Roles of the tumor microenvironment in the resistance to programmed cell death protein 1 inhibitors in patients with gastric cancer

Abstract

Despite the continuous developments and advancements in the treatment of gastric cancer (GC), which is one of the most prevalent types of cancer in China, the overall survival is still poor for most patients with advanced GC. In recent years, with the progress in tumor immunology research, attention has shifted toward immunotherapy as a therapeutic approach for GC. Programmed cell death protein 1 (PD-1) inhibitors, as novel immunosuppressive medications, have been widely utilized in the treatment of GC. However, many patients are still resistant to PD-1 inhibitors and experience recurrence in the advanced stages of PD-1 immunotherapy. To reduce the occurrence of drug resistance and recurrence in GC patients receiving PD-1 immunotherapy, to maximize the clinical activity of immunosuppressive drugs, and to elicit a lasting immune response, it is essential to research the tumor microenvironment mechanisms leading to PD-1 inhibitor resistance in GC patients. This article reviews the progress in studying the factors influencing the resistance to PD-1 inhibitors in the GC tumor microenvironment, aiming to provide insights and a basis for reducing resistance to PD-1 inhibitors for GC patients in the future.

Key Words: Gastric cancer; Tumor microenvironment; Programmed cell death protein 1; Immunotherapy; Drug resistance

Core Tip: The discovery of immunotherapy has changed the management of patients with gastric cancer (GC). Programmed cell death protein 1 (PD-1) inhibitors are revolutionary drugs for the therapeutic approach of GC, but resistance to PD-1 inhibitors often occurs in patients with GC. We herein discuss the effects of the tumor microenvironment on the resistance to PD-1 inhibitors in GC patients, aiming to reduce this resistance in the future.

INTRODUCTION

Gastric cancer (GC) is one of the most prevalent malignancies worldwide. Despite a gradual decrease in its incidence in recent years in China, the sheer size of the population still results in a significant number of GC patients[1]. According to the statistics from the China Cancer Center, GC ranks second among malignant tumors in China, following only lung cancer. There are approximately 400000 to 500000 new cases of GC each year, with the incidence rate in males being more than twice that in females[2].

Immunotherapy is a crucial approach to the treatment of GC patients, with immune checkpoint blockade (ICB) being a revolutionary method. ICB targets T-cell immune checkpoint receptors, like programmed cell death protein 1 (PD-1), or blocks immune checkpoint ligands, like programmed cell death ligand 1 (PD-L1). ICB promotes the activation of the immune cells to kill cancer cells, significantly improving the outcomes of GC patients, prolonging their survival, and enhancing their quality of life. However, it is unfortunate that one-third of the patients still experience drug resistance and recurrence in the advanced stages of treatment[3]. Drug resistance significantly limits the widespread use of ICB in clinical applications and poses a pressing challenge in the field of GC immunotherapy.

The tumor microenvironment (TME) consists of noncancerous cells and their elements found within a tumor, such as molecules produced and released there. Continuous interactions involving the TME and tumor cells play a key role in cancer development, dissemination, and the effects of treatment[4]. The TME can either affect or be influenced by tumor growth. During the early stages, the TME inhibits the tumor growth, primarily through interactions between cells and molecules that restrict tumor cell growth and spread. Evidence suggests that as a tumor progresses, the TME changes, gradually transforming into a procancer environment[5]. The tumor immune microenvironment is usually categorized into two types, namely, immune-excluded and immune-inflamed environments. The latter contains a large number of highly active T cells and myeloid cells that are capable of secreting type I interferon (IFN) and chemokines. It is also known as a “hot tumor” in immunology and is characterized by an abundance of PD-1+ cytotoxic T cells, as well as the upregulation of PD-L1 by white blood cells and tumor cells[5]. Different types of tumors have heterogeneous TMEs, which leads to varying clinical responses to PD-1 blockade and is a significant factor contributing to ICB resistance. As a tumor progresses, it competes metabolically with normal cells, decreases the expression of its own antigens, induces the secretion of a variety of cytokines and extracellular vesicles, suppresses the immune function of normal immune cells in the GC TME, decreases their numbers and distribution, and renders the immune system insensitive to the tumor. This leads to the continued dominance of procancer immune cells in tumors, accelerating tumor progression while weakening the activity of immune cells[6]. The stomach, as an organ with a complex endocrine system and a highly acidic environment, forms a distinctive TME in GC. Therefore, studying the mechanisms by which the TME in GC patients leads to the resistance to PD-1 inhibitors is highly important. To better treat GC, we can target the components of the TME to inhibit the malignant cycle of tumor immune tolerance.

Currently, many mechanisms that lead to PD-1 inhibitor resistance in GC patients have been confirmed and supported by numerous studies. In recent years, research has shown that pathological changes in various components of the TME in GC lead to the resistance to anti-PD-1 monoclonal antibodies. In the following sections, I summarize the research progress on the mechanisms by which multiple components induce resistance to PD-1 inhibitors in the TME of GC. I deliver an overview of recent studies on the impacts of various factors, including immune cells, cytokines, the extracellular matrix (ECM), and microenvironment metabolism, that play roles in resistance to PD-1 inhibitors in GC. This review aims to identify interventions that could alleviate the development of resistance to PD-1 inhibitors in GC patients by studying changes in the TME of GC.

