Skip to main content
NCBI home page
Journal of Cancer Research and Clinical Oncology logoLink to Journal of Cancer Research and Clinical Oncology
. 2024 Oct 28;150(10):480. doi: 10.1007/s00432-024-05996-9

HMGB1/TREM1 crosstalk between heat-injured hepatocytes and macrophages promotes HCC progression after RFA

Bin Xiong 1,2,#, Chunming Li 3,#, Guoqing Hong 1, Junke Li 1, Qing Luo 1, Jianping Gong 1,3, Xing Lai 1,2,✉
PMCID: PMC11513699  PMID: 39465435

Abstract

Purpose

Tumor recurrence after radiofrequency ablation (RFA) affects the survival rate of patients and limits its clinical application. Tumor recurrence around the ablation area may be related to the thermal injury of hepatocytes (HCs) around the tumor, but the specific mechanism is still unclear.

Methods

A liver cancer thermal injury mouse model was established via RFA in the C57BL/6 mice. Primary HCs and Kupffer cells (KCs) were isolated and cultured to assess their sensitivity to thermal injury via the MTT assay. Flow cytometry was used to assess macrophage polarization. Furthermore, Western blotting and co-immunoprecipitation (co-IP) were utilized to evaluate the protein expression of intracellular signaling pathway. Finally, Transwell and wound healing assays was conducted to evaluate the invasion potential of liver cancer cells.

Results

Our findings revealed that RFA-induced liver thermal injury promoted the upregulation and secretion of HMGB1 in HCs. HMGB1 had a protective effect on HCs thermal injury, potentially mediated through autophagy regulation. Heat-injured HCs release HMGB1, which activates the TREM1/JAK2/STAT3 signaling pathway in KCs, thus fostering an immunosuppressive tumor microenvironment (TME). Moreover, HMGB1 secretion by heat-injured HCs exacerbates the migration and invasion of HCC cells by influencing macrophage polarization.

Conclusion

RFA-induced thermal injury triggers HMGB1 release from HCs, driving macrophage M2 polarization and increasing the invasion ability of liver cancer cells. These findings reveal a potential therapeutic target for combating liver cancer recurrence following thermal ablation.

Keywords: Radiofrequency ablation, HMGB1, Thermal injury, Tumor recurrence, Macrophage polarization, HCC

Introduction

Thermal ablation therapy, including radiofrequency ablation (RFA), represents a minimally invasive treatment that has emerged in recent years for solid tumors, particularly primary or metastatic liver cancer(Zhao et al. 2024). It has garnered increasing attention because of its low degree of trauma, high efficacy, and broad applicability. RFA can achieve a curative effect akin to surgical resection for small liver cancer. Moreover, patients with varying degrees of liver cirrhosis often opt for RFA when surgical intervention is not feasible. However, patients who undergo thermal ablation frequently develop new lesions adjacent to the prior ablation area(Visioni et al. 2026). This is because the thermal energy effectively eliminates tumor cells at the energy center but fails to adequately eradicate tumor cells along the temperature decay path. The tumor recurrence around the ablation area may stem from the thermal injury of hepatocytes (HCs) surrounding the tumor, although the precise mechanism remains elusive.

The interactions among various cells within the tumor microenvironment (TME) play pivotal roles in tumor progression. Injured HCs within the TME can influence the activation of immune cells and the biological behavior of malignant cells through the secretion proteins(Bruni et al. 2020). Macrophages, immune cells with strong phenotypic plasticity, play critical roles in the initiation, maintenance, and regression phases of tissue repair(Yan et al. 2024a). M1 macrophages can engulf and eliminate foreign pathogens, secrete pro-inflammatory factors, activate T cell immune responses, and regulate and bolster the Th1 immune response, thereby fostering the initial inflammatory response. On the other hand, M2 macrophages promote tissue repair and wound healing, secrete anti-inflammatory factors, regulate the Th2 immune response, and contribute to disease resolution during the later stages of inflammation(Na et al. 2023). An elevated proportion of M2 macrophages within the TME are linked to more aggressive cancers, as they fuel tumor malignancy through various avenues, including fostering an immunosuppressive TME, promoting angiogenesis, facilitating invasion, and promoting metastasis(Li et al. 2023; Wei et al. 2024). However, the precise polarization state of macrophages under heat stress and its impact on the invasive capacity of tumor cells remain unclear.

