According to the International Agency for Research on Cancer’s World Cancer Statistical Report, approximately 1.8 million deaths occur annually due to lung cancer, followed by rectal cancer, liver cancer, and stomach cancer (1). Lung cancer is most prevalent among male patients and ranks second among female patients (2) due to its initial asymptomatic nature and difficulty in detection (3, 4). Currently, lung cancer holds the highest incidence and mortality rate globally (5). Smoking causes 80% of lung cancer deaths, while other risk factors include radon gas, asbestos, cumulative exposure to air pollution, polycyclic aromatic hydrocarbon emissions, and genetic factors (6).

Lung cancer is categorized based on histology into non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC) (7). NSCLC accounts for 80-85% of all lung cancer cases and includes adenocarcinoma, squamous carcinoma, and other histological subtypes (8). Poor prognosis usually follows an advanced NSCLC diagnosis (9), but new insights into the molecular mechanisms of disease progression and an increased understanding of the disease have allowed for the development of novel treatment options. These treatments include surgery, radiation therapy, chemotherapy, targeted therapy, immunotherapy, interventional therapy, and a combination of Chinese traditional and western medicine, as shown in Figure 1 . Significantly improved survival rates have been observed in lung cancer patients with the continuous improvement of systemic therapy.

Lung cancer treatment has shifted from indiscriminate cytotoxic chemotherapy to more refined targeted agents. The development of small molecule drugs and monoclonal antibodies that target specific components of dysfunctional molecules or immune pathways, as well as mutated genes that target lung cancer, and the development of more personalized combinations based on different conditions, has led to more optimal treatment options for lung cancer (10). Currently, immunotherapy is the most promising clinical treatment for lung cancer (11, 12). The goal of cancer immunotherapy is to elicit a cellular immune response (6, 13). Immunotherapies for lung cancer include tumor-related vaccines, chimeric antigen receptor (CAR)-T, T cell receptor (TCR)-T cell therapy, tumor infiltrating lymphocytes (TILs) therapy, lysing viruses, targeted antibodies for lung cancer, and immune checkpoint inhibitors (ICIs) (6). Among them, ICIs are the most widely used in clinical practice,which have not only revolutionized second-line treatment, but they are now also approved for first-line treatment (14, 15). Cytotoxic T lymphocyte associated antigen 4 (CTLA-4) (3, 16) is the first antibody in immunotherapy to be approved by the U.S. Food and Drug Administration (17). Since the discovery of CTLA-4, several immune checkpoint proteins have been discovered, including programmed death-1 (PD-1), T-cell immunoglobulin and ITIM domain (TIGIT), T-cell immunoglobulin domain and mucin domain-containing molecule-3 (TIM-3), lymphocyte activation gene 3 (LAG-3), V-domain Ig suppressor of T cell activation, B and T cell lymphocyte attenuator and cluster of differentiation 200. Among the most common clinical treatments for NSCLC are PD-1 monoclonal antibodies, including nivolumab and pembrolizumab (18). However, their clinical effectiveness is not high and not all patients benefit from them. The response rate after second-line treatment with nivolumab is approximately 20%. First-line treatment with pembrolizumab is currently limited to patients with a PD-1 ligand (PD-L1) tumor percentage score above 50%, which accounts for approximately one-third of NSCLC patients, and has an response rate of 69% (15). The upregulation of PD-1 expression in TILs is one of the main immunosuppressive mechanisms in the tumor microenvironment (TME). The TILs interact with ligands (PD-L1 and PD-L2), leading to a decrease in CD8⁺ T cells and an increase in regulatory T cells (Tregs), suppressing the function of CD8⁺ T cells or causing immune escape in response to an adaptive response to interferon (IFN) signaling (17, 19). Other factors that affect the effectiveness of treatment include the absence of tumor expression of MHC class I molecules (20, 21), low numbers of CD4⁺ T cell infiltrates in tumor tissue, high numbers of myeloid-derived suppressor cells (MDSC), and low expression of PD-L1 on cancer cells. Based on these potential mechanisms, the antitumor efficacy of immunotherapy can be boosted with the use of other types of treatment. Thus, finding the best combination strategy to expand anti-PD-1/PD-L1-based immunotherapy is currently a hot issue in lung cancer research (22).

