ST6GALNAC1 mediates sialylation of mucins in non-small cell lung cancer to evade immune surveillance.

Lung cancer ranks first among common cancers worldwide and first among causes of cancer death ( 1 ). It mainly originates from the bronchial mucosa, bronchial glands, and alveolar epithelium, with high morbidity and mortality rates ( 2 ). Its incidence rate and mortality are high ( 3 ). Currently, non-small cell lung cancer (NSCLC) patients cannot be completely cured by surgery, and about 77% of NSCLC patients have evidence-based indications for radiation therapy ( 4 ). With the development of immunology and molecular biology, immunotherapy has received increasing attention in the clinical treatment of NSCLC in recent years ( 5 ). However, long-term high-dose radiation therapy can lead to radiation resistance in patients, ultimately leading to cancer recurrence. Therefore, developing effective strategies to overcome radiation resistance in NSCLC treatment is an important challenge ( 6 , 7 ). Immune escape is an important way for tumor progression, so finding appropriate immune surveillance targets has far-reaching clinical significance. Natural killer (NK) cells participate in the body’s innate immunity and have multiple immune functions. It does not require activation by tumor-specific antigens, nor is it restricted by major histocompatibility complex-I (MHC-I) ( 8 ). It kills tumor cells through immune regulation and the inhibition of cell proliferation ( 8 , 9 ). Immune escape is one of the main reasons for the continued development of the disease during tumor treatment ( 10 ). After tumor cells escape the body’s immune surveillance, the biological process accelerates, further leading to tumor proliferation, invasion, and metastasis ( 11 , 12 ). Simply put, “immunity” refers to the ability of the human body to resist diseases and foreign pathogens. The immune system can resist the invasion of bacteria and viruses, clear aging, cancer, and necrotic cells into the body, process metabolic waste into the body, and maintain the stability of the internal environment ( 11 , 12 ). Therefore, the activity and quantity of immune cells can to some extent reflect the strength of the body’s immune capacity. ST6 N-acetylgalactosaminide alpha-2,6-sialyltransferase 1 (ST6GALNAC1) belongs to the sialyltransferase family ( 13 ). Currently, the abnormal expression of ST6GALNAC1 has been found in various tumors (ovarian cancer, esophageal squamous cell carcinoma, colon cancer, bladder cancer, prostate cancer) and other diseases such as ulcerative colitis and endometriosis ( 14 - 16 ). The expression of ST6GALNAC1 is related to prognosis and survival status ( 17 , 18 ). Researchers have found that the overexpression of ST6GALNAC1 leads to enhanced peritoneal metastasis of gastric cancer cells and shortened survival of mice ( 13 ). Here, this study explored the function of ST6GALNAC1 on immune surveillance in NSCLC cells and nude mice. We present this article in accordance with the ARRIVE and MDAR reporting checklists (available at https://jtd.amegroups.com/article/view/10.21037/jtd-2025-660/rc ).