High mobility group protein B1 (HMGB1) is a highly conserved nuclear protein throughout mammalian cells, with low expression in the cytoplasm(Klune et al. 2008). It plays a crucial role in regulating metabolism, inflammation, and tumor progression(Liu et al. 2024; Otoo et al. 2023; Xu et al. 2024; Yao et al. 2024). HMGB1 promotes the production of pro-inflammatory factors such as TNF-α and IL-6 by interacting with receptors such as TLR4, contributing to cellular damage and fostering an inflammatory response(Wyganowska-Swiatkowska et al. 2020). Nonetheless, HMGB1 can also prompt macrophages to transition into the M2 phenotype and exert an anti-inflammatory effect(Biguetti et al. 2019). Triggering receptor expressed on monocytes-1 (TREM1), a principal downstream receptor of HMGB1, plays a pivotal role in modulating inflammatory responses and the growth of tumor cells(Bosco et al. 2016). TREM1 is highly expressed in most solid tumor samples, suggesting the potential significance of the HMGB1/TREM1 signaling axis within the TME (Wang et al. 2015). However, the expression pattern of HMGB1/TREM1 and its implications for tumor recurrence under conditions of heat stress remain uncertain.

In this study, we explored the effects of heat-injured HCs within the TME on macrophages and liver cancer cells and focused on the regulatory role of HMGB1/TREM1 in HCC progression. We aimed to identify potential therapeutic targets for tumor recurrence after RFA.

Methods

Animals

Male C57BL/6 mice (8–10 weeks) were obtained from the Laboratory Animal Center of Chongqing Medical University. All procedures in this study were reviewed by the Ethics Committee of Chongqing Medical University and adhered to the Guidelines for Ethical Review of Laboratory Animal Welfare of the People’s Republic of China. HCC was induced via i.p. injection of DEN (25 mg/kg) at 15 days postpartum. After 2 weeks, 17 weekly injections of CCl4 (0.5 ml/kg i.p.) were subsequently administered(Luo et al. 2021). At 21 weeks after the initial DEN injection, the mice were anesthetized via ether inhalation and secured on a sterile operating table. For the liver thermal injury model, a RFA needle was inserted beneath the xiphoid process to a depth of approximately 0.5–0.7 cm, and the power setting was 5 W for 60 s.

Reagents

Antibodies targeting HMGB1 (ab77302), LC3B (ab51520), TREM1 (ab104413), p-JAK2 (ab219728), JAK2 (ab108596), p-STAT3 (ab76153), STAT3 (ab68153), and gp130 (ab259927) along with their corresponding secondary antibodies, were procured from Abcam. MTT cell proliferation and cytotoxicity assay kits (AR1156), as well as mouse TNF-α (EK0527) and IL-10 (EK0417) enzyme-linked immunosorbent assay (ELISA) kits, were acquired from BOSTE Biological Co., Ltd. HMGB1-shRNA (GCCL3752017236) lentivirus, TREM1-shRNA (GCCL3751098901) lentivirus, HMGB1 plasmid and their related negative controls were purchased from Shanghai Genechem Biological Co., Ltd.