In this paper, we review what is known about the current state of NSCLC research, with a particular emphasis on the TME, the current available drugs for treatment, and the potential for using combined therapies at appropriate doses to improve treatment response while minimizing treatment-related adverse events.

Tumor cells and peripheral cells both coexist and compete with each other. Among the surrounding cells are intrinsic and specific immune cells, resulting in a unique environment that varies by tumor type and is highly adapted to tumor behavior; this is referred to as the TME (21). The TME includes tumor-associated macrophages (TAMs), cancer-associated fibroblasts, tissue-specific mesenchymal cells, endothelial cells, intrinsic and specific immune cells, TILs, neutrophils, eosinophils, MDSCs, cytokines and extracellular matrix (23–26) ( Figure 2 ). The metabolic status of immune cells in the TME is a key factor affecting their immune response (27) and plays an important role in tumorigenesis, disease progression, and treatment response and prognosis (28). Under normal physiological conditions, innate and acquired immune cells capture and destroy cancer cells through immune surveillance (29, 30). However, in the pathological state, tumor cells can shape the immunosuppressive microenvironment through different mechanisms.

The tumor immune microenvironment consists of a diverse array of cell types, including CD4⁺ T cells, CD8⁺ T cells, B cells, TAMs, natural killer cells, CD1c⁺ myeloid and CD141⁺ myeloid dendritic cells, neutrophils, basophils, eosinophils, and mast cells (31). CD4⁺ T cells have different subsets of cells (TH1, TH2, TH17, and Tregs) that perform specialized immunomodulatory functions and secrete different cytokines to enhance or suppress immune responses (32). Tregs are a functional subpopulation of suppressor T cells that express the transcription factor FOXP3 (33). CD8⁺ T cells are activated to secrete IFN-γ and tumor necrosis factor after cell receptors on their surface destroy tumor cells (34). CD4 CTL and CD8⁺ T cells express granzyme and perforin, which are effectors that mediate cytotoxic activity in target cells (32). The activation process of macrophages is highly plastic, and depending on signals in the TME, macrophages can be polarized into M1 or M2 functional phenotypes (35, 36). M1 macrophages secrete IFN, interleukin (IL), nitric oxide synthase, and reactive oxygen species to exert and enhance anti-tumor immunity (35, 37). M2 macrophages are associated with high expression of IL-10, IL-1β and vascular endothelial growth factor (VEGF) in vivo, and form a beneficial survival environment for tumor cells by suppressing immunity and promoting tumor angiogenesis, invasion and distant metastasis (35, 38).

Tumor cells can shape the immunosuppressive microenvironment through nutritional competition, secretion of cytokines, the release of metabolites and modulation of immune cell metabolism to affect immune cell growth, development and differentiation, thereby increasing the function of immune cells toward a pro-tumor phenotype, a process that helps promote the immune escape of tumor cells themselves (27). When PD-L1 on tumor cells binds to PD-1 on T cells, the T cells are unable to specifically recognize the tumor cells, and this also results in immunosuppression (39). If this immunosuppression caused by tumor cells is reversed by drugs, the immune cells can resume their normal function of recognizing and killing tumor cells.