IMPACTS OF IMMUNE CELLS ON THE RESISTANCE TO PD-1 INHIBITORS IN PATIENTS WITH GC

Within the TME, many studies have indicated that the impacts of immune cells are closely related to the mechanism of primary resistance to PD-1 inhibitors. This includes a lack of infiltrating immune-active cytotoxic T lymphocytes due to the reduction in CD8+ T-cell numbers or their functional inhibition. Additionally, the mechanism involves increased activity or numbers of immunosuppressive cells, including regulatory T (Treg) cells, M2 macrophages, myeloid-derived suppressor cells (MDSCs), tumor-associated neutrophils (TANs), etc (Table 1).

Table 1 The relationship between immune cells in the tumor microenvironment and the resistance to programmed cell death protein 1 inhibitors in patients with gastric cancer.

Types	Functions	Mechanism of PD-1 inhibitor resistance	The potential to reduce PD-1 inhibitor resistance in patients with GC
Cytotoxic T cells	Inhibit and eliminate tumor cells	Decrease the quantity and functionality of CD8+ T cells[6]	New intervention methods to enhance the functionality of cytotoxic T cells
M2 macrophages	Inhibit immune responses, accelerate the growth and proliferation of tumor cells	Release a variety of cytokines that can stimulate tumor cell proliferation, reduce the activity of immune cells[11]	Blocking the Th2-cell cytokines, reducing monocytes to differentiate into M2-type macrophages
Treg cells	Play a significant regulatory role in the low-immunity TME	Suppress the effector T cells’ activity and modulate the response of antitumor T cells[13]	Eliminating Treg cells in GC tissue during PD-1 inhibitor therapy
MDSCs	Participate in chronic inflammation, cancer, and autoimmune diseases	Induce T cell exhaustion and lead T cell to lose immune function and proliferation ability[19]	Blocking CXCR2 could reduce the frequency of PMN-MDSCs and enhance the effectiveness of anti-PD-1
TANs	Promote tumor cell proliferation and exhibiting a tumorigenic effect	Promote tumor progression through the GM-CSF-PD-L1 pathway[27]	Utilizing drugs or other treatment methods to inhibit the activity of these pathological neutrophils or suppress the GM-CSF/PD-L1 immune pathway
Others

PD-1: Programmed cell death protein 1; GC: Gastric cancer; MDSCs: Myeloid-derived suppressor cells; Treg cells: Regulatory T cells; TANs: Tumor-associated neutrophils; TME: Tumor microenvironment; Th2-cell: T helper 2 cell; CXCR2: CXC chemokine receptor 2; PMN-MDSCs: Polymorphonuclear myeloid-derived suppressor cells; GM-CSF: Granulocyte-macrophage colony-stimulating factor; PD-L1: Programmed cell death ligand 1.

Cytotoxic cells

T cells are lymphocytes with cellular immune functions and are an essential component of the TME. In the TME, CD8+ T cells are primarily responsible for inhibiting and eliminating tumor cells, and Treg cells are typical CD4+ inhibitory immune cells. In addition, GC immune tolerance is closely related to γδ T cells, memory T cells, helper T (Th) cells and natural killer T cells[6].

Among the various T cells, cytotoxic T cells (CD8+) are crucial in the PD-1 signaling pathway, and immune tolerance in GC is largely due to a decline in the quantity and functionality of CD8+ T cells. Research indicates that in GC with stromal B7-H3 expression, CD8+ T cells are spatially inhibited at the center of the tumor[7]. This leads to a reduction in the quantity of CD8+ T cells in the TME, ultimately resulting in the resistance to PD-1 inhibitors.

Continuous antigen stimulation in resistant tumors or chronic inflammation can lead to T-cell dysfunction, which is characterized by immune dysfunction, reduced cytokine secretion, and the expression of a large number of surface inhibitory antibodies, a condition referred to as exhausted T cells[8]. Prolonged antigen stimulation is the main cause for T-cell exhaustion, and the elevated expression of PD-1 is instrumental in maintaining the dysfunctional state of exhausted T cells. Sustained upregulation of PD-1 indicates continuous impairment of immune function. In contrast to those of memory or functional effector memory T cells, the proliferative capacity and cytotoxic activity of exhausted T cells are significantly reduced, followed by abnormalities and even cytokine deficiency[9]. Additionally, tumor-specific CD8+ T cells (TSTs) are present within solid tumors. Although TSTs possess immune functionality, tumors continue to grow, indicating that the functions of these TSTs may be dysregulated or lost. Initially, the dysfunction of TSTs is reversible but ultimately becomes irreversible, even when dysfunctional T cells are removed from the TME and undergo multiple rounds of cell division[10]. Therefore, increasing the quantity of cytotoxic T cells in the TME and enhancing their immune function play crucial roles in overcoming PD-1 inhibitor resistance in GC patients. It is hoped that future research can develop new methods of intervention to overcome immune escape and resistance in the TME by enhancing the function of cytotoxic T cells.

