Gastric cancer is a significant global health concern, ranking fifth in terms of incidence and fourth in mortality worldwide (1). Certain regions, such as Eastern Asia, Eastern Europe, and South America, have particularly high rates of gastric cancer cases. In mainland China, a considerable number of patients are diagnosed at an advanced stage due to low screening rates and subtle clinical symptoms, resulting in missed opportunities for surgery and poorer prognoses (2). Fortunately, the development of immunotherapy for gastric cancer has shown promising results, changing traditional treatment approaches.

The immune checkpoint is a vital element of the immune system, consisting of receptors found on the surface of immune cells that can either positively or negatively regulate immune responses. For example, PD-1, located on the surface of T cells, functions as a natural brake to control the excessive activity of cytotoxic T effector cells when it binds to its ligand PD-L1. PD-L1 is commonly found in both normal tissues and tumor cells, and their interactions help limit immune-mediated tissue damage and support tumor cells in evading the immune system (3). Immunotherapy using Immune Checkpoint Inhibitors (ICIs) has emerged as a promising approach in the treatment of various cancers. ICIs target the PD-1/PD-L1 pathway to boost the reactivity of anti-tumor T cells. Notably, several PD-1 inhibitors (Nivolumab, Pembrolizumab, Sintilimab, Camrelizumab, Tislelizumab) and PD-L1 inhibitors (Atezolizumab, Avelumab, Durvalumab) have received approval for cancer therapy and have demonstrated effectiveness in an expanding range of malignancies, including gastroesophageal, melanoma, and lung cancers (4). However, despite these significant advancements, a considerable proportion of gastric cancer patients receiving ICIs do not derive therapeutic benefits (5). Numerous clinical studies have been conducted to identify biomarkers that can predict which gastric cancer patients are likely to respond well to ICIs therapy. Some potential biomarkers include PD-L1 expression, tumor mutational burden (TMB), microsatellite instability/mismatch repair (MSI/MMR) status, Epstein-Barr virus (EBV) infection, circulating tumor DNA (ctDNA), and gut microbiota. However, their practical application in day-to-day clinical practice still requires further confirmation (6).

Recent research has been rapidly uncovering the mechanisms linking infection, innate immunity, inflammation, and cancer (7). Cytokines, produced by activated immune cells, play a crucial role in this linkage. Pro-inflammatory cytokines such as IL-1β, IL-8, IL-12, TNF-α, IFN-γ, and anti-inflammatory cytokines like IL-4 and IL-10 have dual functions, activating anti-tumorigenic actions of T cells while also participating in tumor malignant transformation, growth, invasion, and metastasis (8). Cytokines can activate anti-tumorigenic actions of T cells and also contribute to tumor growth, invasion, and metastasis (9). Moreover, systemic inflammatory response (SIR) indicators, such as NLR, PLR, LMR, and TLC, have been reported to be associated with the prognosis of certain cancers (10). Changes in cytokine expression levels and cell composition in the tumor microenvironment (TME) can potentially influence the efficacy of ICIs in various malignancies (11). Therefore, multiplex cytokine and blood cell analysis could yield valuable prognostic assessments in patients.

This study aims to examine the association between baseline and post-treatment peripheral cytokines and blood cells in GC patients who received PD-1 inhibitors combined with chemotherapy. The goal is to identify clinically significant predictive factors for the efficacy of immunotherapy in patients with gastric cancer.