The pathological tissues and serum samples from patients with NSCLC visiting The Fourth Affiliated Hospital of Soochow University as well as their corresponding paracancerous tissues (≥2 cm away from the tumor border) were retrospectively collected. And the tissues were immediately snap frozen in liquid nitrogen and stored at −80 ℃ for further using. The single-cell RNA-seq library construction and sequencing were carried out based on the Li’s work ( 19 ). The study was conducted in accordance with the Declaration of Helsinki and its subsequent amendments. The study was approved by the Ethics Committee of The Fourth Affiliated Hospital of Soochow University (No. 251222) and informed consent was taken from all the patients. Nude mice (24±1 g; aging 10±1 weeks) maintained at 22±2 ℃, 50–60% relative humidity and a 12 h light dark cycle were injected with pentobarbital sodium (40 mg/kg) intraperitoneally, and were injected with A549 cells in the right flank. Mice were randomly divided into two groups according to the random control table: control group (5×10 8 Pfu control virus/mice, once a week, n=6) and ST6GALNAC1 group (5×10 8 Pfu ST6GALNAC1 virus/mice, once a week, n=6). Experiments were performed under a project license (No. 2025-251222) granted by the ethics committee of The Fourth Affiliated Hospital of Soochow University, in compliance with NIH Guide for the Care and Use of Laboratory Animals. Human lung adenocarcinoma A549 cells and mono-nuclear macrophage RAW264.7 cells were performed in compliance with American Type Culture Collection (ATCC) protocols and incubated in cultured with dulbecco minimum essential medium (DMEM; Gibco, Shanghai, China) routinely supplemented with 10% fetal bovine serum (FBS; PAN3000) at a 5% CO 2 atmosphere at 37 ℃. ST6GALNAC1 plasmids were transfected into cells, or si-ST6GALNAC1 plasmids were transfected into RAW264.7 cells using Lipofectamine 2000. Then, in transwell plate (0.4 µM, Corning, New York, USA) with a pore size, RAW264.7 cells (5×10 5 /mL) were loaded on the upper chamber, and A549 cells (2.5×10 5 /mL) were loaded in the lower chamber for 24 h, they were co-cultured in medium without serum lasting for 24 h to produce the co-cultured NSCLC cells. Relative levels of the sample messenger RNA (mRNA) expression were expressed and as 2 −ΔΔCt . According to the Prime-Script TM RT detection kit, qPCR was performed with the ABI Prism 7500 sequence detection system. Lactate dehydrogenase (LDH), iron levels, glutathione (GSH) and propidium iodide (PI) cells were detected, which by using Quantikine ELISA kit. And using a plate reader as a correction wavelength of 450 nm, absorbance at 450 nm was measured. The cellular proliferation was detected using MTT assay according to manufacturer’s instructions after culturing at indicated time. EdU (10 mM) was added to each well, followed by fixation of the cells with 4% paraformaldehyde for 30 minutes. EdU was detected using fluorescent microscope (Olympus, Shanghai, China) after washing. After co-culture of RAW264.7 cells and A549 cells for 24 h, the cell was planted in the upper chamber of transwell chamber. After adding the conditioned medium into the lower chamber, the chamber was cultured in incubator for 24 h. The cells that did not migrate in the upper chamber were wiped off with a cotton swab, and the cells that migrated to the lower chamber were counted under a microscope after being stained with crystal violet. The membranes were incubated with primary antibodies: anti-ST6GALNAC1 (1:1,000, ab69066), anti-Akt (phospho T308) (1:1,000, ab105731), AKT (1:1,000, Cell Signaling Technology, Inc., Shanghai, China), glutathione peroxidase 4 (GPX4; 1:1,000, ab125066), and β-Actin (1:5,000, Santa Cruz Biotechnology, Shanghai, China, sc-69879) after blocking with 5% bovine serum albumin (BSA) in TBS, followed by incubation with peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). Macrophages were sorted out using flow cytometry with CD86 for M1 and CD206 for M2 macrophages marker for further co-culture experiment. Statistical significance was defined as P<0.05 (GraphPad Prism 6). Intergroup comparisons were performed using Student’s t -test for two groups or one-way analysis of variance (ANOVA) with Tukey’s post hoc test for multiple groups.