M2 macrophages

M2 macrophages can suppress immune responses and accelerate the growth and proliferation of tumor cells. These cells release a variety of cytokines that can stimulate tumor cell proliferation, reduce the activity of immune cells, and create a low-immunity TME, leading to the resistance to PD-1 inhibitors.

In some cases, GC patients who are treated with PD-1 inhibitors experience accelerated tumor growth, a phenomenon known as hyperprogressive disease (HPD). Compared with that in primary gastric tumors, the number of PD-L1-positive macrophages (PD-L1+ macrophages) in lymph node metastases is significantly increased after anti-PD-1 treatment. Interleukin-4 (IL-4) and IL-13 are Th2-cell cytokines that play important regulatory roles in immune responses. Both cytokines can induce the differentiation of monocytes into M2 macrophages. Recent studies have revealed that the poor prognosis in a variety of cancers is related to the presence of M2 macrophages in the TME[11]. Studies have shown that M2 macrophages and Treg cells are significantly activated and infiltrate the tumors of HPD patients. Furthermore, there are significant increases in the numbers of CD27+, CD28+, and CD4+ T cells in the blood. Thus, dynamic monitoring of these cells may be useful for detecting such adverse outcomes at an early stage[12-14]. There is a large number of PD-L1+ macrophages in the metastatic lymph nodes of HPD patients, which may promote the tumor growth. In mouse xenograft models derived from HPD tumor samples, treatment with PD-1 inhibitors significantly promoted the infiltration of M2 macrophages and accelerated tumor progression[12].

Tumor development is a complex process that involves the interaction of various factors within the TME. The analysis and understanding of the cancer TME in different tissues require consideration of the natural differences between tissues[11]. Currently, the mechanisms by which anti-PD-1 antibodies activate M2 macrophages are not fully understood, and further research is needed to elucidate the interactions between different cell types in the TME in response to PD-1 antibody treatment.

Treg cells

Treg cells play a significant regulatory role in the low-immunity TME by suppressing the effector T cells’ activity and thus modulating the response of antitumor T-cell. As Treg cells express PD-1 molecules, their activity may be influenced by PD-1 inhibitors[11]. PD-1 inhibitors could stimulate the growth and development of highly suppressive PD-1+ Treg cells in HPD patients, thereby inhibiting the antitumor immune response[13].

A previous study that compared tissue samples from GC patients before and after receiving PD-1 inhibitor treatment revealed that PD-1 inhibitor therapy significantly increased the number of tumor-infiltrating proliferative Treg cells in GC patients with HPD, while the number of Treg cells in patients without HPD decreased. Additionally, in related mouse experiments, genetic elimination of PD-1 or antibody-mediated PD-1 blockade in Treg cells increased their proliferation and suppressed the antitumor immune response[13]. The infiltration of certain types of immune cells is significantly associated with a positive response to ICB. Tumors often harbor Treg cells and MDSCs, thereby inhibiting immune reactions against the tumor and promoting tumor growth and metastasis[15]. An increase in the quantity of Treg cells in the circulating blood or in the TME can induce a refractory state during PD-1 inhibitor therapy[16]. The results of relevant research indicate that PD-1 blockade promotes the cell cycle of Treg cells and enhances Treg cell-mediated immune suppression, possibly through stronger T cell antigen receptor signaling, which could be one of the important factors contributing to the development of HPD in GC patients[13]. In summary, the occurrence of HPD in GC patients during PD-1 inhibitor therapy is closely related to the activation and proliferation of Treg cells in the GC TME. In the future, eliminating Treg cells in GC tissue during PD-1 inhibitor therapy may be an effective approach for treating and preventing HPD, potentially serving as an effective method to overcome the resistance to PD-1 inhibitors in GC patients.

MDSCs

MDSCs are important immunosuppressive cells within the TME and represent a heterogeneous group of immature myeloid cells that play significant roles in chronic inflammation, cancer, and autoimmune diseases. MDSCs can be divided into two phenotypically and functionally distinct subpopulations, mononuclear MDSCs and polymorphonuclear MDSCs (PMN-MDSCs). MDSCs have the ability to inhibit T-cell proliferation through various mechanisms, including the expression of proteins such as inducible nitric oxide synthase 2, NADPH oxidase, arginase-1, and S100A8/A9, thereby allowing tumor cells to evade immune system attacks[17]. Research indicates that the usage of PD-1 inhibitors significantly increases the quantity of CD8+ T cells in peritoneal metastases of GC while concurrently reducing the number of MDSCs[18]. MDSCs are known to induce T-cell exhaustion through various pathways[19]. T-cell exhaustion is characterized by a gradual loss of immune function and the proliferation ability, metabolic disturbances, and high and sustained expression of inhibitory receptors, ultimately leading to reduced clinical effectiveness of immune checkpoint inhibitors (ICIs)[20].

