TOB1 modulates neutrophil phenotypes to influence gastric cancer progression and immunotherapy efficacy

Abstract

Introduction: The ErbB-2.1(TOB1) signaling transducer protein is a tumor-suppressive protein that actively suppresses the malignant phenotype of gastric cancer cells. Yet, TOB1 negatively regulates the activation and growth of different immune cells. Understanding the expression and role of TOB1 in the gastric cancer immune environment is crucial to maximize its potential in targeted immunotherapy.

Methods: This study employed multiplex immunofluorescence analysis to precisely delineate and quantify the expression of TOB1 in immune cells within gastric cancer tissue microarrays. Univariate and multivariate Cox analyses were performed to assess the influence of clinical-pathological parameters, immune cells, TOB1, and double-positive cells on the prognosis of gastric cancer patients. Subsequent experiments included co-culture assays of si-TOB1-transfected neutrophils with AGS or HGC-27 cells, along with EdU, invasion, migration assays, and bioinformatics analyses, aimed at elucidating the mechanisms through which TOB1 in neutrophils impacts the prognosis of gastric cancer patients.

Results: We remarkably revealed that TOB1 exhibits varying expression levels in both the nucleus (nTOB1) and cytoplasm (cTOB1) of diverse immune cell populations, including CD8⁺ T cells, CD66b⁺ neutrophils, FOXP3⁺ Tregs, CD20⁺ B cells, CD4⁺ T cells, and CD68⁺ macrophages within gastric cancer and paracancerous tissues. Significantly, TOB1 was notably concentrated in CD66b+ neutrophils. Survival analysis showed that a higher density of cTOB1/nTOB1⁺CD66b⁺ neutrophils was linked to a better prognosis. Subsequent experiments revealed that, following stimulation with the supernatant of tumor tissue culture, the levels of TOB1 protein and mRNA in neutrophils decreased, accompanied by enhanced apoptosis. HL-60 cells were successfully induced to neutrophil-like cells by DMSO. Neutrophils-like cells with attenuated TOB1 gene expression by si-TOB1 demonstrated heightened apoptosis, consequently fostering a malignant phenotype in AGS and HCG-27 cells upon co-cultivation. The subsequent analysis of the datasets from TCGA and TIMER2 revealed that patients with high levels of TOB1 combined neutrophils showed better immunotherapy response.

Discussion: This study significantly advances our comprehension of TOB1's role within the immune microenvironment of gastric cancer, offering promising therapeutic targets for immunotherapy in this context.

Keywords: TOB1; gastric cancer; immune cells; immunotherapy; neutrophils apoptosis.

Figure 1

Predicting the expression of TOB1 in immune cells of gastric cancers. Boxplots were used to visualize the expression of log (TPM+1) TOB1 in each cell type selected, by CIBERSORT (A) and quanTIseq (B) algorithms.

Figure 2

Expression profiles of TOB1 and immune biomarkers in gastric cancers. mIF images of representative gastric cancer sections analyzed for panel 1 (A) and panel 2 (B). Paired boxplots were employed to show the density of nTOB1⁺ (C) and cTOB1⁺ (D) cells between cancer and adjacent paracancerous tissues by the paired Wilcoxon test analysis. Moreover, the box-violin plots depicted the densities of CD8⁺ T cells (E), CD4⁺ T cells (F), FOXP3⁺ Tregs (G), CD20⁺ B cells (H), CD68⁺ macrophages (I), and CD66b⁺ neutrophils (J) between gastric cancer and the corresponding adjacent tissues. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001, ns, not significant.

Figure 3

The expression of TOB1 in various immune cell types between gastric cancer and adjacent paracancerous tissues. TOB1 expression of CD8⁺ T cells (A, B), CD66b⁺ neutrophils (C, D), CD4⁺ T cells (E, F), FOXP3⁺ Tregs (G, H), CD20⁺ B cells (I, J), and CD68⁺ macrophages (K, L). The densities of double positive cells of each immune marker with nTOB1 (M) or cTOB1 (N) analyzing by pairwise Wilcoxon rank sum test. Bar graphs were employed to illustrate the expression proportions of nTOB1 (O) and cTOB1 (P) within various immune cells, with an ANOVA analysis conducted for assessment. ^**P < 0.01, ^***P < 0.001, ns, not significant.