Firstly, the expression levels of ST6GALNAC1 in patients with NSCLCs were investigated. Serum ST6GALNAC1 mRNA expression was up-regulated in patients with NSCLCs ( Figure 1A ). Meanwhile, the overall survival (OS) and disease-free survival (DFS) of high expression of ST6GALNAC1 in patients with NSCLCs were lower than low expression of ST6GALNAC1 in patients with NSCLCs ( Figure 1B,1C ). In tumor tissue of patients with NSCLCs, ST6GALNAC1 protein expression was also increased ( Figure 1D ). ST6GALNAC1 expression in patients with non-small cell lung cancer. ST6GALNAC1 mRNA expression (A), DFS (B) and OS (C), ST6GALNAC1 protein expression (D) in patients with non-small cell lung cancer. **, P<0.01 compared with the normal group. DFS, disease-free survival; mRNA, messenger RNA; NSCLCs, non-small cell lung cancers; OS, overall survival; ST6GALNAC1, ST6 N-acetylgalactosaminide alpha-2,6-sialyltransferase 1. Then, we determined the effects of ST6GALNAC1 on the mice model of NSCLCs. It was found that ST6GALNAC1 virus increased Caspase-3/9 activity levels, promoted tumor weight and volume, induced tumor necrosis factor-alpha (TNF-α) mRNA expression and cyclooxygenase-2 (Cox-2) in tumor tissues of mice model of NSCLCs, when compared with the control group ( Figure 2 ). ST6GALNAC1 promoted cell growth in mice model. Tumor volume and weight (A,B). Images of tumors (C). Cox-2 and TNF-α mRNA expression (D,E). Caspase-3/9 activity levels (F,G). **, P<0.01 compared with the control group. Cox-2, cyclooxygenase-2; mRNA, messenger RNA; ST6GALNAC1, ST6 N-acetylgalactosaminide alpha-2,6-sialyltransferase 1; TNF-α, tumor necrosis factor-alpha. In RAW264.7 cells co-cultured with A549 cells, ST6GALNAC1 plasmid increased ST6GALNAC1 expression in RAW264.7 cells, ST6GALNAC1 up-regulation promoted cell growth, migration of A549 cells ( Figure 3A-3D ). si-ST6GALNAC1 treatment would reduce ST6GALNAC1 expression in RAW264.7 cells, and suppress cell growth, migration of A549 cells ( Figure 3E-3H ). ST6GALNAC1 promotes the invasion and migration of NSCLCs. ST6GALNAC1 expression (A), cell growth (B), EdU positive cells (C), migration (D) in A549 cells of RAW264.7 cells by ST6GALNAC1 up-regulation. ST6GALNAC1 expression (E), cell growth (F), EdU positive cells (G), migration (H) in A549 cells of RAW264.7 cells by ST6GALNAC1 down-regulation. *, P<0.05; **, P<0.01 compared with the control groups. CXCL10, C-X-C motif chemokine 10; EdU, 5-ethynyl-2'-deoxyuridine; IFN-γ, interferon-gamma; IL-12, interleukin-12; NSCLCs, non-small cell lung cancers; ST6GALNAC1, ST6 N-acetylgalactosaminide alpha-2,6-sialyltransferase 1; TGF-β, transforming growth factor-beta. In vivo , single-cell data in Figure 4A showed that ST6GALNAC1 was possible to be expressed in macrophages of patients with NSCLCs. The CD86/CD206 rate was significantly reduced in the ST6GALNAC1 group than the control group ( Figure 4B ). Meanwhile, in the ST6GALNAC1 group, the interleukin-12 (IL-12), C-X-C motif chemokine 10 (CXCL10), interferon-gamma (IFN-γ) levels were evidently decreased, but the IL-10, transforming growth factor-beta (TGF-β) levels were significantly increased (all P<0.01, Figure 4C-4J ). ST6GALNAC1 promoted immune surveillance in mice model. model. IL-12, CXCL10, IFN-γ levels (A-C); IL-10, TGF-β levels (D,E) in RAW264.7 cells by ST6GALNAC1 up-regulation; IL-12/CXCL10/IFN-γ levels (F-H), IL-10/TGF-β levels (I,J) in RAW264.7 cells by ST6GALNAC1 down-regulation. **, P<0.01 compared with the si-nc or negative group; IL-12, interleukin-12; nc, normal control; NSCLC, non-small cell lung cancer; ST6GALNAC1, ST6 N-acetylgalactosaminide alpha-2,6-sialyltransferase 1; TGF-β, transforming growth factor-beta. In RAW264.7 cells co-cultured with A549 cells, ST6GALNAC1 up-regulation reduced IL-12, CXCL1, IFN-γ levels