The accumulation of MDSCs primarily results from cytokine recruitment by tumor cells[21]. Researches have shown a strong correlation between the number of PMN-MDSCs and the level of C-X-C motif ligand 1 (CXCL1) in tumor tissues in GC patients. GC cell-derived CXCL1 promotes the migration of PMN-MDSCs, and PMN-MDSC accumulation creates a positive feedback loop by directly promoting CXCL1 expression through the S100A8/A9/Toll-like receptor 4 (TLR4)/p38 mitogen-activated protein kinases/nuclear factor-kappa B (NF-κB) pathway. Furthermore, research has shown that PMN-MDSCs induce CD8+ T-cell depletion via the S100A8/A9/TLR4/AKT/mechanistic target of rapamycin (mTOR) pathway, which results in reduced efficacy of or resistance to PD-1 inhibitors. In mouse GC model experiments, blocking CXC chemokine receptor 2 reduced the frequency of PMN-MDSCs and enhanced the effectiveness of anti-PD-1 treatment. Therefore, inhibiting CXC chemokine receptor 2 may reduce the accumulation of PMN-MDSCs, enhance antitumor immune responses, and reduce resistance to PD-1 inhibitors[22]. Hence, therapeutic approaches aimed at reducing the recruitment of MDSCs to the TME may help eliminate resistance to PD-1 inhibitors in GC patients.

TANs

TANs can be divided into tumor-suppressive N1 cells and tumor-promoting N2 cells according to their different functions, and transforming growth factor-beta (TGF-β) can induce the transformation of N1 cells to N2 cells[23]. TANs polarize Th-cell subpopulations that produce IL-17A via the B7-H2/extracellular signal-regulated kinase (ERK) signaling pathway, thereby enhancing GC cell growth and exhibiting a tumorigenic effect[24].

Relevant experiments, including immunohistochemistry, showed that TANs might induce a local immunosuppressive TME by inhibiting T-cell growth and reproduction and enhancing PD-L1 expression[25]. Current research suggests that TANs play a crucial role in the formation of an low-immunity TME by remodeling the ECM, promoting angiogenesis, generating neutrophil extracellular traps, secreting cytokines, depleting essential metabolites, stimulating the expression of immune checkpoint molecules, and recruiting other immunosuppressive cell types[26]. There are several therapeutic approaches that target TANs to alleviate immune tolerance. However, reducing neutrophil levels may increase the risk of infections, posing a challenge to this strategy. Studies have created models of progressive immune suppression within GC. In the first step, gastric tumors secrete granulocyte-macrophage colony-stimulating factor (GM-CSF), leading to proinflammatory effects. In the second step, GM-CSF that is released by GC cells induces the activation of neutrophils within the tumor, simultaneously activating PD-L1 expression on these cells through the Janus kinase/signal transducer and activator of transcription 3 (STAT3) signaling pathway. In the third step, these activated immunosuppressive neutrophils in the GC microenvironment exert a protumor and immune tolerance effects by inhibiting T-cell function in a PD-L1/PD-1-dependent manner[27].

In summary, neutrophils can promote tumor progression through the GM-CSF/PD-L1 pathway. Therefore, the curative effect of PD-1 inhibitors in GC may be significantly associated with a marked increase in the number of PD-L1+ neutrophils. Future developments may include drugs or other treatment methods to inhibit the activity of these pathological neutrophils or suppress the GM-CSF/PD-L1 immune pathway, thus providing new strategies to reduce the resistance to PD-1 inhibitors in GC patients.

IMPACTS OF NONCELLULAR TME COMPONENTS ON PD-1 INHIBITOR RESISTANCE IN PATIENTS WITH GC

Various components of the TME include the ECM and cytokines, among others. Recent research has confirmed a close relationship between PD-1 inhibitor resistance and noncellular components in the TME (Table 2). In the future, these components hold promise as new targets for reducing ICI resistance in GC patients.

Table 2 The relationship between noncellular tumor microenvironment components and the resistance to programmed cell death protein 1 inhibitors in patients with gastric cancer.

Types	Functions	Mechanism of PD-1 inhibitor resistance	The potential to reduce PD-1 inhibitor resistance in patients with GC
POSTN	Regulate a variety of biological processes	It can promote the chemotaxis of macrophages indirectly via the Akt signaling pathway, thereby contributing to resistance to PD-1 inhibitor[30,31]	Targeting POSTN+FAP+ eCAFs to reduce resistance to PD-1 inhibitor therapy in GC patients
FAP	An essential factor in the progression of cancer	It can increase proliferation and migration abilities in vitro, enhance tumor growth[34]	Targeting POSTN+FAP+ eCAFs to reduce resistance to PD-1 inhibitor therapy in GC patients
Inflammatory cytokines	Pro-inflammatory and anti-inflammatory effects	GCMSC secretes IL-8 and activates the Akt pathway resulting in the nuclear localization of the key glycolytic enzyme HK2. Phosphorylated HK2 binds to HIF-1α, supporting GC cell proliferation. GCMSC-induced excessive lactate production impairs CD8+ T-cell function[43]	The use of CXCR antagonists and IL-8-neutralizing antibodies can reverse GCMSC-mediated immune suppression, restoring the sensitivity of patients with GC to antitumor effects of PD-1 antibodies
TGF-β	Participate in various physiological processes, including cell growth, differentiation, reproduction, and immune homeostasis	It can promote tumor growth via EMT, genomic instability, angiogenesis, cancer cell activity, immune escape, and metastasis, and create a suitable microenvironment for cancer cell dissemination and further worsening of cancer[46]	Blocking TGF-β could improve anti-PD-1/PD-L1 responses, decrease the tumor phenotype, inhibit tumor development and promote patient outcomes
LPP	Participate in cell cytoskeleton organization, cell movement, and mechanical sensing	High LPP expression was found to correlate with decreased infiltration of resting CD4+ memory T cells and enhanced infiltration of activated CD4+ memory T cells[50]	LPP could be used as a target to forecast the effects of PD-1 inhibitors in patients with GC
Others