Isolation and culture of primary cells

After anesthesia, the portal vein of each mouse was sufficiently exposed and catheterized. Following in situ liver perfusion with PBS, the liver tissue was isolated and rinsed in a 10-cm dish with 5 ml of PBS. After the gallbladder and surrounding connective tissue were excised, a portion of the liver tissue was fixed in 5 ml 4% paraformaldehyde (maikun, shanghai). The remaining fresh liver tissue was utilized to isolate primary HCs and Kupffer cells (KCs). The liver was finely minced and combined with 4 ml of collagenase IV (0.1%, Gibco) and 4 ml of phosphate-buffered saline (PBS, Hyclone). This mixture was then transferred to a 15 ml centrifuge tube, and incubated at 37℃ for 30 min, with intermittent mixing for 2 min at 8 min intervals. The cell suspension was introduced to 5 ml culture medium, sequentially filtered through 200-mesh and 100-mesh metal meshes, and allowed to stand for 10 min. The cells were resuspended in 20 ml culture medium and centrifuged at 50 g for 2 min to isolate HCs. The cell pellets were resuspended in 20 ml culture medium and seeded in pre-coated Matrigel (BD) culture dishes (Matrigel: DMEM = 1:9) and placed in an incubator. The supernatant was centrifuged at 300 g for 5 min, and after removing the supernatant, the cells were resuspended in 10 ml culture medium and centrifuged again for three times. The cell precipitate was resuspended in 8 ml medium and seeded into four wells of a 6-well plate. After culture for 2 h, the medium was replaced for subsequent experiments.

Hematoxylin-eosin (HE) and immunohistochemical (IHC) staining

The fixed liver tissues were embedded in paraffin, then sliced, deparaffinized, and subjected to staining procedures. For HE staining, the sections were stained with hematoxylin for 5 min, followed by differentiation with 1.5% alcoholic hydrochloric acid for 2 min. The samples were subsequently rinsed with distilled water for 10 min and stained with eosin for 2 min. The sections were then dehydrated, dried, sealed, and examined under an inverted microscope. For IHC staining, the sections were immersed in a citric acid solution for antigen retrieval at high temperature and pressure for 2 min. After cooling to room temperature, the sections were washed three times with PBS for 5 min each. Endogenous peroxidase activity was blocked by treatment with 3% H2O2 for 10 min, followed by another three washes with PBS. Following blocking with a 5% goat serum solution for 1 h, the primary antibody solution (HMGB1) was applied to the slides, which were then incubated overnight at 4℃. The sections were subsequently incubated at room temperature for 1 h with the secondary antibody solution, followed by DAB color development. After counterstaining with hematoxylin, dehydration, and clearing, the slides were sealed with neutral mounting medium and examined under an inverted microscope.

Cell culture, transfection, and establishment of a thermal injury model

The cells utilized in this study were cultured in DMEM high glucose medium supplemented with 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (Hyclone). To perform lentivirus transfection, the cells were seeded into 6-well plates (2 × 105 cells/well) and cultured for 24 h. Next, 6 µl transfection reagents (Invitrogen) and 2 µl shRNA or empty lentivirus were added to each well. The cells were then cultured for 24 h and changed the medium. Afterward, the cells were screened with 5 µg/ml puromycin to select stable cell lines where the target gene was successfully silenced. These stable cell lines were chosen for subsequent experiments. To establish the cell thermal injury model, a previously described method was adopted with some modifications(Jiang et al. 2019). Briefly, different cells were subjected to water baths at temperatures of 37℃, 40℃, 42.5℃, 45℃, 47.5℃, 50℃, 52.5℃, 55℃, and 57.5℃ for 30 s each. Subsequently, the treated cells were seeded into 6-well plates (5 × 104 cells/well) and continued to be cultured for subsequent experiments.

Western blot

The protein concentration was determined via the BCA (BOSTER) method. Loading buffer (Beyotime) was added to the protein samples, which were subsequently boiled for 5 min. The upper layer of the concentrated gel was electrophoresed under 80 V, while the lower layer of the separated gel was run at 110 V. The proteins were then transferred to a PVDF membrane (Merck) using a constant current of 250 mA for an appropriate duration. The membrane was blocked with 5% BSA at room temperature for 1 h. The primary antibody (diluted at 1:1000) was incubated overnight at 4 °C. Following three washes with Tris buffered saline tween-20 (TBST), the membrane was incubated with the secondary antibody (diluted at 1:5000) for 1 h. Subsequently, the membranes were washed again with TBST and incubated with an ECL solution (MCE). The membranes were visualized and photographed using a Bio-Rad gel imaging system, and the bands were analyzed using Image Lab software.

