77). This uptake is regulated by varying levels of glucose transporters (GLUT) expression on tumor and immune cells (78). The high expression of GLUT1 facilitates the polarization of macrophages towards M2-type TAMs in LUSC, associated with unfavorable survival and pathological responses to immunotherapy combined with chemotherapy (79). Consequently, the high glucose uptake rate of tumor cells significantly hampers the efficiency of glucose uptake by other immune cells.

Lactate, produced by both aerobic and anaerobic glycolysis, creates an acidic environment that enables malignant cells to thrive and metastasize, detrimental to the survival of immune cells (80-82). Lactate variably impacts the metabolism of TAMs in different subgroups, impeding the tricarboxylic acid cycle of M1-type TAMs while promoting the oxidation and glycolytic metabolism of M2-type TAMs. Additionally, lactate hampers effector T cell proliferation and sustains the immunosuppression of Tregs (83). Classifying LUSC subtypes according to lactate metabolism levels can better guide individual treatment (82). The application of spatial metabolomics has shown that stratifying patients based on metabolic differences in conjunction with their immune characteristics can advance personalized therapy (84), providing a robust basis for future metabolism-guided therapy.

Currently, key metabolic steps that regulate the TIME include hypoxia, high glucose uptake by tumor cells, and lactate accumulation. Ivonescimab is poised to become one of the first-line treatment options in the future.

The regulation of TIME by epigenetics

Epigenetic aberrations are closely related to the squamous differentiation and occurrence and development of tumors (85,86). The features of epigenetics are deregulation in LUSC (87). The combination of epigenetic therapy and immunotherapy is being widely studied in LUSC to restore drug activity or reverse acquired resistance (86). DNA methylation, histone modifications, and non-coding RNAs are key epigenetic regulatory modes, integral to the immunosuppressive network of the TIME.

DNA methylation

DNA methylation is an epigenetic process regulated by DNA methyltransferases (DNMTs) (88). DNMTs catalyze the methylation at the 5-carbon position of cytosine (5-mC) to form a stable epigenetic structure. However, the epigenetic aberration of 5-mC exhibit a poor response to ICIs (89). And DNMT1 suppresses the expression of CXCL9 and CXCL10 in Th1 cells, thereby impeding the activity of effector T cells (90).

Histone modifications

Histone methylation and acetylation are the primary histone modifications in LUSC, constituting significant epigenetic regulation mechanisms influencing tumorigenesis and the core of TIME (91). Lysine specific demethylase 1 (LSD1), nuclear receptor binding SET domain protein 1 (NSD1), nuclear receptor binding SET domain protein 3 (NSD3), and enhancer of zeste homolog 2 (EZH2) are prospective epigenetic targets in LUSC.

LSD1 inhibits the proliferation, activation, and cytotoxicity of CD8⁺ T cells in head and neck squamous cell carcinoma (HCCNS) (92). Inhibitors of LSD1 have been shown to upregulate the expression of chemokines CXCL5, CXCL10, transforming growth factor beta (TGF-β), and PD-L1, thereby increasing the infiltration of CD8⁺ T cells and improving immunotherapy efficacy (93). However, due to the excessive toxicity observed during phase I clinical trials (94), anti-LSD1 therapies have not been approved for clinical use.

NSD1 and NSD3, as histone methyltransferases, exert a similar impact as LSD1. NSD1 shows reduced methylation activity in both LUSC and HCCNS, indicating an immune-cold tumor phenotype (92,95). NSD3 is considered a key factor in tumorigenesis (17). Xu et al. confirmed that the amplification of NSD3 gene leads to the downregulation of critical cytotoxic components and impairment of antigen presentation capability (96). Bromodomain and extra-terminal domain (BET) inhibitors have effectively restrained tumor growth in mouse xenografted models (97), suggesting the feasibility of BET inhibitors in phase I clinical trials.

EZH2, another histone methyltransferase, is overexpressed in NSCLC with dual regulatory effects on the tumor itself and TIME. In tumor cells, EZH2 interacts with ARID1A, a member of the ATP-dependent polymorphic BRG/BRM-associated factor chromatin remodeling unit, enhancing the immune escape phenotype of the tumor through the IFN-γ response. Additionally, the survival of effector T cells is regulated by EZH2 through the miRNA-EZH2-NOTCH axis, highlighting the biological role of epigenetic aberrations in reprogramming the TIME (98). Several phase I/II clinical trials involving EZH2 are in progress (NCT04407741, NCT04104776, NCT05467748).