The detection of cytokines and blood cell parameters is preferable to other biomarkers due to their widespread use and minimally invasive sampling technique. In this study, we had access to two groups of patients treated either with immunochemotherapy or solely with chemotherapy. This allowed us to assess the biological impacts of incorporating Immune ICIs. Only associations that were notably significant in Cohort 1, distinct from those observed in Cohort 2 (chemotherapy-only group), were considered indicative of the effects related to ICIs. We employed statistical analysis to determine whether baseline levels and variations in cytokines and blood cell parameters could predict the efficacy of immunotherapy across different treatment outcomes. Initially, we compared baseline cytokine levels of GC patients against those found in healthy subjects. One significant observation was the universally elevated cytokine levels in GC patients, with the exceptions being IL-2 and IL-4. Notably, levels of IL-6, IL-10, and TNF-α were markedly increased in the GC patient group. As depicted in Figure 1 , there were no significant differences in the levels of IL-2, IL-4, INF-γ, and IL-17A between GC patients and healthy controls. IL-2 is primarily secreted by T cells (14), and our study observed a decrease in the total lymphocyte count among gastric cancer patients. Consistent with our findings, Mohammad et al. (15), reported no significant differences in IL-2 levels between gastric cancer patients and healthy controls. Furthermore, another study noted that patients with gastric cancer stage III or IV exhibited elevated levels of IL-2, while there was no distinction in the serum levels of IL-2 between patients with gastric cancer stage I or II and healthy controls (16), which aligns with our findings in Table S7 . Increased IL-4 levels have been frequently observed in various types of cancers. However, the evidence regarding the pro- or antitumoral role of IL-4 is conflicting, and this function is closely linked to IL-4 levels and its interaction with other immunological modulators (17). IFN-γ, produced by numerous immune cell subsets (including T cells, natural killer cells, B cells, and others), possesses both pro-tumor and anti-tumor activities (18). Nitu et al. reported that no significant differences existed in the concentration of IFN-γ between patients and healthy controls (19), which is consistent with our findings. Norma et al. also identified that circulating levels of IL-6 and IL-10 were discernibly higher in GC patients compared to a healthy control group (20), aligning with our results. Numerous studies indicate the pivotal role of IL-6 in a variety of malignancies (21–23). Elevated serum IL-6 concentrations have been documented in several solid tumors, including those of the lung, breast, pancreas, and stomach (24). The STAT3 pathway, when activated by IL-6, up-regulates the expression of cyclins and down-regulates the expression of the cyclin-dependent kinase (Cdk) inhibitor p21. This mechanism consequently promotes tumor cell cycle progression, leading to metastasis and tumor cell proliferation (25). Additionally, IL-6 has been reported to prevent cellular senescence by increasing telomerase activity, thereby promoting tumor growth (26). Studies indicate that IL-10 primarily inhibits the differentiation and antigen-presenting properties of DCs (dendritic cells) during the early stages of immune response (27). As a result, IL-10 significantly suppresses the production of IL-2 from antigen-presenting cells. In the absence of Th1-associated cytokines (like IL-2), the T-cell-mediated response is inevitable (28). While TNF-α, a pro-inflammatory cytokine, has been linked to promoting tumor metastasis and correlated with advanced cancer stages (29–31), its presence in cancers has also been associated with immune suppression. Animal model research further supports TNF-α’s role in promoting tumor growth and malignancy (32–35). Conversely, there are reports suggesting the benefits of the potent pro-inflammatory cytokine (TNF-α) in cancer treatments, especially given its recognition as a major factor in the anti-tumor activities of Coley’s toxins (36). In this study, both NLR and PLR were statistically elevated in GC patients compared to healthy controls. The neutrophil-to-lymphocyte ratio in peripheral blood reflects the balance between systemic inflammation and immunity. Consistent with our results, Mishra et al. discovered that the NLR is higher in cancer patients and its elevated level is linked to a worse.

In SIR studies, elevated NLR levels after ICI treatment have been linked to reduced survival rates in advanced esophagus cancer and lung cancer (10, 37). Consistent with these findings, our patients with a pre-treatment NLR above the median demonstrated a notably worse PFS. This negative correlation may be indicative of the interplay between intense inflammation and compromised immune function (38). While some studies suggest that a higher PLR corresponds to a worse prognosis in lung cancer patients (38), there is a dearth of research examining whether PLR, TLC, and LMR values differ between cancer patients and healthy individuals.