More than 100 chemotherapeutic drugs have entered clinical use since 1948; they are divided into four main categories: 1) alkylating agents such as cyclophosphamide, cisplatin, and oxaliplatin; 2) antimetabolites such as pemetrexed, gemcitabine, and fluorouracil; 3) botanicals such as vincristine and paclitaxel; and 4) antibiotics such as doxorubicin and bleomycin. Chemotherapeutic drugs are thought to produce anti-proliferative or cytotoxic effects during cell division (40), selectively killing cells that are proliferating rapidly in the body. Thus, while killing tumor cells, bone marrow suppression may also occur with a decrease in neutrophils, lymphocytes, platelets and hemoglobin (41). In addition, adverse skin reactions to chemotherapy occur in 30-40% of patients (41). High doses of chemotherapy drugs can also cause significant liver and kidney damage and gastrointestinal complications, side effects that many patients do not tolerate well (42). Thus, chemotherapy combined with immunotherapy seems to be somewhat contradictory because chemotherapy can kill anti-tumor immune cells. To further improve the treatment efficacy in clinical treatment of lung cancer with a combination of chemotherapy drugs and PD-1 monoclonal antibody, the chemotherapy dose may be reduced to reduce the killing effect on immune cells (43).

Recent evidence indicates that some chemotherapy drugs at low doses have anti-angiogenic and sometimes even immunomodulatory effects (44). Several new studies have demonstrated that small doses of gemcitabine combined with cisplatin can not only cause immunogenic death of lung cancer tumor cells but can also directly activate NK cells and increase IFN-γ secretion, thus inhibiting tumor growth. The optimal antitumor outcome was observed in in vivo experiments when administering a low dose of gemcitabine (30 mg/kg) (45) ( Table 1 , Figure 3 ).

Conventional chemotherapy attempts to maximize efficacy and directly eradicate tumor cells using doses close to the maximum tolerated dose (MTD) and has been the standard of care in lung cancer treatment (51, 52). Even with the use of intensive chemotherapeutic agents, remission rates as well as survival remain poor for lung cancer patients. However, metronomic chemotherapy (MET) is dosed at one-tenth to one-third the MTD (52) and defined as (53) the rhythmic chemotherapy of low-dose cytotoxic drugs with short or no drug-free breaks over prolonged periods (51). MET have shown promising anti-angiogenic properties (53, 54), as well as anti-tumor immune activation, while limiting side effects (54–56). Zhong et al. conducted a study to investigate the effects of three different rhythmic chemotherapy regimens on tumor growth in C57BL mice. The researchers injected 10⁶ cells into the right abdomen of the mice and after four days, administered different doses of cyclophosphamide for a period of three weeks. The three regimens used were MTD, which consisted of three doses of 150 mg/kg during the first week only; Met-1, which was 170 mg/kg given every six days; and Met-2, which was 25 mg/kg given every other day. The results of the study revealed that continuous administration of low-dose cyclophosphamide (Met-2) had a significant impact on the tumor microenvironment. Specifically, there was an increase in the number of CD4⁺ and CD8⁺ T cells, while the number of Tregs decreased. Low doses of cyclophosphamide also decreased the expression of transforming growth factor (TGF-β) receptor 2 by up-regulating the expression of E-calcineurin and down-regulating the expression of N-calcineurin (49) ( Table 1 , Figure 3 ). TGF-β can act as an immunosuppressive factor, and tumors with high expression of TGF-β can escape the surveillance of the immune system and inhibit expression of CD86 by TAMs. As CD86 is a tumor suppressor (57, 58), this implies that inhibition of TGF-β may enhance the antitumor immune response. In one study, co-administration of a Toll-like receptor 9 agonist and TGF-β2 inhibitor not only effectively exerted anti-tumor effects, but also led the TME to have T and NK cell enrichment and improved immunosuppression (59). In addition, sustained regular low-dose cyclophosphamide administration exerts anti-angiogenic effects by inhibiting the expression of VEGF, which can have a sustained tumor suppression effect and has the advantages of less toxic side effects and less drug resistance than conventional MTD administration. A prominent feature of this anti-angiogenic effect is tumor stabilization, not rapid tumor destruction (60).