Histone modifications impact the core component of the TIME, namely CD8⁺ T cells, shaping an immune-excluded type TIME. Recent research has demonstrated that histone lysine methyltransferase 2D (KMT2D) significantly impact LUSC tumorigenesis, and its effects on the TIME require further exploration (99).

Non-coding RNAs

Previous studies have demonstrated a close association between non-coding RNAs and tumor development, with recent investigations further elucidating their role in the TIME of LUSC (100-102). In LUSC, circular RNAs (circRNAs) regulate downstream miRNAs to interfere with the immune response. The expression of HMGB2-associated circRNA (circHMGB2) is upregulated in LUSC, leading to the inactivation of the IFN response pathway mediated by miR-181a-5p (103). Additionally, circASCC3 sponges miR-432-5p, increasing levels of the complement C5a and disrupting the immune system (104). Intriguingly, miRNAs can interact with immune-related pathways to affect the TIME. MiR-455-5p can induce the expression of PD-L1 by activating the IFN-γ response pathway (105). Furthermore, the miR-7-5p/TGF-β axis shows a strong correlation with the acidic tumor microenvironment in metastasis (80). Thus, circRNAs and miRNAs can reshape the microenvironment through a cascade of downstream epigenetic regulatory pathways.

Epigenetics influences key components within the TIME in LUSC, particularly T cells. A plethora of studies have emerged in the field of epigenetic regulation within the TIME of solid tumors, highlighting epigenetic therapeutic targets such as NSD3 and EZH2 for future treatment strategies. The integration of epigenetics and immunotherapy may offer promising prospects for enhancing patient outcomes.

The regulation of TIME by genomics

In LUSC, although there are currently no effective drugs targeting genomic events, genomic aberrations are closely associated with immune infiltration and patient prognosis (106,107), signifying genomic events as key factors in the regulation of the TIME.

KEAP1-NRF2 signal pathway

The KEAP1-NRF2 pathway plays a vital role in preserving cellular redox balance and shielding cells from oxidative stress and toxic damage (108,109). In NSCLC, loss of function mutations in KEAP1 and gain of function mutations in NFE2L2 (which encodes NRF2) allow tumor cells to evade immune responses, leading to resistance to immunotherapy and chemotherapy (110). These mutations co-occur infrequently in LUSC, with an incidence of about 10% for KEAP1 deletion mutations and approximately 28% for NFE2L2 activation mutations (111,112). KEAP1 mutations in LUSC are strongly linked to reduced CD8⁺ T cell density, while NRF2 overactivation correlates with diminished IFN-γ levels, decreased MHC-I expression, limited T cell infiltration, poor prognosis, and reduced efficacy of anti-PD-L1 therapy (113-115).

PI3K/AKT/mTOR pathway

The PI3K/AKT/mTOR pathway is associated with oncogenesis and tumor progression, with about 68% likelihood of alterations in LUSC (116,117). This pathway exhibits two prominent mutations: PIK3CA alterations and PTEN deletions, both strongly associated with reduced immune infiltration (107,113). Spatial omics suggests that high infiltration of TANs in LUSC is associated with PI3KCA mutations (23). PIK3CA mutations correlate with low PD-L1 expression, significantly impacting the ORR in NSCLC patients treated with atezolizumab (106,118). The PTEN gene, which acts as a tumor suppressor, when deleted, results in hyperactivation of the PI3K/AKT/mTOR pathway, contributing to increased tumor proliferation, metabolism, and survival, with an incidence ranging from 10% to 15% in LUSC (119). PTEN deletion, as shown in mouse studies, upregulates TGF-β and CXCL10 expression within the TIME, facilitating the conversion of CD4⁺ T cells into Tregs and impeding the antitumor effects of CD8⁺ T cells, NK cells, and M1-type TAMs (120).

WNT/β-catenin pathway

The WNT/β-catenin signaling pathway is crucial in regulating the TIME and the therapeutic efficacy of ICIs (107,121). An animal study showed that in NSCLC with high TMB, activation of the WNT/β-catenin pathway reduces CCL4 levels, disrupts dendritic cell (DC) infiltration, and promotes an immune-excluded microenvironment. In this environment, CD8⁺ T cells targeting neoantigens are predominantly found in peripheral blood rather than within the TIME (122). Combining targeting of the WNT/β-catenin pathway with anti-PD-1 therapy enhances the immunomodulatory effect within the TIME (123), offering a promising therapeutic strategy.

