Gastric cancer (GC) is the fifth most common cancer worldwide, with nearly one million new diagnoses every year, and is the third leading cause of cancer-related deaths (1, 2). Most patients have the middle- or late-stage disease when diagnosed, and have very short survival and high mortality (3). Globally, GC is characterized remarkable by two major topographical subsites, cardia (CGC) and non-cardia (NCGC) (4). CGC and NCGC have different clinical biological characteristics in different geographical, ethnic and socioeconomic groups. Therefore, CGC and NCGC prognoses remain controversial. Some studies have reported that patients with CGC have a worse prognosis, while others have found no significant differences between the two subtypes (5, 6). It is of great importance to study the relationship between the pathogenesis and location of GC, as these factors can contribute to the development of new therapeutic strategies to improve patient prognosis.

Tumor-associated macrophages (TAMs) are macrophages that infiltrate tumor tissues and are among the most abundant immune cells in the tumor microenvironment (TME). Two main functional subtypes of macrophages have been described: M1 and M2. The function of M1-like macrophages include antigen presentation and tumor cell destruction, while M2-like macrophages promote extracellular matrix remodeling and angiogenesis and exhibit immunomodulatory characteristics (7). The anti-inflammatory characteristics of TAMs are controlled by tumor cells, and this is of great significance in treatment strategies for patients with GC, especially for combination therapies that target cancer cells and macrophages, which can have synergistic effects (8). Moreover, the efficacy of PD-L1/PD-1 antibodies in GC requires M1-like TAMs because they recruit more infiltrating CXCR3⁺CD8⁺ T cells by releasing CXCL9, 10, and 11 (9). M1-like TAMs not only improve the objective response rates of GC but also increase the application of PD-L1/PD-1 antibodies. This suggests that TAMs play a key role in GC development and provide a good target for anticancer therapy (9, 10). Previous studies have shown that high levels of TAM infiltration relate to the aggressive characteristics of GC and are independent poor prognostic factors for patients with GC (11, 12). Nevertheless, no prognostic difference was observed between CD68 density and overall survival (OS) in another study (13) and the M2-like macrophage signature has also been associated with improved survival (14). This could be because TAMs at different locations within the tumor differentially impact GC progression (15). Therefore, we speculated that the number and distribution of TAMs are key factors that affect the coevolution of cancer cells and TAMs.

In tumors, TAMs are usually stimulated by environmental factors to differentiate into M2-like macrophages. Due to the scavenging capabilities of M2 cells, the scavenger receptor CD163 has been proposed as a marker for M2-like macrophages. CD68 is considered the gold standard marker for human macrophages (16). Thus, in this study, we selected CD68 (pan-macrophage marker) and CD163 (M2-like macrophage) labeled TAM to explore the relationship between TAM infiltration at different locations and the prognosis of patient with GC.

Helicobacter pylori (H. pylori) is the most common chronic bacterial infection, affecting approximately 50% of the world’s population, and is a major risk factor for the development of GC (17). It was previously reported that H. pylori urease-activated mucosal macrophages can produce proinflammatory cytokines, which result in H. pylori-related mucosal inflammation (18). In mice with colitis-associated cancer, H. pylori infection was found to reduce TAM infiltration, especially the infiltration of M2-like TAMs (19). Che et al. also demonstrated that H. pylori infection-induced upregulation of activated mesenchymal–epithelial transition factor in exosomes influences the tumor-promoting effect of TAMs (20). These findings suggest a connection between H. pylori and TAMs.

In this study, we explored the relationship between TAM infiltration in different locations and the prognosis of patients with GC, and also whether TAM infiltration associates with H. pylori infection. We found that the distribution of TAMs has distinct characteristics in CGCs and NCGCs. In CGC, CD68⁺ and CD163⁺ macrophages were distributed at the invasive margin (MI) in most samples, while they mainly existed at the core (CT) in the NCGC group. The relationship between TAMs in different locations and the prognosis of patients with GC is conflicting. We thus utilized the difference to distinguish GC into central and marginal GC, to determine the relationship more accurately between macrophages and the prognosis of patients with GC. Moreover, a positive H. pylori status in central GC was significantly associated with better prognosis and TAM infiltrations. Taken together, these results suggest that TAMs and H. pylori are independent prognostic and predictive biomarkers for GC, and this finding might shed light on a new potential target for immunotherapeutic approaches for treating GC.