GC: Gastric cancer; PD-1: Programmed cell death protein 1; POSTN: Periostin; FAP: Fibroblast activation protein; TGF-β: Transforming growth factor-β; LPP: Lipoma preferred partner; eCAFs: Extracellular matrix cancer-associated fibroblasts; GCMSC: Gastric cancer mesenchymal stem cell; HK2: Hexokinase 2; IL: Interleukin; CXCRs: CXC chemokine receptors; HIF-1α: Hypoxia-inducible factor 1α; EMT: Epithelial-mesenchymal transition; PD-L1: Programmed cell death ligand 1.

Periostin

Periostin (POSTN) is an ECM protein produced by the periosteum and periodontal ligaments and classified as a secreted ECM-associated protein. ECM proteins regulate a variety of biological processes. A recent study has shown that ECM proteins play a crucial role in the TME-induced resistance to ICIs[28]. Cancer-associated fibroblasts (CAFs) represent a significant proportion of the stromal cell population in the TME and involved in tumor initiation, cancer stem cell renewal, metastasis, chemotherapy resistance, and immune escape[29]. Studies have demonstrated a negative correlation between the abundance of ECM CAFs (eCAFs) and the overall response rate to PD-1 inhibitors therapy in The Cancer Genome Atlas of Stomach Adenocarcinoma (TCGA-STAD) and real-world GC cohorts[30]. Previous researches have shown that POSTN secreted by glioma cells activates the phosphorylation of Akt in macrophages through αvβ3 integrin signaling, resulting in chemotaxis of M2 macrophages[31]. Another study has confirmed that the chemotaxis-activating ability of CAF-secreted POSTN also depends on the Akt signaling pathway[30]. Therefore, POSTN secreted by CAFs promotes the chemotaxis of macrophages indirectly via the Akt signaling pathway, thereby contributing to the resistance to PD-1 inhibitors. These studies provide a reference for the biological mechanisms of CAFs in PD-1 inhibitor resistance and highlight the potential value of targeting POSTN+ fibroblast activation protein (FAP)+ eCAFs to reduce resistance to PD-1 inhibitor therapy in GC patients.

FAP

FAP is one of the type II transmembrane serine proteases that is highly upregulated at remodeling sites in pathological tissues, such as those involved in fibrosis, arthritis, and cancer[32]. Moreover, FAP is an essential factor in the progression of cancer and has emerged as a novel target for cancer diagnosis and therapy[33]. A study has shown that coculturing GC cells with FAP-expressing fibroblasts leads to increased proliferation and migration abilities of GC cells in vitro, enhanced tumor growth in vivo, and resistance to PD-1 inhibitor therapy[34].

A study has investigated the relationships between FAP levels, POSTN levels, and Tumor Immune Dysfunction and Exclusion (TIDE) scores by utilizing the TCGA-STAD database. High upregulation of FAP or POSTN was shown to be positively associated with high TIDE scores, suggesting the poorer effects of ICB therapy[30]. A retrospective analysis at the Union Hospital revealed a positive correlation between the high expression of FAP or POSTN and a poorer treatment response in GC patients receiving immunotherapy[30].

Research conducted in a GC model revealed that the plant-derived compound polyphyllin significantly inhibited the proliferation of CAFs in vitro by downregulating FAP, which resulted in the suppression of in vivo tumor growth[35]. Previous research has revealed the presence of the POSTN+FAP+ eCAF subpopulation in GC tissue and in the TCGA database, with FAP and POSTN primarily expressed in GC stromal cells[30]. Many laboratories are conducting research on the combination therapy of anti-FAP antibodies and drugs to enhance the effectiveness of ICB therapy[36], although the clinical benefits to patients remain unclear. These findings confirm the associations between POSTN+FAP+ eCAFs and resistance or a poor response to ICB therapy in GC patients. Targeting FAP in the future may be key to alleviating resistance or a poor response to PD-1 inhibitor therapy.

Inflammatory cytokines

In terms of inflammatory responses, cytokines play pivotal roles in both proinflammatory and anti-inflammatory processes[37]. Emerging research highlights the expression of inflammatory cytokines by inflammatory cells, particularly the induction of IL-1β expression during Helicobacter pylori infection. This induction can activate NF-κB and concurrently promote the upregulation of tumour necrosis factor α and IL-6. These molecular events contribute to pathological alterations in the stomach, fostering the genesis, progression, and metastasis of GC[38].