NOTCH pathway

The NOTCH signal pathway primarily maintains the homeostasis of immune cells within the TIME. Zefi et al. revealed that NOTCH signaling pathway is significantly associated with smoking (124). Gene set enrichment analysis (GSEA) demonstrated significant enrichment of aberrant NOTCH signal pathway in LUSC (125). The NOTCH1 gene, often with loss-of-function mutations in LUSC, acts as a tumor suppressor associated with a reduction in both innate and adaptive immune components (126). Additionally, the NOTCH signal pathway reshapes the TIME through cascading interactions with the fibroblast growth factor (FGF) and WNT signaling pathways to promote CSC stemness properties (127). However, there are enhanced immunogenicity and CD8⁺ T cell infiltration in NSCLC with NOTCH4 gene mutations (128). Therefore, the regulation of the NOTCH pathway to the TIME remains controversial and requires active exploration in the future.

Multiple genomic events collectively influence the immune infiltration of CD8⁺ T cells, illustrating that targeting a single pathway to improve prognosis is not feasible. Thus, targeting genomic events presents a significant challenge in enhancing immunotherapy (86).

As a result, metabolomics creates a hypoxic environment by promoting lactate production, contributing to tumor growth, while epigenetics and genomics regulate the activity of T cells and the infiltration of other cells in the TIME through various mechanisms. Exploring the regulatory mechanisms underlying the TIME is critically important for improving immunotherapy outcomes (Figure 3).

Figure 3 The regulatory mechanism of LUSC’s TIME. Metabolomics induces lactate accumulation through aerobic and anaerobic glycolysis, promoting M2-type TAMs polarization and impairing CD8⁺ T cell function. Epigenetics and genomics primarily regulate the cytotoxicity of CD8⁺ T cells by modulating cytokines and chemokines secretion. IFN, interferon; LUSC, lung squamous cell carcinoma; PD-L1, programmed death-ligand 1; TAM, tumor-associated macrophage; TGF, transforming growth factor; TIME, tumor immune microenvironment; Treg, regulatory T cell.

Future directions for immunotherapy

Despite advances in ICIs, such as anti-PD-1/PD-L1 therapies, a significant proportion of LUSC patients exhibit limited or transient responses, underscoring the need for novel immunotherapeutic strategies (129). Recent research has identified key immunomodulatory molecules, cellular components, and signaling pathways within the TIME that contribute to immunosuppression. We highlight emerging therapeutic strategies, including anti-CD73, T cells activation, targeting Tams and Tregs, assessing their application prospects and challenges in reshaping the TIME of LUSC.

Focusing on immunomodulatory molecule

CD73 is identified as an immunomodulatory agent in the resistance to durvalumab (42). Targeting CD73 can reduce adenosine production, diminish immunosuppression in the TIME, and enhance the effectiveness of immunotherapies synergistically. Mupadolimab, a monoclonal antibody against CD73 stimulate the activation of B cells to produce specific antibodies against specific antigens, which has shown high tolerability at various dosages in humanized mouse models and phase I clinical trials (130). The phase II COAST trial evaluated the efficacy and safety of durvalumab combined with oleclumab (anti-CD73 antibody) as consolidation therapy in patients with unresectable stage III NSCLC without progression after concurrent chemoradiotherapy (cCRT). Compared to durvalumab alone, the combination showed a higher confirmed ORR (30.0% vs. 17.9%) and a prolonged 12-month progression-free survival (PFS) rate (62.6% vs. 33.9%). Grade ≥3 treatment-emergent adverse events (TEAEs) were higher in the oleclumab-durvalumab combination group (40.7%) compared with the durvalumab monotherapy group (39.4%) (131). The phase II NeoCOAST trial, neoadjuvant oleclumab combined with durvalumab demonstrated a modestly higher major pathological response (MPR) rate (19.0% vs. 11.1%) and pathological complete response (pCR) (9.5% vs. 3.7%) compared with durvalumab. Treatment-related adverse events (TRAEs) were observed in 57.1% of patients in the durvalumab plus oleclumab group, with one patient of grade ≥3 TRAE reported in this cohort. Notably, IHC revealed a reduction in CD73 expression levels following treatment with durvalumab plus oleclumab. Concurrently, increased infiltration of NK cells and GZMK⁺CD8⁺ T cells and decreased adenosine were observed in the TIME (132).