Cytokines represent a broad category of intercellular signaling proteins that play a pivotal role in almost every aspect of human immunology. However, the interaction of cytokine signaling activities is highly complex due to the redundancy and pleiotropy exhibited by cytokines. Moreover, there exists an intricate network of “cytokine cascades,” wherein the expression of a specific cytokine gene is invariably influenced by other cytokines (39). Cytokines are subject to regulation through various mechanisms. For instance, the anti-inflammatory cytokine IL-10 can suppress the expression of TNF-α and IFN-γ, a process referred to as feedback inhibition (40). IL-4, on the other hand, can suppress the production of IFN-γ by T cells, a phenomenon known as antagonism (41), IL-2, conversely, can enhance the production of IFN-γ (42), and IL-17A can synergistically stimulate TNF-α-induced IL-8 production (43).

To explore the prognostic and predictive role of cytokines, we examined the baseline and variations in cytokine levels and assessed their influence on patient outcomes across both cohorts. Cohort 2 had a higher number of patients in the early stages, and more had undergone radical surgery, which is traditionally considered a positive indicator for survival. However, the better OS in Cohort 1 implies that immunotherapy plays a more pivotal role in enhancing survival. A comparative analysis of the two cohorts allowed us to discern the specific effects associated with ICIs. Based on our findings, IL-2 can be perceived as a predictor of favorable response to ICIs. Higher baseline levels of IL-2 correlated with a significantly extended PFS and OS in Cohort 1, a distinction not observed in Cohort 2. IL-2 is a cytokine important in T-cell proliferation and promoting immune responses, as well as in increasing the activity of natural killer cells (44). Garrelds et al. identified that mice deficient in IL-2 are more prone to gastrointestinal inflammation, resembling human ulcerative colitis (45). Ren et al. documented that combining IL-2 with anti-PD-1 helps overcome tumor resistance to ICIs in mice by reactivating intratumoral CD8+ T cells rather than CD4+ Treg cells (46). Similarly, Ewan A et al. reported a two-year remission resulting from combined anti-PD-1 and intralesional IL-2 therapy in two patients with locoregional metastatic melanoma. This impressive response was partly due to an altered tumor microenvironment, including increased PD-L1 expression and CD8 T cell infiltration (47). Moreover, as shown in Figure 5 , patients of Cohort-1 whose IL-2 increased more than 20% from baseline as a response, had a longer OS, which conforms to our preceding view.

IL-6 seems to be a predictor of resistance to ICIs, as patients with higher levels of this factor were found to have significantly worse OS. These observations perfectly agree with the study by Yu et al., who reported that increased circulating levels of IL-6 are associated with poor outcomes in liver cancer patients who received therapy with PD-1 inhibitors (48). IL-6 is a pro-inflammatory cytokine that may contribute to tumor progression by stimulating angiogenesis, invasion, and metastasis (8, 49). In some studies, increased IL-6 serum levels were reported to be associated with metastasis and poor prognosis in prostate, ovarian, and gastrointestinal cancers (21, 50, 51). Tsukamoto et al. indicated that increased IL-6 levels could indicate decreased efficacy of PD-1 blockade in patients with melanoma, and IL-6 blockade augments PD-L1 expression on tumor cells (52). Consistently, a study using IL-6-deficient mice bearing a murine colon cancer cell line found that the lack of IL-6 enhances the induction of effector T cells and inhibits tumorigenesis. Additionally, PD-L1 expression levels on tumor cells were significantly increased in the IL-6-deficient mice compared with wild-type mice (53). These findings strongly indicate the negative immune role of IL-6, especially in patients receiving ICIs.