but increased IL-10, TGF-β levels (all P<0.01, Figure 5A-5E ). ST6GALNAC1 down-regulation increased IL-12, CXCL10, IFN-γ levels but reduced IL-10, TGF-β levels in RAW264.7 cells co-cultured with A549 cells (all P<0.01, Figure 5F-5H ). ST6GALNAC1 promoted immune surveillance in vitro model. ST6GALNAC1 expression (A), cell growth (B), EdU positive cells (C), migration (D) in A549 cells of RAW264.7 cells by ST6GALNAC1 up-regulation; ST6GALNAC1 expression (E), cell growth (F), EdU positive cells (G), migration (H) in A549 cells of RAW264.7 cells by ST6GALNAC1 down-regulation. *, P<0.05; **, P<0.01 compared with the negative or si-nc groups. CXCL10, C-X-C motif chemokine 10; DAPI, 4',6-diamidino-2-phenylindole; EdU, 5-ethynyl-2'-deoxyuridine; IFN-γ, interferon-gamma; IL-12, interleukin-12; nc, normal control; OD, optical density; ST6GALNAC1, ST6 N-acetylgalactosaminide alpha-2,6-sialyltransferase 1; TGF-β, transforming growth factor-beta. Next, the function of ST6GALNAC1 on ferroptosis of macrophage in cell model of NSCLCs was explored. It was observed that ST6GALNAC1 up-regulation increased PI positive cells and LDH activity levels, enhanced iron concentration, and suppressed GPX4 protein expression in RAW264.7 cells (all P<0.01, Figure 6A-6D ). ST6GALNAC1 down-regulation reduced PI positive cells and LDH activity levels, inhibited iron concentration, and increased GPX4 protein expression in RAW264.7 cells (all P<0.01, Figure 6E-6H ). Then, ST6GALNAC1 up-regulation reduced mitochondrial damage in RAW264.7 cells and also suppressed JC-1 and Calcien-AM/CoCl2 levels (all P<0.01, Figure 6G-6I ). ST6GALNAC1 down-regulation reduced mitochondrial damage in RAW264.7 cells and increased JC-1 and Calcien-AM/CoCl2 levels (all P<0.01, Figure 6J-6L ). In abdominal macrophages of mice model of NSCLCs, ST6GALNAC1 virus reduced GPX4 protein expression (P<0.01, Figure 6M ). ST6GALNAC1 promotes ferroptosis of macrophage. PI positive (A), LDH activity (B), iron concentration (C), GPX4 protein expression (D) in RAW264.7 cells by ST6GALNAC1 up-regulation. PI positive (E), LDH activity (F), iron concentration (G), GPX4 protein expression (H) in RAW264.7 cells by ST6GALNAC1 down-regulation. JC-1 levels (I), Calcien-AM/CoCl2 (J), mitochondrial damage (electron microscopic images; magnification: ×40,000) (K) in RAW264.7 cells by ST6GALNAC1 up-regulation. JC-1 levels (L), Calcien-AM/CoCl2 (M), mitochondrial damage (electron microscopic images; magnification: ×40,000) (N) in RAW264.7 cells by ST6GALNAC1 down-regulation. GPX4 protein expression in abdominal macrophages of mice model of NSCLC (O). **, P<0.01 compared with the negative or si-nc groups. DAPI, 4',6-diamidino-2-phenylindole; GPX4, glutathione peroxidase 4; JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetramethylbenzimidazolylcarbocyanine iodide; LDH, lactate dehydrogenase; nc, normal control; NSCLC, non-small cell lung cancer; PI, propidium iodide; ST6GALNAC1, ST6 N-acetylgalactosaminide alpha-2,6-sialyltransferase 1. We further examined the potential mechanism for ST6GALNAC1 on ferroptosis of macrophage. ST6GALNAC1 up-regulation induced ST6GALNAC1 protein expression, and suppressed p-Akt protein expression in RAW264.7 cells (P<0.01, Figure 7A ). ST6GALNAC1 down-regulation suppressed ST6GALNAC1 protein expression, and induced p-Akt protein expression in RAW264.7 cells (P<0.01, Figure 7B ). Immunofluorescence showed that ST6GALNAC1 up-regulation suppressed p-Akt protein expression, and induced ST6GALNAC1 protein expression in RAW264.7 cells ( Figure 7C ). Then, ST6GALNAC1 virus suppressed p-Akt protein expression in abdominal macrophages of mice model of NSCLCs (P<0.01, Figure 7D ). ST6GALNAC1 suppressed p-Akt expression of macrophage to promote ferroptosis of macrophage. ST6GALNAC1 and p-Akt protein