Therefore, CD73 indeed modestly improve the efficacy of immune monotherapy. The ongoing phase III PACIFIC-9 trial (NCT05221840) aims to further validate these results in a larger cohort. Notably, further investigation is required to determine whether CD73 can serve as a predictive biomarker in clinical applications and whether CD73 inhibitors exhibit efficacy as monotherapy.

T cells activation regulation

T-cell engagers (TCE)

TCE are engineered to target both CD3 on human T cells and specific tumor cell antigens, enhancing the proximity between T cells and tumor cells for more precise tumor eradication (133).

5T4, a widely expressed carcinoembryonic antigen, promotes tumor migration and spread by interfering with the WNT/β-catenin and CXCR4/CXCL12 pathways (134,135). The bispecific antibody targeting CD3 and 5T4 has demonstrated the capacity to induce T cell cytotoxicity in preclinical studies of solid tumors, showing promising therapeutic potential (136). GEN1044, a bispecific TCE targeting CD3 and 5T4, was evaluated in a phase I clinical trial involving 48 patients with solid tumors conducted in July 2020, revealing acceptable safety despite some participants experiencing grade ≥3 adverse events (AEs) (NCT04424641).

B7-H4, a member of the B7 family, is significantly upregulated in various tumor cells and TAMs, playing a crucial role in tumorigenesis and immune regulation. It has a dual regulatory effect on T cell function, suppressing effector T cell activity while enhancing the immunosuppression of Tregs (137). In a humanized mouse model of breast cancer, the bispecific antibody targeting CD3 and B7-H4 facilitated CD8⁺ T cell infiltration into the tumor, displaying potent antitumor effects (138). A phase I clinical study involving 500 patients with solid tumors is assessing the safety of targeting CD3 and B7-H4 TCE GEN1047, with results eagerly anticipated in LUSC (NCT05180474).

TCEs bridges T cells and tumor cells, as demonstrated by preclinical and early clinical studies targeting antigens like 5T4 and B7-H4. However, the identification of optimal tumor-associated antigens for LUSC and the mitigation of T cell exhaustion represent the most significant challenges in current research.

Activating co-stimulatory molecules

Co-stimulatory receptors such as 4-1BB and OX40, members of the tumor necrosis factor receptor superfamily, are gaining interest as potential immune checkpoints. Their activation enhances the tumor immune response, analogous to an “accelerator” in a car (139).

4-1BB is primarily expressed on T cells and NKs, whose activation can stimulate antigen presentation by DCs and reduce Treg infiltration in the TIME (140). Ongoing research into bispecific immunotherapy targeting PD-L1 and 4-1BB includes a preclinical study that showed effective antitumor activity induction by GEN1046. A phase I clinical trial indicated that GEN1046 is manageable safety-wise, with most treatment-related AEs being grade 1–2, grade 3 AEs occurring at about 9.8%, and no grade 4 AEs. Of the six NSCLC patients previously treated with anti-PD-(L)1, two achieved an unconfirmed partial response (PR), and while stable disease (SD) is the optimal response for GEN1046, its significant disease control rate suggests clinical relevance for patients with limited treatment options (141). INBRX-105, another bispecific antibody targeting 4-1BB and PD-L1, combined with pembrolizumab, is currently in a phase II clinical study in advanced solid tumors, meriting close attention to upcoming results (NCT03809624).

OX40 is an additional co-stimulatory molecule expressed on T cells, primarily involved in regulating the action of effector and memory T cells. Porciuncula et al. proved that the expression of OX40 in LUSC is higher than 80%, while the expression of its ligand OX40L is only about 15% in LUSC (142). Accordingly, OX40 agonists are likely to demonstrate significant therapeutic potential in individuals expressing both OX40 and OX40L. Preclinical investigations have verified that these agonists elevate levels of CCL4, CXCL10, and IFN-γ in effector and memory T cells within PTEN-deficient melanomas, indicating immune activation in the TIME (143). In a phase Ib clinical trial involving 17 HNSCC patients, the OX40 agonist MED16469 exhibited a favorable safety profile and underscored the efficacy of CD103⁺CD39⁺CD8⁺ T cells as biomarkers for evaluating the clinical impact of OX40 agonists (144). Furthermore, an ongoing phase I clinical study is assessing the safety, tolerability, pharmacokinetics, and initial antitumor efficacy of the OX40 monoclonal antibody BGB-A445, used in conjunction with anti-PD-1 therapy, across various solid tumors, including NSCLC, with outcomes highly anticipated (NCT04215978).