Existing studies have revealed that inflammatory cytokines can be used to assess the impact of immunotherapy on GC patients and have predictive value for immunotherapy efficacy. Elevated levels of cytokines suggest increased sensitivity to anti-PD-1 antibodies[39]. Qi et al[40] investigated the correlations between the levels of twelve different cytokines (including IL-4, IL-8, tumour necrosis factor α, IFN-γ, etc.) in GC patients and the therapeutic effectiveness of PD-1 ICIs combined with chemotherapy. The findings indicated that even after immunotherapy, the cytokine levels in good responders remained higher than those in poor responders, suggesting potential correlations between cytokine levels and immunotherapy outcomes. The serum level of IFN-γ could be regarded as a marker for assessing the effectiveness of PD-1 inhibitor treatment[40].

Hou et al[41] have suggested that a PD-1 inhibitor coupled with chemotherapy has an effect on serum cytokine levels in GC patients. IL-2, IL-6, IL-17A, and the neutrophil-to-lymphocyte ratio have the potential to serve as reliable circulatory predictive biomarkers, aiding in the identification of patients who are likely to benefit from PD-1 inhibitors in combination with chemotherapy[41]. Several studies have explored the mechanisms through which inflammatory cytokines affect the efficacy of PD-1 inhibitors. For instance, Liu et al[42] demonstrated that IL-17A was able to inhibit the expression of miR-15b-5p by enhancing the expression of nuclear respiratory factor 1. Decreased expression of miR-15b-5p increases PD-L1 levels, promoting the occurrence of colorectal cancer. In addition, the high expression of miR-15b-5p sensitizes tumors to anti-PD-1 treatment, thereby improving the efficacy of PD-1 inhibitors. Inhibiting IL-17A can enhance the effectiveness of PD-1 inhibitors in a microsatellite stability colorectal cancer mouse model[42]. However, the impact of IL-17A in GC immunotherapy requires further investigation.

In GC, GC mesenchymal stem cells (GCMSCs) are closely associated with immunotherapy tolerance. GCMSCs secrete IL-8 and activate the AKT pathway, which results in the nuclear localization of the key glycolytic enzyme hexokinase 2. Phosphorylated hexokinase 2 binds to hypoxia-inducible factor 1α, promoting PD-L1 transcription and supporting GC cell proliferation. Additionally, GCMSC-induced excessive lactate production impairs CD8+ T-cell function, leading to the resistance to PD-1 ICIs. The use of CXC chemokine receptor antagonists and IL-8-neutralizing antibodies can reverse GCMSC-mediated immune suppression, restoring the sensitivity of patients with GC to antitumor effects of PD-1 antibodies[43]. Therefore, inflammatory cytokines are considered to be correlated with the efficacy of PD-1 inhibitors against GC.

TGF-β

TGF-β is a multifunctional cytokine belonging to the TGF superfamily. TGF-β plays a role in various physiological processes, including cell growth, differentiation, reproduction, and immune homeostasis[44]. TGF-β is also involved in the emergence and development of cancer and can inhibit tumorigenesis early by inhibiting cancer cell cycle progression and inducing apoptosis[45]. In addition, TGF-β signaling is associated with early embryonic development, tissue organogenesis, immune surveillance, tissue repair, and adult tissue homeostasis. As an anticancer factor, TGF-β effectively inhibits cancer cell proliferation. However, in the later stages of cancer, TGF-β promotes tumor growth via epithelial-mesenchymal transition, promotes genomic instability, angiogenesis, cancer cell activity, immune escape, and metastasis, and creates a suitable microenvironment for cancer cell dissemination and further worsening of cancer[46].

TGF-β is a critical factor in the initiation and progression of cancer. In the early stages of tumor development, it inhibits cell cycle progression and stimulates apoptosis to suppress tumorigenesis. Elevated TGF-β levels correlate with adverse outcomes in several types of cancer[45]. However, the use of a single drug to inhibit TGF-β signaling in clinical trials has not yielded satisfactory results. The likely reason is that inhibiting TGF-β signaling alone can promote the expression of PD-L1 and PD-L2 on tumor cells and induce the recruitment of MDSCs, thus enhancing resistance to antitumor effects of ICIs. Treatment with PD-1 inhibitors alone can significantly increase the level of phosphorylated homolog of Drosophila mothers against decapentaplegic 3 and increase the CD4+ Treg/CD4+ T-cell ratio in tumor cells, thereby promoting cancer cell evasion and immune resistance. The use of TGF-β inhibitor antibodies can effectively reduce these adverse effects[47]. Studies have revealed that blocking TGF-β could improve anti-PD-1/anti-PD-L1 responses, decrease the tumor phenotype, inhibit tumor development and improve patient outcomes[48]. In conclusion, inhibiting TGF-β expression with drugs may help reduce resistance to PD-1 inhibitor therapy in GC patients.