expression (A) in RAW264.7 cells by ST6GALNAC1 up-regulation; ST6GALNAC1 and p-Akt protein expression (B) in RAW264.7 cells by ST6GALNAC1 down-regulation. Immunofluorescence (magnification: ×100) for ST6GALNAC1 and p-Akt protein expression (C) in RAW264.7 cells by ST6GALNAC1 up-regulation. p-Akt protein expression in abdominal macrophages of mice model of NSCLCs (D). **, P<0.01 compared with the negative or si-nc groups. DAPI, 4',6-diamidino-2-phenylindole; nc, normal control; NSCLCs, non-small cell lung cancers; p-Akt, phosphorylated protein kinase B; ST6GALNAC1, ST6 N-acetylgalactosaminide alpha-2,6-sialyltransferase 1. Based on the above findings, the effect of ST6GALNAC1/p-Akt pathway on the immune surveillance in NSCLCs attracted our attention. Akt inhibitor (25 nM uprosertib) reduced the effects of ST6GALNAC1 on p-Akt/GPX4 protein expression in RAW264.7 cells ( Figure 8A ). Akt inhibitor reduced the effects of ST6GALNAC1 on the migration of A549 cells ( Figure 8B-8D ). Akt inhibitor also reduced the effects of ST6GALNAC1 on the ferroptosis and immune surveillance of RAW264.7 cells ( Figure 8E-8J ). Akt agonist (10 µM SC79) reduced the effects of si-ST6GALNAC1 on p-Akt/GPX4 protein expression in RAW264.7 cells ( Figure 9A ). Akt inhibitor reduced the effects of ST6GALNAC1 on the migration of A549 cells ( Figure 9B-9D ). Akt inhibitor also reduced the effects of ST6GALNAC1 on the ferroptosis and immune surveillance of RAW264.7 cells ( Figure 9E-9J ). Akt inhibitor reduced the effects of ST6GALNAC1 on immune surveillance. P-Akt and GPX4 protein expression (A) in RAW264.7 cells by ST6GALNAC1 up-regulation + Akt inhibitor. Cell growth (B), migration (C), EdU positive cells (magnification: ×10) (D) in A549 cells of RAW264.7 cells by ST6GALNAC1 up-regulation + Akt inhibitor; PI positive (E), LDH activity (F), iron concentration (G), JC-1 levels (H), Calcien-AM/CoCl2 (I), IL-12/CXCL10/IFN-γ/IL-10/TGF-β levels (J) in RAW264.7 cells by ST6GALNAC1 up-regulation + Akt inhibitor. *, P<0.05; **, P<0.01 compared with the negative group; # , P<0.05; ## , P<0.01 compared with the ST6GALNAC1 group. CXCL10, C-X-C motif chemokine 10; DAPI, 4',6-diamidino-2-phenylindole; EdU, 5-ethynyl-2'-deoxyuridine; GPX4, glutathione peroxidase 4; IFN-γ, interferon-gamma; IL-12, interleukin-12; JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetramethylbenzimidazolylcarbocyanine iodide; LDH, lactate dehydrogenase; nc, normal control; OD, optical density; p-Akt, phosphorylated protein kinase B; PI, propidium iodide; ST6GALNAC1, ST6 N-acetylgalactosaminide alpha-2,6-sialyltransferase 1; TGF-β, transforming growth factor-beta. Akt agonists reduced the effects of si-ST6GALNAC1 on immune surveillance. P-Akt and GPX4 protein expression (A) in RAW264.7 cells by ST6GALNAC1 down-regulation + Akt agonists. Cell growth (B), migration (C), EdU positive cells (magnification: ×10) (D) in A549 cells of RAW264.7 cells by ST6GALNAC1 down-regulation + Akt agonists; PI positive (E), LDH activity (F), iron concentration (G), JC-1 levels (H), Calcien-AM/CoCl2 (I), IL-12/CXCL10/IFN-γ/IL-10/TGF-β levels (J) in RAW264.7 cells by ST6GALNAC1 down-regulation + Akt agonists. *, P<0.05; **, P<0.01 compared with the si-nc group; # , P<0.05; ## , P<0.01 compared with the si-ST6GALNAC1 group. CXCL10, C-X-C motif chemokine 10; DAPI, 4',6-diamidino-2-phenylindole; EdU, 5-ethynyl-2'-deoxyuridine; GPX4, glutathione peroxidase 4; IFN-γ, interferon-gamma; IL-12, interleukin-12; JC-1, 5,5',6,6'-tetrachloro-1,1',3,3'-tetramethylbenzimidazolylcarbocyanine iodide; LDH, lactate dehydrogenase; nc, normal control; OD, optical density; p-Akt, phosphorylated protein kinase B; PI, propidium iodide; ST6GALNAC1, ST6 N-acetylgalactosaminide alpha-2,6-sialyltransferase 1; TGF-β, transforming growth factor-beta.