Although co-stimulatory receptors such as 4-1BB and OX40 enhance T cell activation, the research is still in the early stages of exploration. Challenges include optimizing target engagement and managing immune-related toxicities.

Targeting TAMs

Earlier discussions have emphasized that TAMs significantly suppress the cytotoxicity and infiltration capabilities of CD8⁺ T cells. CD163, a critical immunosuppressive receptor on TAMs, has emerged as a significant therapeutic target. OR2805, a monoclonal antibody specifically targeting CD163, has shown potential in preclinical studies to reduce the suppressive effects of M2-type TAMs and enhance the release of IFN-γ, thereby restoring the effector function of T cells (145). A phase I/II clinical trial involving 172 participants is evaluating the safety, tolerability, and antitumor activity of OR2805, both as a monotherapy and in combination with cemiplimab or docetaxel in solid tumors. Although results are pending, they are eagerly anticipated (NCT05094804).

Targeting Tregs

CCR8, expressed by Tregs within the tumor region, facilitates the formation of an immunosuppressive TIME. Kim et al. identified CCR8 as a potential therapeutic target following omics-integrated comprehensive analysis in LUSC (146). Preclinical studies have suggested the benefit of targeting CCR8 to augment the efficacy of anti-PD-1, proposing that anti-CCR8 may act as a complementary therapy to ICIs (147). A current phase I clinical trial is exploring the safety of combining the CCR8 monoclonal antibody BAY 3375968 with PD-1/PD-L1 inhibitors in a cohort of 270 patients with solid tumors (NCT05537740).

The future of immunotherapy focuses on enhancing the precision and effectiveness of T cells activation through innovative approaches, including bispecific TCE, co-stimulatory molecules, and targeting immunosuppressive elements. However, early clinical trials have revealed notable challenges regarding safety and efficacy. Ongoing research is critical to optimize these therapies and reduce associated adverse effects, improving their clinical viability (Table 1).

Conclusions

Although immunotherapy has transformed the treatment landscape, its efficacy in patients with LUSC is still constrained by TIME. This may be attributed to the fact that the majority of patients (approximately 80%) exhibit immuno-negative and immune-deficient TIME types, while only a minority of patients (around 20%) demonstrate an immune-positive type heightening activation of MHC-II molecules and the IFN pathway in response to anti-PD-1/PD-L1. In the dissection of specific TIME, CD8⁺ T cells is limited by their functional status and spatial distribution, which curtails their anti-tumor activity. And the interactions with other cellular components also affect their cytotoxic capabilities. However, multi-omics technology still faces great challenges in clinical application due to its high price, technical complexity and difficulty in obtaining samples. It is noteworthy that ivonescimab may prolong the survival of LUSC patients in the future. Additionally, anti-CD73 antibodies show modest improvement as treatment strategies. TCEs and co-stimulatory receptors such as 4-1BB and OX40 may reveal therapeutic potential in the future, yet challenges including antigen specificity, T-cell exhaustion, and toxicity management persist. Future efforts must prioritize biomarker-driven patient selection, novel engineering to mitigate on-target/off-tumor effects, and rational combinatorial approaches that balance efficacy with tolerability.

Acknowledgments

We gratefully acknowledge Figdraw (www.figdraw.com) for providing the platform to create the graphical abstracts and illustrations presented in this work.

Footnote

Peer Review File: Available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-350/prf

Funding: This work was supported by the Natural Science Foundation of Zhejiang Province (grant No. LY22H160037) and the Natural Science Foundation of China (grant No. 82473290).

Conflicts of Interest: All authors have completed the ICMJE uniform disclosure form (available at https://tlcr.amegroups.com/article/view/10.21037/tlcr-2025-350/coif). The authors have no conflicts of interest to declare.

Ethical Statement: The authors are accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Open Access Statement: This is an Open Access article distributed in accordance with the Creative Commons Attribution-NonCommercial-NoDerivs 4.0 International License (CC BY-NC-ND 4.0), which permits the non-commercial replication and distribution of the article with the strict proviso that no changes or edits are made and the original work is properly cited (including links to both the formal publication through the relevant DOI and the license). See: https://creativecommons.org/licenses/by-nc-nd/4.0/.

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