Lipoma preferred partner

Lipoma preferred partner (LPP) belongs to the zyxin protein family and modulates cytoskeletal formation, cellular movement, and adhesion. LPP is commonly considered a crucial tumor inducer associated with tumor migration, proliferation, and drug resistance. Located inside adhesions, LPP promotes the formation of invadopodia, acting as a tumor metastasis accelerator[49]. Immunohistochemistry data from The Human Protein Atlas indicated that the LPP was primarily located in stromal fibroblasts within GC tumor tissues, in contrast, it was almost absent in healthy gastric tissues. Consistently, high LPP expression was found to correlate with decreased infiltration of resting CD4+ memory T cells and enhanced infiltration of activated CD4+ memory T cells[50].

A recent study researched the relationship between LPP expression and the reaction to PD-1 inhibitor therapy in 45 GC patients. The results indicated that responders had lower LPP expression than nonresponders, and the therapeutic response rate was lower in patients with elevated LPP expression compared to those with reduced LPP expression. Additionally, the TIDE score could be employed to estimate the efficacy of ICB in GC patients within the TCGA-STAD dataset. The outcomes revealed that patients with elevated LPP expression exhibited higher TIDE scores, and based on their TIDE scores, GC patients were categorized into responder and nonresponder groups. The responder group had lower LPP expression than the nonresponder group, suggesting that tumors with elevated LPP expression tend to have reduced infiltration of resting CD4+ memory T cells and greater infiltration of activated CD4+ memory T cells. Patients with strong expression of PD-L1 or IFN-γ also exhibited enhanced infiltration of activated CD4+ memory T cells in the TME, which led to an unfavorable prognosis. These consistent findings suggest a potential mechanistic theory that fibroblast-derived LPP is related to the infiltration of activated CD4+ memory T cells in the TME of GC patients with elevated IFN-γ/PD-L1 expression, possibly due to a complex intercellular crosstalk leading to immune suppression[50]. In summary, GC patients with elevated LPP expression exhibit significantly greater immune evasion and resistance to immune therapy than those with reduced LPP expression. However, further evidence is required to fully clarify the effects of LPP in mediating resistance to PD-1 inhibitors in GC patients.

IMPACTS OF METABOLIC PATHWAYS ON PD-1 INHIBITOR RESISTANCE IN PATIENTS WITH GC

In the TME of GC, different types of cellular components have distinct metabolic patterns. Competition for metabolites results in metabolic reprogramming, thereby influencing the functions of diverse cellular components within the TME. This can contribute to immune therapy resistance[51].

Glucose metabolism

Glucose metabolism is a crucial pathway for cell survival. In the TME, GC cells exhibit significantly greater glucose uptake than other cells because of nutrient deprivation. This glucose scarcity induces metabolic reprogramming in other TME cells, leading to their dedifferentiation[51]. In experiments involving mouse and human tumor-infiltrating T cells, the loss of mitochondria in tumor-infiltrating T cells and their reliance on glycolysis were observed. Therefore, the functional effects of these T cells were suppressed in a low-glucose TME, contributing to immune escape by tumors[52]. Recent studies have indicated that unrestrained glycolysis can impair the expression of Foxp3 and the suppressive function of Tregs. In mouse models, blocking glycolysis pathways or fatty acid synthesis could enhance the development of Treg cells[53]. The transcription factor Foxp3 in Tregs is able to reprogram metabolism in T cells by enhancing oxidative phosphorylation and suppressing glycolysis, which enables Treg cells to adapt to a low-glucose, high-lactate environment, potentially compromising antitumor immunity[54].

The hypoxia-induced elevation in the levels of glycolysis can lead to a decrease in the numbers of M1 macrophages in GC, highlighting the importance of glucose metabolism reprogramming in the TME[55]. Furthermore, PMN-MDSCs proliferate in GC and suppress glycolysis in CD8+ T cells via the S100A8/A9/TLR4/AKT/mTOR axis, causing the exhaustion of CD8+ T-cell and rendering resistance to PD-1 inhibitors in GC patients[56]. The activation of mTOR also promotes the elevated expression of the glucose transporter glucose transporter type 1 in the membrane of GC cells, resulting in increased glucose uptake. However, in the TME, GC cells have a greater ability than T cells to take up glucose and undergo aerobic glycolysis, leading to the suppression of T-cell effector functions and allowing GC cells to evade immune cytotoxicity[57].

GC cells can suppress the uptake and metabolism of glucose by CD8+ T cells through the CD155/T cell immunoreceptor with immunoglobulin and ITIM domain signaling pathway, thereby suppressing their effector functions and leading to reduced antitumor immune responses[58]. Inactivation of glycogen synthase kinase 3β upregulates the expression of FasL, Lamp1, GrB, and IFN-γ in activated CD8+ T cells, increases the penetration of CD8+ T memory stem cells in GC tumors, and enhances their antitumor immunity[59]. In the TME, a high level of glycolysis results in the accumulation of lactate, promoting the acidification of the TME and inhibiting the functionality of CD8+ T cells[60]. Additionally, high lactate levels can induce M2 macrophage polarization and increase the functionality of Treg cells via stabilization of hypoxia-inducible factor 1α and activation of the ERK/STAT3 signaling pathway, indirectly inhibiting the efficacy of PD-1 inhibitors[61]. Regulating glucose metabolism in the TME is an important approach to overcoming PD-1 inhibitor resistance.