NSCLC incidence rate accounts for about 80% of lung malignancies, and its incidence rate and mortality rate rank first among all types of malignancies in many regions ( 20 ). The current treatment options such as radiotherapy, chemotherapy, and surgery have shown good efficacy in inhibiting the metastasis of malignant tumors and improving prognosis ( 21 , 22 ). However, during the treatment process, patients often suffer from toxic side effects and drug resistance. Therefore, it is particularly urgent to search for drugs with low toxicity and good tolerance, which has important significance for clinical treatment. In this study, serum ST6GALNAC1 mRNA and protein expression was up-regulated in patients with NSCLCs. Luo et al. showed that ST6GALNAC1 promoted the invasion and migration of breast cancer ( 15 ). Thus, these results suggest that ST6GALNAC1 might exert protective functionality for treatment with NSCLCs. With the change of lifestyle, the incidence of malignant tumors has shown a trend of regionalization and youthfulness, bringing heavy economic and mental burden to many families ( 23 ). The causes of tumors are complex, but closely related to our lifestyle and environment ( 23 ). Clinical studies have shown that the rapid growth and metastasis of malignant tumors are accompanied by a decrease in the body’s immune system, and the cellular immunity mediated by T lymphocyte subsets is one of the main ways for the body to resist tumors ( 24 ). Regulatory T cells include helper T cells, which is distinguished by the cell surface marker CD4 + ( 25 ) and the main function of it is to activate B lymphocytes and assist in completing humoral immunity. Effector T cells include cytotoxic T cells, which is distinguished by the cell surface marker CD8 + and it can directly kill target cells through pathways such as perforin ( 26 ). NK cells have the function of non-specific killing of tumor cells, and their anti-cancer function is mainly achieved by secreting some cytokines such as IFN-γ, IL-2, etc. ( 27 ). We found that ST6GALNAC1 promoted cell growth and immune surveillance in mice model of NSCLCs. Yao et al. showed that ST6GALNAC1 regulated immunity of inflammatory effect ( 14 ). Therefore, ST6GALNAC1 could promote cell growth of NSCLCs through immune surveillance. Compared with the healthy population, the T cell subsets and various indicators of malignant tumor patients detected in this study showed significant variability, indicating significant individual differences in immune function after cancer ( 28 ). This suggests that clinical practice can evaluate patient immunity by introducing immune cell monitoring, and adopt personalized and precise diagnosis and treatment based on this ( 29 ). IFN-γ and IL-4, as immune promoting factors in the tumor microenvironment, and IL-10, as an immunosuppressive factor, have important regulatory roles in the immune escape of cancer cells ( 30 , 31 ). This study found that, in RAW264.7 cells co-cultured with A549 cells, ST6GALNAC1 up-regulation in RAW264.7 cells promoted cell growth, invasion and migration of A549 cells. Sánchez-Martínez et al. reported that ST6GALNAC1 ameliorated inflammation in intestinal barrier integrity ( 16 ). Therefore, ST6GALNAC1 might promoted cell growth in NSCLCs through immune surveillance. Ferroptosis, an immunogenic form of cell death firstly reported by Dixon in 2012, can release a series of damage-associated molecules to trigger an inflammatory response ( 32 ). The regulatory mechanisms of ferroptosis have been a hot research topic in recent years. Under the catalysis of iron and hydrogen peroxide, the excessive reactive oxygen species (ROS) reacts with polyunsaturated fatty acids (PUFAs) on the cell membrane, causing membrane rupture and ultimately cell death ( 33 ). Macrophages are polarized into M1 or M2 types under the stimulation of different tissue microenvironments, and different cell types produce different modes of glucose metabolism. After