IL-17A is a prominent member of the IL-17 family of pro-inflammatory cytokines. Prior research has reported its upregulation in the serum and tumors of GC patients. Kang et al. suggested that IL-17A promotes gastric carcinogenesis by regulating the IL-17RC/NF-κB/NOX1 pathway (54). However, it is worth noting that Karl et al. (55) found decreased IL-17A levels in esophageal adenocarcinoma patients when compared to healthy controls. In our study, we observed a less pronounced elevation of IL-17A in GC patients in comparison to healthy controls (as shown in Figure 1 ). Furthermore, our study revealed that GC patients with lower levels of IL-17A experienced improved OS, as demonstrated in Figures 2C , 3B . Interestingly, IL-17A exhibited a noticeable decline from baseline to the point of maximum tumor remission. Accumulating evidence indicates that IL-17A activity may contribute to resistance to anti-tumor immunity and play a role in therapeutic failure. It is reported that the IL-17A signaling pathway can enhance the immunosuppressive activity of regulatory T cells (Tregs), leading to tumor growth and development (56). Liu et al. revealed that IL-17A increases PD-L1 expression through the p65/NRF1/miR-15b-5p axis, thereby promoting resistance to anti-PD-1 therapy. Blocking IL-17A improved the efficacy of anti-PD-1 treatment in murine models of MSS CRC (57). Another clinical analysis suggested that the activation of IL-17A signaling is associated with the failure of anti-PD-1 therapy in patients with colorectal cancer (58).

Prior research has shown that tumor cells release cytokines, vascular endothelial growth factors, and chemokines, which attract neutrophils into tumors. These neutrophils facilitate vascular invasion and contribute to the metastatic potential of tumor cells (59). Neutrophils also participate in creating an immunosuppressive microenvironment by releasing myeloperoxidase and arginase-1, and upregulating PD-L1. This, in turn, reduces the number of tumor-infiltrating lymphocytes (TIL) and leads to decreased effectiveness of immunotherapy (60). The correlation between peripheral blood NLR and clinical outcomes may be explained by the association between tumor-infiltrating lymphocytes and neutrophils, which results in reduced anti-tumor T-cell responses (61, 62).

As depicted in Figure 4 , we observed changes in cytokine levels after treatment in both Cohorts. Cancer cells are the primary sources of cytokines, so successful treatment can lead to reductions in specific cytokines, as observed for IL-2, TNF-α, IFN-γ, and IL-17A in Cohort 2. However, patients treated with chemotherapy alone exhibited stabilization or an increase in levels of IL-4, IL-6, and IL-10 cytokines, which may suggest that the crucial cell compartments contributing to the presence of these cytokines might not be affected by chemotherapy, such as M2 macrophages in the tumor microenvironment (63, 64). Furthermore, the addition of ICIs increased concentrations of cytokines after treatment globally and appeared to counteract the effect of chemotherapy, which typically decreases cytokine levels. It is believed that cytokine levels reflect the immunosuppressive state to some extent, where a high level of cytokines indicates that the body is more sensitive to PD-1 antibodies (65). This finding is consistent with our observation that GC patients in Cohort 1 with more than 20% variation in IL-2 from baseline to the point of maximum remission had better OS.

In conclusion, ongoing studies are actively investigating the predictive role of peripheral blood indicators in the effectiveness and prognosis of immunotherapy. However, comprehensive data on the use of Immune Checkpoint Inhibitors (ICIs) in advanced gastric cancer patients, both domestically and internationally, are still limited. Therefore, further prospective validation is required. To sum up, serum cytokines have varying significance in assessing the response of gastric cancer (GC) patients to anti-PD-1 therapy. Baseline levels of IL-2, IL-6, IL-17A, and Neutrophil-to-Lymphocyte Ratio (NLR), as well as changes in IL-2 levels over time, may serve as convenient predictive biomarkers for identifying GC patients who are likely to benefit from the addition of anti-PD-1 monoclonal antibodies to chemotherapy.

The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation.

The studies involving humans were approved by Research and Clinical Trial Ethics Committee of the First Affiliated Hospital of Zhengzhou University. The studies were conducted in accordance with the local legislation and institutional requirements. The ethics committee/institutional review board waived the requirement of written informed consent for participation from the participants or the participants’ legal guardians/next of kin because The study was retrospective, with anonymous patient information obtained through the hospital record system, and all patients were deceased.

YH: Data curation, Formal Analysis, Writing – original draft. XL: Validation, Visualization, Writing – original draft. YY: Investigation, Software, Writing – review & editing. HS: Conceptualization, Writing – review & editing. SW: Investigation, Writing – review & editing. MG: Funding acquisition, Methodology, Writing – review & editing.