Downregulation of triggering receptor expressed on myeloid cells 1 inhibits invasion and migration of liver cancer cells by mediating macrophage polarization

. Triggering receptor expressed on myeloid cells‑1 (TREM1) is a cell‑surface protein expressed on tumor‑asso‑ ciated macrophages (TAMs), the predominant inflammatory cells in the tumor microenvironment; however, the mecha‑ nisms for the influence of TREM1 on TAM polarization during liver cancer progression have not been investigated. In the present study, 20 patients diagnosed with hepatocellular carcinoma (HCC) who underwent surgery were enrolled, and TREM1 expression on M1/M2 macrophages and on M2 macro‑ phages was assessed by immunohistochemical staining. Human leukemia monocytic cells (THP‑1) were differentiated into M2 macrophages using phorbol 12‑myristate 13‑acetate, IL‑4 and IL‑13. A specific short hairpin RNA was used to knockdown TREM1 expression. To investigate the effects of TREM1 downregulation in macrophages on the migration and invasion of liver cancer cells, HepG2 and MHCC97H cell lines were co‑cultured with specific conditioned media. Reverse transcription‑quantitative PCR and western blot analyses were used to detect M1 and M2 macrophage marker expression. The expression levels of proteins of the PI3K/AKT/mTOR signaling pathway were analyzed by western blotting, revealing that TREM1 expression in HCC tissues was significantly elevated compared with that in adjacent normal tissues, and TREM1 was highly expressed on the cell membranes of M2 macrophages in tumor tissues compared with in adjacent normal tissues. The present results demonstrated that TREM1 downregulation in macrophages shifted M2 macrophages towards an M1 pheno‑ type, as defined by higher expression levels of M1‑associated markers and decreased expression levels of M2‑associated markers. In addition, TREM1 downregulation in macrophages suppressed migration and invasion of HepG2 and MHCC97H cells. Furthermore, TREM1‑knockdown in macrophages inhibited PI3K/AKT/mTOR activation in the polarization of M2 macrophages. In conclusion, downregulation of TREM1 expression in macrophages shifted M2 macrophages towards a M1 phenotype via inhibiting PI3K/AKT signaling. In addi‑ tion, migration and invasion of HepG2 and MHCC97H cells were inhibited when this signaling pathway was blocked. The present findings suggest TREM1 as a novel potential thera‑ peutic target for liver cancer management. knockdown PCR assay western blot non‑specific shRNA (5'‑TTCTCCGAACGTGTCACGT‑3') a negative control (sh‑ctrl).


Introduction
Hepatocellular carcinoma (HCC) is one of the most notorious solid tumors and has become a rapidly increasing cause of cancer-associated death in the USA (1)(2)(3). Based on the 2018 Global Cancer Statistics, liver cancer has an incidence of 4.7% and a mortality rate of 8.2% (4). Although surgery, local ablation, trans-arterial chemoembolization and systemic therapy have greatly advanced treatment of patients with HCC (5), distant and intrahepatic metastasis and postoperative recurrence remain a concern and result in poor outcomes in these patients (6,7). Therefore, identifying or discovering novel early diagnostic biomarkers and elucidating the underlying mechanisms of cancer metastasis has received much focus for improving treatment efficacy and prognosis in patients with liver cancer.
HCC is an inflammation-associated cancer (8,9), with chronic inflammation closely associated with disease progression. Tumor-associated macrophages (TAMs) are predominantly inflammatory cells in the tumor microenvironment (TME) that serve a pivotal role in tumor initiation, metastasis, angiogenesis and suppression of adaptive immunity (10). In the TME, TAMs exhibit high plasticity and heterogeneity in response to different stimuli, existing as classically activated macrophages (M1 macrophages) and alternatively activated macrophages (M2 macrophages) (11). In response to inflammatory mediators, M1 macrophages express inducible nitric oxide synthase (iNOS), which uses L-arginine as a substrate to produce nitric oxide (12), and exhibit pro-inflammatory and anti-tumorigenic properties. Conversely, M2 macrophages constitutively express the enzyme arginase-1 (Arg-1), which hydrolyzes L-arginine to L-ornithine (12) and demonstrates anti-inflammatory and pro-tumor characteristics. TAMs predominantly exhibit M2 properties with a pro-tumor phenotype (13), promote tumor cell proliferation, invasion, metastasis, neovascularization and suppression of the adaptive immune response in the TME (14,15). Therefore, identifying mechanisms through which macrophages are polarized in the TME is crucial for understanding their roles in tumor progression.