In an in vivo study, rhythmical treatment with vincristine combined with Endo (Met NVB+Endo) gave better results than a maximum tolerated dose of vincristine combined with Endo (MTD NVB+Endo) in terms of anti-tumor responses, reduction of CD31, VEGF, HIF-1α and CEPS expression, as well as reduction of toxic side effects and induction of apoptosis. In this experiment, mice were administered vincristine at the MTD of 10 mg/kg, with the MET ranging from 1/10 to 1/3 of the maximum daily dose. In addition, the combination showed better antitumor effects than either drug alone (50). VEGF expression, detected by western blotting, indicated reduced VEGF expression levels in the Met NVB+Endo group and MTD NVB+Endo group ( Table 1 , Figure 3 ). The potent antitumor effect of MET in combination with anti-angiogenic drugs through enhanced inhibition of tumor-associated angiogenesis is consistent with previous findings (50, 61). Despite the observed positive outcomes in terms of tumor control and reduced side effects, conclusive Phase III trial results are yet to be established. Moreover, patient drug dosage and dosing intervals are currently determined empirically, and inter-individual variances necessitate a criterion for patient categorization (62).

Low-dose chemotherapy drugs combined with ICIs have a synergistic effect in the treatment of tumors (44). Li Zhou et al. (42) showed carboplatin activated the STING/TBK1/IRF3 signaling pathway and the STING-NF-κB signaling pathway, then experimentally verified that low dose of carboplatin could increase PD-L1 expression in lung cancer cells. In addition, a low dose (20 mg/kg) of carboplatin also increased the infiltration of cytotoxic CD8⁺ T cells and the secretion of the chemokines CXCL10 and CCL5 compared to a high dose (75 mg/kg) (42) ( Table 1 , Figure 3 ). Low-dose carboplatin in combination with PD-1 monoclonal antibody significantly improved the anti-tumor effect compared to both PD-1 monoclonal antibody alone and carboplatin alone without significant toxic side effects (42).

Lu et al. (46) found that sublethal doses of pemetrexed (100nM) and 5-fluorouracil (5-FU) could upregulate PD-L1 expression and regulate TIL activity in NSCLC cells, and found that pemetrexed or 5-FU elevated PD-L1 protein levels in a dose-dependent manner. In vivo experiments, the combination of pemetrexed (100 mg/kg) with a PD-1/PD-L1 blocker (3 mg/kg) further enhanced the antitumor immune response. The antimetabolite pemetrexed induced PD-L1 upregulation by activating the ROS-NF-κB signaling pathway through inactivation of thymidylate synthase and thus in combination with PD-1 monoclonal antibody activation of CD4⁺ T cells and CD8⁺ T cells provides a more favorable immune microenvironment for tumor growth inhibition (46) ( Table 1 , Figure 3 ).

Sen et al. found that low-dose gemcitabine (40 mg/kg, first day of the week) in combination with SRA737+ (100 mg/kg, first and second day of the week) and anti-PD-L1 (300 μg, third day of the week) combination therapy in the treatment of tumor-bearing mice had significantly better antitumor effects than single-agent or two-by-two dosing regimens. This triple therapy increased T-cell infiltration, decreased T-cell depletion, and significantly increased antigen-presenting cell subpopulations. This was demonstrated by a significant increase in the number of CD8⁺ cytotoxic T cells, dendritic cells, and M1 macrophages and a significant decrease in the number of immunosuppressive M2 macrophages and MDSC. Triple therapy also increased the expression of the type 1 interferon gene, IFN-β, and the chemokines CCL5 and CXCL10 (47) ( Table 1 , Figure 3 ).

He et al. demonstrated that low-dose high-density DTX (11 mg/kg) or PTX (11 mg/kg) indirectly activated the killing effect of T cells on tumor cells by activating the PI3K/AKT/NF-κB signaling pathway, promoting the exposure of antigen on the surface of the tumor cells, and further activating the antigen-presenting function of antigen-presenting cells. Combined with PD-1/PD-L1 monoclonal antibody, it can increase the expression of type 1 macrophages, dendritic cells (DCs) and cytotoxic CD8⁺ T cells (48) ( Table 1 , Figure 3 ).