activation in tumor or inflammatory environments, macrophages undergo glucose metabolism reprogramming, whether it is the glycolysis and pentose phosphate pathways preferred by M1 macrophages, or the tricarboxylic acid (TCA) dependent pathway by M2 macrophages ( 33 ). These metabolic intermediates or pathways often exhibit significant differences. Not only does macrophage glucose metabolism affect iron death, but iron death inducers can also affect macrophage polarization, affect macrophage metabolic reprogramming, and lead to different sensitivity of macrophages to iron death ( 34 ). We found that ST6GALNAC1 promoted ferroptosis of macrophage. Therefore, ST6GALNAC1 could enhance ferroptosis of macrophage to evade immune surveillance in NSCLCs. Akt is a key regulatory pathway that regulates cellular functions by sensing cellular metabolic status, including apoptosis and ferroptosis ( 35 , 36 ). Akt activation can encourage ferroptosis and oxidative stress in liver cancer cells by regulating SREBP1 mediated adipogenesis, and PI3K inhibitors can block this effect. The Akt signaling pathway can negatively regulate glycogen synthase kinase-3 beta (GSK-3β) activity, which is attributed to the fact that GSK-3β can be activated by phosphorylated Akt and lose its activity, while the phosphorylated GSK-3β can promote Nrf2 expression and nuclear transcription ( 33 , 35 ). Membrane iron transporter protein is the main protein that regulates the extracellular transport of iron, maintains the homeostasis of intracellular iron, prevents iron overload induced oxidative damage to cells, and is also regulated by nuclear factor-erythroid 2-related factor 2 (Nrf2). In hepatoma cells, Akt activation can also boost ferroptosis and oxidative stress by regulating sterol regulatory element-binding protein 1 (SREBP1)-mediated adipogenesis, which could be blocked through PI3K inhibitors ( 31 , 36 ). In this study, ST6GALNAC1 was discovered to suppress the p-Akt expression in macrophage to promote ferroptosis of macrophage. Wang et al. pointed out that ST6GALNAC1 induced Akt signaling pathway of ovarian cancer stem cells ( 15 ). These findings indicate that ST6GALNAC1 has capability of inhibiting p-Akt protein expression in macrophage to evade immune surveillance in NSCLCs. However, the current research may have a limited sample size and fail to fully consider the heterogeneity of patients with NSCLC. There are differences in tumor microenvironment, genetic background and immune status in different patients, which may affect the mechanism of ST6GALNAC1 in immune escape. Although sialylation mediated by ST6GALNAC1 can promote immune escape, immune escape is a complex multi-factor process, involving the interaction of multiple signal pathways and cell types. The current research may not fully reveal the synergistic effect of ST6GALNAC1 and other immune escape related factors. In the future, the expression level of ST6GALNAC1 in different subtypes of NSCLC and its correlation with prognosis and immune escape need to be verified through large-scale clinical sample analysis. Else, how sialylation mediated by ST6GALNAC1 interacts with other signal pathways in tumor cells (such as Wnt/β-catenin, PI3K/Akt, etc.) is necessary to fully understand its role in tumor progression.

The expression of ST6GALNAC1 in patients with NSCLCs was up-regulated. ST6GALNAC1 suppressed p-Akt activity to evade immune surveillance in model of NSCLCs through the promotion of ferroptosis in macrophage ( Figure 10 ). This infers that ST6GALNAC1 is potential target to be used in the treatment of NSCLCs. ST6GALNAC1 mediates sialylation of mucins to evade immune surveillance in NSCLC by Akt signal pathway. Red arrows: invasion and migration. Green arrows: immune surveillance. Akt, protein kinase B; GPX4, glutathione peroxidase 4; NSCLC, non-small cell lung cancer; ST6GALNAC1, ST6 N-acetylgalactosaminide alpha-2,6-sialyltransferase 1.

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