Triggering receptor expressed on myeloid cells (TREM) proteins are a family of immunoglobulin cell surface receptors expressed on myeloid cells that consist of a single extracellular immunoglobulin-like domain, a trans-membrane region and a short cytoplasmic tail (16). The TREM gene cluster is located on human chromosome 6p21, and includes genes that encode TREM1 and TREM2; on mouse chromosome 17c3, it encodes for TREM3 (16). TREM1 activation is associated with downstream signaling adaptor DNAX activation protein of 12 kDa (17) and peptidoglycan recognition protein-1 (18). TREM1 acts as a potent amplifier of inflammatory responses and leads to the increased secretion of pro-inflammatory cytokines, including monocyte chemotactic protein-1, TNFα, IL-1α, IL-1β, IL-6, IL-8 and macrophage colony-stimulating factor (19). In human non-small cell lung cancer, high expression levels of TREM1 on TAMs are associated with tumor recurrence and poor prognosis (20), suggesting that TREM1 may play an important role in cancer progression. In experimental xenograft mouse models of pancreatic cancer, inhibiting TREM1 attenuates tumor growth and prolongs survival (21). Similarly, in a chemically induced model of HCC, TREM1 serves a role in the connection between the activation of Kupffer cells and tumorigenesis (22). However, the role of TREM1 in the regulation of macrophage polarization in tumor-associated inflammation and its microenvironment has not been established. The present study aimed to investigate the influence and possible signaling pathway of TREM1 on macrophage polarization. Additionally, the effects of TREM1 downregulation in macrophages on the migration and invasion of liver cancer cells were explored.

Materials and methods
Patients and specimens. Incisional biopsy samples (tumor and adjacent normal tissues) were collected from 20 clinically diagnosed patients with HCC between August 2014 and April 2016, and were sent for histological confirmation according to classical histopathological features. The distance of the adjacent tissue from the tumor was >5 cm. Patients with other systemic tumors, infections or incomplete clinical data were excluded. All samples were obtained from the Department of Hepatobiliary Surgery of The First Affiliated Hospital of Fujian Medical University (Fuzhou, China). The mean age of the patients was 53 years (age range, 41-65 years). Clinical and laboratory parameters of patients such as disease severity were evaluated using the Child-Pugh score (23). Tumor staging was based on the Barcelona-Clinic Liver Cancer system (24 Immunohistochemistry. For immunohistochemistry, three areas of tumor specimens and adjacent normal tissues from 20 patients with HCC were fixed with 10% formalin for 24 h at 37˚C and embedded in a paraffin block. Subsequently, the samples were de-paraffinized in two changes of xylene for 5 min each at 37˚C and rehydrated in 100, 95, 70 and 50% alcohol for 5 min each at 37˚C, and finally sectioned into 5-µm-thick slices. For antigen retrieval, samples were treated with 10 mM citrate buffer (pH 6.0) at 100˚C for 10 min. After quenching endogenous peroxidase with 3% hydrogen peroxide for 30 min at 37˚C, 0.5% bovine serum albumin (BSA; cat. no. ST025-5 g; Beyotime Institute of Biotechnology) was used for 1 h at 37˚C to block non-specific binding. The sections were then incubated overnight at 4˚C with polyclonal rabbit anti-human TREM1 antibody (1:200; cat. no. YT5133; ImmunoWay Biotechnology Company). Subsequently, the sections were incubated with the appropriate goat anti-rabbit IgG secondary antibody (1:1,000; cat. no. ab6721; Abcam) for 60 min at 37˚C according to the manufacturer's instructions. Images of the labeled specimens were captured with an Olympus CX41 light microscope (Olympus Corporation; magnification, x400) and analyzed using ImageJ software (v1.4; National Institutes of Health). All experiments were performed in triplicate. Cell culture and chemicals. The human leukemia monocytic cell line (THP-1), the 293T cell line and the HepG2 and MHCC97H cell lines were purchased from The Cell Bank of Type Culture Collection of The Chinese Academy of Sciences. THP-1 cells were cultured in RPMI-1640 medium (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal calf serum (Gibco; Thermo Fisher Scientific, Inc.) and 0.05 mM β-mercaptoethanol (Sigma-Aldrich; Merck KGaA). 293T, HepG2 and MHCC97H cell lines were cultured in high-glucose DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 10% fetal calf serum. All cell lines were cultured with 5% CO 2 at 37˚C.