In summary, low-dose chemotherapy drugs have vascular and immunomodulatory effects, and their combined application with ICIs has a synergistic effect. One of the important tasks ahead is to conduct more research to further determine the optimal dose, frequency, and sequence of chemotherapy drugs that achieve the best antitumor efficacy with ICIs.

Current targeted therapy for lung cancer includes the targets VEGF, EGFR, ALK, ROS1, MET, BRAF, NTRK, RET, and RAF (63). As research continues, many other oncogenic drivers, such as HER2 exon 20 insertion mutations are being identified, and the efficacy data of targeted therapies are constantly being updated (64). Targeted therapies are undoubtedly a milestone in the development of cancer therapy. They play a key role in early disease detection and increase our understanding of tumor evolution and treatment resistance. Targeted therapies represent one of the future directions of precision oncology approaches.

With the identification of alterations in the targeted oncogene, advanced lung cancer can be treated with greater precision (64). Targeted agents are increasingly available as a first-line choice of lung cancer treatment and have improved prognosis and reduced toxicity compared to chemotherapy (97). EGFR mutations and ALK fusions are the most common targeted alterations (98, 99). In the analysis of specific cell populations, different TME modifications were detected in EGFR-positive and ALK-positive tumors compared to EGFR/ALK-negative tumors: TME in EGFR-positive tumors had decreased numbers of CD8⁺ T cells; TME in ALK-positive cases had increased numbers of Tregs. This suggests that in the development of lung cancer different immune cell responses occur (98, 100). In addition, targeted oncogenic alterations were also associated with PD-L1 expression, and upregulated by activation of MAPK, PI3K-AKT-mTOR and JAK-STAT3 signaling pathways in NSCLC cells with altered KRAS, EGFR and ALK activating genes or PD-L1 expression (19, 101–106).

Immunotherapy is a complex and intriguing area of cancer research (116). How to optimize it is currently a hot topic in cancer research, including how to further improve response rates, expand the population that can benefit, and reduce the incidence of treatment-related adverse events (92). Increasingly, studies indicate that reducing the dose of chemotherapy and adjusting the dosing regimen can not only lead to better anti-tumor effects but can also reduce drug toxicities and regulate the immune microenvironment by modifying the number of immune cells and cytokines to further achieve anti-tumor immunity. Chemotherapeutic drugs can upregulate PD-L1 expression to create favorable conditions for combining PD-1/PD-L1 monoclonal antibodies and allow low-dose chemotherapy combined with immunotherapy to work better. Currently, low-dose chemotherapy combined with immunotherapy has been validated in both in vitro and in vivo experiments, but clinical studies are limited to date, and more research is needed. Similarly, reducing the dose of anti-angiogenic drugs and adjusting the dosing regimen can improve the anti-tumor effect and activate immunity, which has also been demonstrated in some animal studies. Low-dose anti-angiogenic agents are able to modulate multiple immune checkpoints other than PD-L1, such as TIGIT and LAYN, but all lack substantial experimental data to support this. In a clinical study, the trinity of low-dose chemotherapy plus anti-angiogenic plus immune inducer was found to be more effective than the combination of any two drugs or a single drug (91). For targeting oncogenes, the most common are EGFR mutations and ALK fusions. Studies have shown that normal doses or high doses of EGFR-TKI and ALK-TKI can alter the immune microenvironment in lung cancer, but whether immune activation or immune suppression occurs needs to be further explored.

In conclusion, for the long-term treatment of lung cancer patients, an appropriate dose and targeted combination and dosing regimen based on individual patient differences and tolerance to the drug that provides the best combination strategy to expand anti-PD-1/PD-L1-based immunotherapy can greatly improve the prognosis and quality of life for a patient.

LW: Writing – original draft, Writing – review & editing. ZW: Writing – review & editing. LC: Writing – review & editing. CD: Writing – review & editing BJ: Supervision, Writing – review & editing.