Enzyme-linked immunosorbent assay (ELISA)

An ELISA kit (BOSTER) was used for the experiment according to the instructions. In brief, the diluted cell supernatant was dispensed into the 96-well plates at a volume of 100 µl per well, with 8 wells allocated for standards to construct the standard curve. The plate was incubated at 37 °C for 1.5 h, and the contents of the plate were discarded. 100 µl of the pre-prepared primary antibody working solution was added to each well, followed by an incubation period of 1 h at 37 °C. Subsequently, the contents were removed from the wells, which were then washed three times with PBS and dried by patting. Next, 100 µl of ABC solution was added to each well and incubated at 37 °C for 30 min. The wells were washed five times with PBS before they were patted dry. To each well, 90 µl TMB chromogenic solution was added and incubated at 37 °C for 7 min. Then, 100 µl termination solution was added to each well. The optical density (OD) value of each well was measured using a microplate reader (Bio-Tek). The standard curve was generated from the standard protein concentration, and the actual concentration of the sample well was determined.

Co-immunoprecipitation (Co-IP)

Following the extraction of total protein, 5 µg indicated antibody was added, and the mixture was incubated overnight at 4 °C. Protein G magnetic beads were then introduced and incubated for 4 h at 4 °C. After washing with TBST, protein loading buffer was added, and the mixture was boiled before Western blot analysis.

Cell proliferation assay (MTT assay)

The cells were plated into 96-well plates at a density of 3000 cells per well and incubated for 24 h. Then, 10 µl of MTT solution (5 mg/ml) was added to each well and incubated for 4 h at 37 °C. The culture medium was removed, and 100 µl of DMSO solution was added to dissolve the formazan. A microplate reader was then used to measure the absorbance at 490 nm.

Flow cytometry

Isolated KCs were digested, centrifuged, and washed with PBS. Subsequently, the cells were stained with appropriate antibodies for 30 min on ice in the dark. All flow cytometry assays were performed on a flow cytometer (Bio-Tek) and analyzed with FlowJo software (Tree Star).

Transwell

The migration and invasion capabilities of the HCC cell lines were evaluated using a Transwell assay. For cell migration, 1 × 105 HCC cells were added to the upper chamber, while 1 × 105 µl of KCs was added to the lower chamber. For cell invasion, 60 µl diluted Matrigel solution (1:8) was added to the upper chamber and incubated at 37 °C for 30 min to polymerize into a gel. 2 × 105 HCC cells were added to the upper chamber, while 1 × 105 µl of KCs were added to the lower chamber. The chambers were then incubated for 48 h. Subsequently, the cells were fixed with 4% paraformaldehyde (maikun, shanghai) for 30 min, and stained with crystal violet (maikun, shanghai) for 10 min. After washing with PBS, the cells in the upper chamber were gently removed with a cotton swab. Following air drying, the cells in the lower chamber were observed and counted using a microscope.

Wound healing assay

Briefly, 5 × 105 HepG2 cells were grown in six-well plates until complete confluence. After washing with PBS, a 200 µl pipette tip was used to scratch the cells. After 0 and 48 h, the cells were photographed by a microscope, and the area of the wound was measured by ImageJ.

Statistical analysis

Measurement data are presented as mean ± standard deviation (SD). The Shapiro-Wilk method was used to check if the data conformed to a normal distribution, and the F-test was performed to estimate the variation within each group of data. All the data were normally distributed, and the variance was similar between the groups, and the ANOVA and unpaired t-test were conducted using SPSS 20.0 statistical software. p < 0.05 was considered statistically significant.