Conditioned medium preparation. After incubating M2 and shTREM1 macrophages with fresh RPMI-1640 medium for an additional 24 h at 37˚C, the cells' conditioned media (CM) was harvested, centrifuged at 3,000 x g for 15 min at 37˚C, filter-sterilized through a 0.22-µm nylon filter (Nalgene; Thermo Fisher Scientific, Inc.) and finally stored at -80˚C for further experiments.

Double-labeling cells immunofluorescence.
For immunof luorescence double labeling, cells were fixed in 4% paraformaldehyde for 20 min at 37˚C and permeabilized with 0.1% Triton X-100 for 10 min at 37˚C. After blocking non-specific binding with 0.1% BSA for 30 min at 37˚C, cells were incubated with primary antibodies against CD163 (cat. no. ab156769; Abcam) and TREM1 antibody (1:200; cat. no. YT5133; ImmunoWay Biotechnology Company) at a 1:500 dilution overnight at 4˚C. Subsequently, the samples were incubated with Alexa Fluor ® 488-labeled donkey anti-rabbit IgG antibody (1:1,000; cat. no. A-21206; Thermo Fisher Scientific, Inc.) and Alexa Fluor ® 546-labeled donkey anti-mouse IgG antibody (1:1,000; cat. no. A-10036; Thermo Fisher Scientific, Inc.) for 30 min at room temperature, and 1 µg/ml DAPI (Invitrogen; Thermo Fisher Scientific, Inc.) was used for nuclear labeling for 5 min at room temperature. Images of the labeled specimens were captured with a Zeiss confocal laser scanning microscope (Carl Zeiss AG) at x2,000 magnification and analyzed using ImageJ software. All treatments were performed in triplicate.
Cell migration/invasion co-culture assay. Transwell chambers (8-µm; EMD Millipore) were used to evaluate cancer cell migration and invasion as previously described (27). A total of 80 µl high-glucose DMEM (Gibco; Thermo Fisher Scientific, Inc.) supplemented with 2% fetal calf serum (Gibco; Thermo Fisher Scientific, Inc.) containing 2x10 5 HepG2 and MHCC97H cells was added into the upper chamber inserts. Serum-free RPMI-1640 medium, M2 macrophage-CM, shTREM1 macrophage-CM and sh-ctrl macrophage-CM were added into the bottom chambers to evaluate their influence on cancer cell migration. Before seeding HepG2 and MHCC97H cells, Matrigel (100 µl; Becton, Dickinson and Company) was placed into the upper chamber for invasion assays for 4 h at 37˚C, and the remaining steps were the same as for migration. After a 24-h incubation at 37˚C, the upper inserts were washed three times in PBS, fixed with 4% formaldehyde for 20 min at 37˚C and stained with crystal violet for 2 h at 37˚C. Five fields of view were randomly photographed with an Olympus CX41 light microscope (Olympus Corporation; magnification, x100) and the numbers of migrated and invaded cells per field were determined using ImageJ software.
Gap closure assay. A total of 2x10 5 HepG2 and MHCC97H cells were seeded into culture inserts (Ibidi GmbH); the culture inserts were then removed, and a 500-µm wide gap was generated. Cells were incubated with serum-free RPMI-1640 medium, M2 macrophage-CM, shTREM1 macrophage-CM and sh-ctrl macrophage-CM for 24 h at 37˚C. Five fields of view were randomly photographed with an Olympus CX41 light microscope (Olympus Corporation; magnification, x100) and the gap closure rate was estimated by calculating the gap area with ImageJ software.