Lipid metabolism

Clinical patients with GC often exhibit tumor-associated macrophages with an increased lipid content, which significantly impacts the polarization of macrophage function[62]. Tumor-associated macrophages with a higher lipid content display characteristics that are similar to those of M2 macrophages, including a reduced phagocytic capability and upregulated PD-L1 expression. This polarization weakens the response of antitumor T cells and enhances the immunosuppressive function of GC cells[62]. The activation of M2-polarized macrophages is crucial for fat breakdown after triglyceride intake. M2 macrophages rely on fatty acid oxidation to promote their proliferation and support their functions. Disruption of this process can inhibit the functionality of M2 macrophages[63].

Genomic analysis has revealed that RHOA mutations in tumor cells, which enhance tumor development, activate the phosphoinositide 3-kinase/AKT/mTOR signaling pathway, resulting in elevated levels of free fatty acids. Treg cells consume free fatty acids more efficiently than Teff cells, which enables Treg cells to better utilize these free fatty acids and enhances their function. Immune suppression caused by RHOA mutations is one of the underlying mechanisms of ICB resistance. Studies have shown that in GC patients with the RHOA Y42 mutation, increased concentrations of free fatty acids in the TME enhance the activity and immunosuppressive function of Treg cells, even in a low-glucose environment, thereby suppressing the antitumor effects of PD-1 inhibitors[64]. In summary, lipid metabolism has a significant impact on the resistance to PD-1 Inhibitors in GC patients, and the specific underlying mechanisms require further study.

Amino acid metabolism

The indoleamine 2,3-dioxygenase (IDO)/kynurenine (Kyn) pathway is a widely recognized immunoinhibitory mechanism. Kyn produced by GC cells can enhance the infiltration ability of Treg cells and promote the secretion of IL-10 by Treg cells, ultimately activating the STAT3/BCL2 pathway and causing chemotherapy resistance in GC patients[65]. IDO is a critical enzyme in the first rate-limiting step of tryptophan (Trp) metabolism in the Kyn pathway, converting the essential amino acid L-Trp into the major metabolite Kyn[66]. It has been demonstrated that ginseng polysaccharides can enhance the antitumor effect of αPD-1 mAbs by increasing the level of the intestinal microbiota metabolite valeric acid and decreasing the L-Kyn level and the Kyn/Trp ratio. Trp metabolism is related to the resistance to PD-1 ICI therapy in cancer[67]. Botticelli et al[68] discovered that a high level of IDO activity, represented by the Kyn/Trp ratio, could serve as a predictor for resistance to PD-1 inhibitor therapy.

Research by Nguyen et al[69] indicated that Kyn played a critical role in Treg cell induction. Previous studies revealed that patients with GC who developed chemotherapy resistance had elevated serum levels of Kyn, leading to a significant increase in the proportion of Tregs, which promoted chemotherapy resistance in GC patients[65]. The Kyn/AhR signaling pathway plays a selective role in tumors that overexpress IDO/TDO and is associated with the resistance to ICB by genetically regulating Foxp3 expression, inducing Treg cell generation and promoting their function[70]. Furthermore, via the mechanism of elevated IDO expression, IFN-γ can induce tumor immune suppression by promoting the production of IDO, the expression of human leukocyte antigen-G and PD-L1, and the generation of MDSCs[71]. Relevant studies support the link between the upregulation of anti-PD-1 antibodies and IDO in the TME, demonstrating that IFN-γ may also promote immune escape of tumor cells by inducing the upregulation of IDO, which leads to the resistance to ICI therapy[72]. This resistance can be overcome by IDO inhibitors[73]. However, the therapeutic effectiveness of IDO inhibitors in patients with GC resistant to PD-1 inhibitors still requires further confirmation.

CONCLUSION

Immunotherapy is a key treatment for patients with GC, and ICB is a revolutionary approach to cancer treatment. ICB targets immune checkpoints on T cells, activating the immune system to attack and kill cancer cells. ICB has significantly improved the prognosis of GC patients, prolonging their survival and enhancing their quality of life. Most patients who respond to ICB therapy maintain long-term disease control. However, unfortunately, one-third of patients still experience treatment-resistant relapses in the late stages of therapy[3]. Resistance largely limits the widespread application of ICB in clinical practice, and thus, overcoming resistance is a pressing new challenge in the field of GC immunotherapy. As immunotherapy continues to evolve, the role of the TME in immunotherapy has become increasingly crucial. Clinical research on the association between the TME and resistance to ICIs actively continues worldwide.

Despite the progress in studies on the mechanisms of resistance to PD-1 inhibitors due to the various aforementioned TME factors, there are still many mechanisms that need further investigation. These include the specific mechanisms underlying the effects of cytokines on PD-1 inhibitor resistance and the specific effects of metabolites and signaling pathways on the functionality of immune cells. However, many countries are already conducting related studies. I believe that with the further development of immunotherapy techniques, research on the impact of the TME on PD-1 inhibitor resistance in GC patients will eventually increase. In the future, targeting the TME will alleviate or overcome acquired immune resistance to PD-1 inhibitors, bringing new hope for GC patients who are resistant to immunotherapy.