GC, currently ranked fourth in global cancer-related mortality worldwide, is often diagnosed when it reaches an advanced stage after distant metastasis (23). The importance of TAMs in the development of GC is gaining an increasing interest (24). However, the precise role of TAMs in GC remains unknown and even somewhat contradictory. Here, we revealed a clear effect of different TAM infiltration phenotypes at the CT and MI on GC with topographical subsites. We found a discrepancy in the relationship between TAMs at different locations and the prognosis of patients with GC. Most importantly, positive H. pylori infection in central GC was significantly associated with better prognosis and TAM infiltrations. These findings contribute to a more complete understanding of the correlation between TAMs and the prognosis of patients with GC and have important implications for clarifying their potential role as therapeutic targets.

The densities and prognostic effects of tumor-infiltrating lymphocytes differ in relation to tumor locations within the stomach (25). There are also contrasting prognostic effects of the Foxp3/CD4 ratio in CGC and NCGC (25), which suggests that location may be an important factor for tumor progression. It is possible that neglecting the location during risk assessment could account for the differential findings in previous studies (26, 27). Indeed, accumulating evidence has demonstrated distinct molecular and pathophysiological mechanisms of carcinogenesis in CGC and NCGC (28, 29). Different staging systems have also been used to assess CGC and NCGC (30). Moreover, significant advances have been made in studies of the impact of TAMs on clinical outcomes, and the clinical significance of TAMs can be affected by their number, phenotypes, and distributions at each pathological stage. Therefore, we sought to determine the impact of different macrophages infiltration phenotypes at the CT or MI on the prognosis of patients with CGC or NCGC.

Our findings revealed the distinct TAMs distribution characteristics in CGCs and NCGCs. In most samples of CGC, CD68⁺ and CD163⁺ macrophages were distributed at the MI, whereas they mainly existed at the CT in the NCGC samples. The Kaplan–Meier analysis showed that a high number of CD68⁺ macrophages at the CT in CGCs was associated with a better prognosis. Conversely, a high number of CD68⁺ macrophages at the MI in NCGCs was associated with a poor prognosis. These data indicate the differential effects of CD68⁺ macrophage infiltration at different sites on the prognosis of GC. This may also partly explain why certain prior studies have shown that a high density of CD68⁺ TAMs predicts a poor prognosis in GC (31), while other studies have demonstrated no prognostic difference of CD68 density on OS (13). TAMs at different sites in GC tissue might represent a distinct significance and prognostic value (32).

In addition, the specific localization of TAMs is affected by the environment at different areas in the tumor tissue, and TAM functions have been inconsistent, such as in the presence of hypoxia (33). In breast cancer, tumor nest-associated macrophages have been found to promote angiogenesis to a greater extent than macrophages in the tumor stroma (34). Similarly, our results found that tumor nest-associated CD68⁺ macrophages and tumor stroma-associated CD68⁺ macrophages have different prognostic values for patients with GC, which suggests that macrophages infiltration at the CT or MI have different TME roles. For another, CD68⁺ macrophages at the CT or MI whether have more complex phenotypes. The M1/M2 paradigm represents two extreme TAM activation states, which may neglect that the flexible, rather than static, adaptation driven by environmental signals in the TME. To distinguish the unique role of TAMs under various conditions, it is urgent to redefine TAM subsets and their function in TMEs. A better understanding of how TAM subsets are affected by conditions in specific regions will certainly benefit the related treatments.