Cell proliferation assay. HepG2 and MHCC97H cells were seeded into 96-well plates at a density of 1x10 4 cells/well. Cells were incubated with serum-free RPMI-1640 medium, M2 macrophage-CM, shTREM1 macrophage-CM and sh-ctrl macrophage-CM for 24 h at 37˚C. According to the instructions of a BrdU Cell Proliferation Assay kit (cat. no. 6813; Cell Signaling Technology, Inc.), BrdU was added to wells and incubated for 12 h at 37˚C. Subsequently, cells were fixed and denatured using 100 µl Fix/Denature solution (part of the aforementioned kit) for 30 min at 37˚C. Cells were washed with cold PBS three times. Cells were incubated with 100 µl anti-BrdU antibody (part of the aforementioned kit) for 1 h at 37˚C and were then washed three times with cold PBS. Subsequently, HRP-conjugated secondary antibody (part of the aforementioned kit) was added for 1 h at 37˚C. A total of 100 µl Substrate Solution was then added and incubated for 5 min at 37˚C. Finally, 100 µl Stop Solution was added to stop the enzyme reaction, and the levels of BrdU incorporation was determined using an ELISA reader at a wavelength of 450 nm (Bio-Rad Laboratories, Inc.).
Statistical analysis. All continuous variables were presented as the mean ± SD. The paired Student's t-test was used for comparative analysis of paired cancerous and adjacent normal tissues. The unpaired Student's t-test was applied for comparisons between 2 groups for the other assays, and one-way ANOVA followed by Tukey's multiple comparison test was used when >2 groups were evaluated. Two-sided P<0.05 was considered to indicate a statistically significant difference. All statistical analysis was performed using SPSS version 19.0 (IBM Corp.).

High TREM1 expression on M2 macrophages in HCC.
The clinical characteristics of the study population are described in Table SI. Immunohistochemistry was performed to determine the expression levels of TREM1 in HCC tissues and adjacent normal tissues. The percentage of TREM1 + cells per field in HCC tissues was significantly higher than that in adjacent normal tissues (11.23±0.8840 vs. 2.253±0.8662%; P<0.01; Fig. 1A-C). Immunohistochemical double staining was used to detect the tumor-associated macrophage phenotype in HCC by analyzing CD206 and Arg-1 expression. The results revealed that the infiltration of M2 macrophages in liver cancer tissues was significantly increased compared with that in adjacent normal tissues (3.50±0.5000 vs. 0.5100±0.1813%; P<0.01; Fig. 1D-F). No double-positive cells of CD16 and iNOS (M1-associated markers) were observed, indicating no M1 macrophage infiltration in liver cancer tissues and adjacent normal tissues (Fig. 1G-I).
In addition, immunohistochemical double staining with the M2 macrophage surface markers CD206 and TREM1 revealed that TREM1 was highly expressed on the surface of M2 macrophages in HCC tissues (16.51±0.9327 vs. 6.644±1.221%; P<0.01; Fig. 1J-L). The current results suggested that M2 macrophages mainly infiltrate HCC tissues, while no infiltration of M1 macrophages was observed in both HCC and adjacent normal tissues. M2 macrophages in HCC tissues highly express TREM1. These results indicated that TREM1 expression on M2 macrophages may serve an important role in the progression of HCC.

M2 macrophages induced from human leukemia monocytic cells.
To establish a model of human M2 macrophages, human leukemia monocytic cells (THP-1) were stimulated using PMA, IL-4 and IL-13. Double-immunofluorescence labeling was used to identify co-expression of CD163 and TREM1 in M2 macrophages ( Fig. 2A). RT-qPCR and western blot analysis revealed that TREM1 expression in M2 macrophages was significantly higher than in THP-1 cells (Fig. 2B and C). The mRNA expression levels of iNOS, CD16 and IL-12 (M1 macrophage-associated markers) were not significantly different between THP-1 cells and M2 macrophages, while the expression levels of CD163, Arg-1 and IL-10 (M2 macrophage-associated markers) were significantly increased in M2 macrophages compared with in THP-1 cells (Fig. 2D and E). Similarly, protein expression levels of iNOS, CD16 and IL-12 exhibited no significant difference between THP-1 cells and M2 macrophages, while protein expression levels of CD163, Arg-1 and IL-10 were significantly upregulated in M2 macrophages compared with in THP-1 cells (Fig. 2F). Overall, the present data indicated that M2 macrophages were successfully induced from the THP-1 monocytic cell line.

Downregulation of TREM1 inhibits M2 macrophage polarization and promotes a shift towards the M1 phenotype.