Figure 4

The correlation of TOB1 expression and immune cell infiltration in gastric cancer tissues. Correlation analysis between densities of nTOB1 or cTOB1 and each immune marker positive cells, with the method of Spearman. The lower left section of the figure displays scatter plots depicted the correlations between each pair of indicators. The upper right section provides correlation coefficient (R) values and significance levels. The diagonal section shows the distribution of each individual indicator in bar graphs. ^*P < 0.05, ^**P < 0.01, ^***P < 0.001.

Figure 5

Kaplan-Meier analysis in gastric cancers. The prognosis of gastric cancer patients with the densities of cTOB1⁺ cells (A), nTOB1⁺ cells (B), CD66b⁺ neutrophils (C), cTOB1⁺CD66b⁺ neutrophils (D), nTOB1⁺CD66b⁺ neutrophils (E), CD68⁺ macrophages (F), cTOB1⁺CD68⁺ macrophages (G), and nTOB1⁺CD68⁺ macrophages (H). Highlighted lines represent subgroups with high (red line) and low (blue line) densities.

Figure 6

TOB1 expression in neutrophils and PBMCs of gastric cancer patients and healthy controls. (A) TOB1 mRNA of neutrophils in blood of gastric cancer patients and healthy control was examined by RT-qPCR. (B) The correlation between TOB1 mRNA expression level in neutrophils and TNM clinical stage (I+II vs III+IV) of gastric cancer patients was assessed using the Mann-Whitney test. The expression of TOB1 mRNA in both neutrophils and PBMCs from gastric cancer patients (C) and healthy controls (D) was assessed using the Wilcoxon matched-pairs signed rank test. ^***P < 0.001.

Figure 7

Evaluating the role of TOB1 in the antitumor function and apoptotic regulation of neutrophils. The TOB1 mRNA (A) and protein (B) expression levels in neutrophils following stimulation with NTCS or TTCS were assessed using RT-qPCR and immunofluorescence. (C) Neutrophils were cultured with NTCS or TTCS for 12 hours, and apoptosis was assessed using flow cytometry. (D) The data from three independent apoptosis experiments were collected and analyzed by paired t-tests. (E) After 4 days of DMSO induction, a notable upregulation of CD11b mRNA was observed in HL-60 cells. (F) No discernible alteration of TOB1 mRNA was demonstrated by comparative analysis before and after the induction of HL-60 cells by DMSO. (G) HL-60N cells were transfected with si-NC or si-TOB1 for 48h, mRNAs of TOB1 were detected by RT-qPCR. HL-60N transfected with si-TOB1 and si-NC were co-cultured with neutrophils and gastric cancer cell lines AGS and HGC-27 to evaluate their proliferation (H), migration (I), and invasion (J). In figure H, pink represents proliferating cells, while blue represents cell nuclei. HL-60N cells were transfected with si-NC or si-TOB1 for 48h, cell apoptosis (K), Caspase3, Bax, and Bcl2 (L) were detected by a apoptotic and necrosis assay kit and RT-qPCR, respectively. Additionally, quantitative analysis of apoptosis (M) was performed by randomly selecting three images from each of the three biological replicates per group (a total of nine images per group), counting the percentage of apoptotic cells, and conducting a t-test. Bright blue cells indicated by the arrows represented apoptotic cells, while red cells indicated necrotic cells. ^*P< 0.05, ^**P < 0.01, ^***P< 0.001, ns, not significant.

Figure 8

Evaluating the impact of TOB1 expression in neutrophils on immunotherapy in gastric cancer patients. (A, B) The boxplots of TMB differences between the high-TOB1 and low-TOB1 subgroups from the high-neutrophil and low-neutrophil groups, respectively. (C, D) The boxplots of TIDE differences between the high-TOB1 and low-TOB1 groups combined neutrophils. (E, F) The boxplots of TOB1 expression differences between immunotherapy response and non-response groups combined neutrophils in gastric cancer patients.