H. pylori infection greatly promotes the carcinogenic effects of GC. Interestingly, many studies have reported that a positive H. pylori infection status predicts the survival of patients with GC, with a favorable effect. A prospective study showed that patients with GC with H. pylori infections had better OS and DFS after radical resection (35). A meta-analysis (36) of 2,454 patients also showed that H. pylori infection is an independent protective factor for GC progression, and this protective effect applies to different ethnicities (36). These findings are also consistent with our results, which show that positive H. pylori status in central GC was significantly associated with better prognosis. However, further experiments are required to verify this mechanism (37).

The suppressive effect of H. pylori on GC progression may be due to the induction of an improved immune response against the tumor (35, 38). It has been suggested that H. pylori components simulate surface molecules or specific receptors on gastric epithelial cells, and autoantibodies can induce a cross-reaction against GC cells (39). Another possible explanation is that the true prognostic significance of H. pylori status may be suspected, as a negative H. pylori infection status may only represent a more advanced tumor status. Hobsley et al. (39) proposed that with GC progression, most or all parietal cells become destroyed in advanced GC, which results in the stomach becoming alkaline and negative for H. pylori infection, while H. pylori is positive in patients with early and milder GC. Nevertheless, in our study, we selected patients with stage III advanced GC, and the H. pylori infection positivity rate was 67.6%, which is relatively high. Our results also showed that H. pylori infection is closely related to macrophages infiltration. Considering previous reports and our current research, we are more inclined to hypothesize that H. pylori status has strong prognostic significance. Since H. pylori in the stomach can continuously release bacterial components into the gastrointestinal tract, H. pylori components in the cavity may interact with immune cells (40). Indeed, a number of studies have demonstrated the effect of H. pylori infection on macrophages polarization through in vivo and in vitro experiments. H. pylori not only prevents chronic colitis by promoting M2 polarization (41), but also promotes M1 polarization of human and mouse gastric macrophages, resulting in the occurrence of H. pylori-related atrophic gastritis (42). In addition, Lu et al. (43) confirmed that a low H. pylori multiplicity of infection (MOI) of promotes the M1 and M2 phenotypes, while a high MOI suppresses the M2 phenotype. Intriguingly, our current research focuses on a novel perspective that the degree of H. pylori infection relates to the CD68⁺ and CD163⁺ macrophage infiltration at different locations in GC tissue. However, the mechanism underlying this effect remains to be elucidated. Towards better understanding the underlying mechanism, and verifying the influence of H. pylori infection on the number, location, and polarization of tumor tissue infiltrating macrophages, an animal model of GC has been established. Furthermore, in vitro experiments are underway to determine whether H. pylori is itself a key factor. We hope that our future research will address these outstanding questions.

In summary, our research differs from prior studies in that it focuses on the role of H. pylori and TAM infiltration on GC according to the topographic locations of tumors within the stomach. Our results suggest a new classification method based on CD68⁺ macrophage infiltration at the CT to evaluate the prognosis of patients more accurately with GC. We found that CD68⁺ macrophage infiltration in the CT and a positive H. pylori status were independent protective factors in central GC. These findings indicate that tumor location along with the location of infiltrating cells within the stomach should be considered when evaluating individualized patient prognosis. Furthermore, elucidating the detailed connection between H. pylori and TAMs will facilitate the development of new therapeutic strategies.

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

The studies involving human participants were reviewed and approved by the Ethics Committee of Tianjin Medical University Cancer Institute and Hospital. The patients/participants provided their written informed consent to participate in this study.

QS and XR conceived and designed the experiments. ML, LZ, QZ, and YW performed the experiments. XR provided some suggestions. Data analysis was performed by YW and ML. Writing, reviewing, and manuscript editing was done by ML, QS, and XR. All authors contributed to the article and approved the submitted version.

This work was supported by grants from the National Key R&D Program of China (2018YFC1313400), the National Natural Science Foundation of China (81974416, 81872166, U20A20375 and 81802873), the Tianjin Natural Science Foundation (19JCYBJC27600 and 18JCQNJC81300), and the Scientific Research Program of Tianjin Education Commission (2019KJ185).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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