To discover possible functions of TREM1 in macrophage polarization, shRNAs were used to knock down TREM1 expression. As shown in Fig. 3A, shTREM1-2 was the most effective at TREM1 silencing and stable regulation of TREM1 in THP-1 cells and was therefore used for subsequent experiments. Western blot analysis revealed that transfection with shTREM1-2 resulted in a significant downregulation of TREM1 expression compared with sh-ctrl-transfected cells (Fig. 3B). RT-qPCR and western blot analysis demonstrated that iNOS, CD16 and IL-12 expression was significantly upregulated in shTREM1 macrophages, while CD163, Arg-1 and IL-10 expression was significantly downregulated in shTREM1 macrophages compared with in sh-ctrl macrophages ( Fig. 3C-E). Overall, the present results indicated that TREM1 downregulation inhibited M2 macrophage polarization and promoted a shift towards the M1 phenotype.

Downregulation of TREM1 in macrophages inhibits the migration and invasion of tumor cells.
A co-culture system was used to investigate the effect of shTREM1 macrophages on the migration and invasion of cancer cells. The results revealed that M2 macrophage-CM (M2-CM) significantly increased cell gap closure (Fig. 4A), as well as migration and invasion ( Fig. 4B), in HepG2 and MHCC97H cell lines compared with RPMI-1640 medium (1640); meanwhile, shTREM1 macrophage-CM (shTREM1-CM) significantly decreased this effect. The present results indicated that downregulation of TREM1 can inhibit the migration and invasion of tumor cells.

Downregulation of TREM1 in macrophages inhibits epithelialmesenchymal transition (EMT) and the proliferation of tumor cells.
A co-culture system was used to investigate the effect of shTREM1 macrophages on EMT of cancer cells. The results revealed that M2-CM significantly increased the protein expression levels of snail and significantly decreased those of E-cadherin in HepG2 (Fig. 5A) and MHCC97H (Fig. 5B) cells compared with RPMI-1640 medium; meanwhile, shTREM1-CM reversed this effect. These results indicated that downregulation of TREM1 inhibited the EMT of tumor cells. A BrdU kit was used to investigate the effect of shTREM1 macrophages on the proliferation of cancer cells.
The results demonstrated that M2-CM significantly increased proliferation of HepG2 and MHCC97H cells compared with RPMI-1640 medium; meanwhile, shTREM1-CM reversed this effect (Fig. 5C). The present results indicated that downregulation of TREM1 inhibited the proliferation of tumor cells.
Downregulation of TREM1 inhibits PI3K/AKT signaling in the M2 polarization of macrophages. To discover the molecular mechanisms of the effects of silencing TREM1 in macrophages and inhibition of M2 polarization of macrophages, western blotting was used to determine the protein expression levels of p-PI3K (p85), PI3K, p-AKT, AKT, p-mTOR and mTOR. As illustrated in Fig. 6, the ratios of p-PI3K/PI3K, p-AKT/AKT and p-mTOR/mTOR were significantly upregulated in M2-polarized  macrophages, while downregulation of TREM1 in macrophages reversed this effect. These results indicated that the PI3K/AKT/mTOR signaling pathway may be involved in the regulation of macrophage polarization by TREM1.

Discussion
The TME is a complex and dynamic cellular system that is associated with cancer occurrence, progression and response to therapy (28). TAMs are predominant inflammatory components in the TME and can exist as classically activated macrophages (M1 macrophages) and alternatively activated macrophages (M2 macrophages) in response to different stimuli within the TME (10). In numerous types of cancer, such as ovarian, breast and pancreatic cancer, TAMs tend to exhibit M2 polarization characteristics (10,13). The functions of M2 macrophages in cancer are complex, including promotion of angiogenesis, enhancing cell proliferation, migration and invasion, and suppression of the immune response in the TME (29). Yeung et al (14) revealed that M2 macrophages promote tumor growth and invasiveness in HCC through CCL2-induced EMT.
HCC is a well-known and extensively investigated inflammation-associated cancer (8,9). TREM1 is regarded as an effective amplifier of inflammatory responses in the immunoglobulin superfamily (30). Previous studies have investigated TREM1 expression in HCC cells and hepatic stellate cells (31,32). Duan and Wang (31) reported that the influence of TREM1 expression on HCC cell proliferation, invasion and apoptosis was not mediated by monocytes or macrophages, indicating that TREM1 had a direct pro-tumor effect in cancer cells. The present study confirmed that TREM1 expression was elevated in tumor tissues of patients with HCC. Furthermore, the current results revealed that TREM1 was highly expressed in M2 macrophages in HCC tissues through immunohistochemical double-staining with the surface markers CD206 and TREM1. This result indicated that TREM1 may have an indirect effect on HCC mediated by macrophages. The activation of TREM1 expression in Kupffer cells (resident macrophages in liver tissue) stimulates tumorigenesis during diethylnitrosamine-induced hepatocarcinogenesis (22). Therefore, TREM1 expression on macrophages may serve an important role in HCC formation.
Recruited monocyte-derived macrophages are thought to be more plastic and prone to switch from an antitumor to a pro-tumor phenotype under the influence of the growing tumor cells (33). These phenotypes are usually defined by the M1/M2 polarization classification and different functional patterns. As a pro-inflammatory receptor, TREM1 protein expression on macrophages may serve as a regulator to switch the phenotype of macrophages and change their function (34). The cross-talk between TREM1 and macrophages in the TME during liver cancer development remains poorly understand. To explore the mechanisms by which TREM1 influences macrophage polarization, the present study knocked down TREM1 expression in macrophages by shRNA, revealing that M2 polarization markers were inhibited and M1 macrophage-associated markers were upregulated in shTREM1 macrophages. The present results suggested that downregulation of TREM1 may promote a shift in shTREM1 macrophages towards the M1 phenotype, presenting as iNOS upregulation and Arg-1 downregulation. Additionally, the CM from TREM1-knockdown macrophages was co-cultured with liver cancer cells, as well as the CM from M2 macrophages used as a control group. Blocking TREM1 shifted the polarization of macrophages to inhibit migration, invasion, EMT and proliferation of liver cancer cells. Similarly, a recent study has found that lncRNA cox-2 small interfering RNA decreases the ability of M1 macrophages to inhibit tumor cell proliferation, invasion, migration and EMT, while strengthening the ability of M2 macrophages to promote tumor cell proliferation and inhibit apoptosis (35). Therefore, the present results provided evidence for macrophage polarization in the TME, which increases the current understanding of the role of TAMs in cancer progression. The current study further defined phenotypic transition as one of the major mechanisms by which TREM1 signaling may act on activated M2 macrophages to drive hepatocarcinogenesis. Notably, TAMs are being identified as increasingly complex, with several studies suggesting that distinct populations may have different roles in tumorigenesis (22,31).
The PI3K signaling pathway is a classical signaling pathway that converges inflammatory and metabolic signals during phagocytosis, autophagy and cell metabolism (36,37). Class IA PI3K includes a p110 catalytic subunit, which interacts with an SH2 domain-containing p85 regulatory subunit (36,37). The p85 regulatory subunit representative of PI3K phosphorylation was assessed in the present study. Phosphorylation of PI3K results in the activation of downstream signal effectors, including AKT and mTOR; PI3K/AKT and its downstream effectors are regarded as central mediators of the inflammatory response and polarization of macrophages (38). In bone marrow-derived macrophages, the PI3K/AKT signaling pathway promotes M2 polarization (39), while inhibition of either PI3K or AKT attenuates the expression levels of M2-associated genes (39,40), illustrating the importance of the PI3K/AKT signaling pathway in M2 polarization. The present findings suggested that TREM1 regulated macrophage polarization through the PI3K/AKT signaling pathway. Upregulated expression levels of markers of PI3K/AKT signaling pathway activation were detected in the current study, suggesting that the M2 macrophage polarization depended on this activation. In addition, downregulation of TREM1 in macrophages blocked the activation of PI3K/AKT signaling in M2 polarization. Although the present results do not formally prove a cause-effect association, they clearly support that the PI3K/AKT signaling pathway may partially mediate an M1/M2 phenotypic switch in the TME. A limitation of the present study is that it was only demonstrated that TREM1 inhibited liver cancer cells by regulating macrophage polarization. The function of TREM-1 + TAMs and their relevant mechanisms of immunosuppression in the tumor microenvironment require further investigation.
In conclusion, the present study provided novel evidence that knocking down TREM1 expression may shift M2 macrophages towards a M1 phenotype, likely through PI3K/AKT signaling. These findings enhance the understanding of TREM1 expression on M2 macrophages and its role in the pathogenesis of liver cancer. Finally, the present study supports targeting TREM1 and/or TAMs as a therapeutic strategy against cancer to improve the efficacy of current systemic therapy.