Results

Thermal injury promotes the expression and secretion of HMGB1 in HCs

In our study, we mimicked local thermal injury models through RFA in mice left liver (Fig. 1A). Histological examination via HE staining revealed extensive necrosis of HCs, accompanied by pronounced infiltration of immune cells in the RFA zone (Fig. 1B). IHC analysis further demonstrated that the expression of HMGB1 in HCs was significantly greater near the necrotic region than in distant areas (Fig. 1B). Subsequently, we employed mouse primary HCs, KCs, and the liver cancer cell line HepG2 to establish thermal injury models at different temperatures. Through MTT cell proliferation assays, we discerned that HCs presented the greatest susceptibility to thermal injury. At 47.5 °C, approximately half of the HCs exhibited a decline in viability, whereas at 50 °C, nearly all HCs died. In contrast, the viability of KCs and HepG2 cells demonstrated decreased viability at 52.5 °C and 57.5 °C, respectively (Fig. 1C). These findings underscore the heightened vulnerability of HCs to thermal injury, suggesting their pivotal role as the initiating factor in the ensuing pathological cascade after thermal injury.

Fig. 1.

Fig. 1

Open in a new tab

RFA-mediated heat injury promotes the expression and secretion of HMGB1 in HCs. (A) Representative gross liver images of mice before and after radiofrequency ablation (RFA) (n = 6). (B) Effects of RFA-mediated heat damage on pathological changes were measured using HE staining (left panel), and HMGB1 expression after RFA was measured using immunohistochemical (IHC) staining (right panel). (C) MTT assay was used to detect the cell viability of HCs, KCs and HepG2 cells stimulated at different temperatures. (D) Western blot was used to detect the protein expression and distribution of HMGB1 in HCs. (E) ELISA detected the level of HMGB1 in HCs supernatants stimulated at different temperatures. Data are presented as mean ± SD; significance determined by Two-way ANOVA (C) and One-way ANOVA with Tukey’s multiple comparisons test (E). *p < 0.05, ***p < 0.001

We examined the expression of HMGB1 in HCs under different temperatures and found that HMGB1 was mainly expressed in the nucleus at temperatures below thermal injury (37–45 °C), and that its expression was not significantly different from that without thermal stimulation. However, upon reaching the critical temperature for thermal injury (47.5 °C), HMGB1 notably translocated from the nucleus to the cytoplasm, accompanied by a significant increase in total HMGB1 expression (Fig. 1D). In addition, we found that the expression of HMGB1 in the culture medium of heat-injured HCs was increased, suggesting that thermal injury promoted the secretion of HMGB1 (Fig. 1E).

HMGB1 enhances HCs tolerance to thermal injury by enhancing autophagy

To investigate the effect of HMGB1 on HCs to thermal tolerance, we assessed HCs viability under various temperature stimuli by knockdown or overexpression in HCs (Fig. 2A, B). HMGB1 overexpression markedly increased, whereas HMGB1 knockdown greatly decreased the temperature threshold for thermal injury and total cell death of HCs, indicating a protective role of HMGB1 against HCs thermal injury (Fig. 2C, D). Studies have shown that autophagy plays a role in regulating cell tolerance to heat stress(Zhang et al. 2023). Co-IP experiments demonstrated an interaction between HMGB1 and LC3 during heat injury stimulation (Fig. 2E). We observed that heat stimulation upregulated the expression of the autophagy marker protein LC3II, whereas HMGB1 silencing notably attenuated this heat-induced upregulation (Fig. 2F). Chloroquine, an autophagy inhibitor, dramatically attenuated HMGB1-overexpression-mediated protection against thermal injury (Fig. 2G). These findings revealed that HMGB1 enhances HCs tolerance to thermal injury by boosting autophagy.

Fig. 2.

Fig. 2

Open in a new tab

HMGB1 enhances HCs tolerance to thermal injury by enhancing autophagy. (A) HCs were transfected with a non-targeting shRNA Control (Ctrl) or shRNA targeting HMGB1 (shHMGB1), and Western blot was used to detect the expression of HMGB1. (B) HCs were transfected with HMGB1 plasmid (Flag-HMGB1) or empty plasmid (Vector) for 48 h, and Western blot was used to detect the expression of HMGB1. (C and D) MTT assay was used to detect the cell viability of HCs cells under different temperature stimulation after silencing or overexpression HMGB1. (E) Co-IP was used to detect the interaction between HMGB1 and LC3 in HCs. (F) HCs were transfected with shHMGB1 with or without heat stimulation, and Western blot was used to detect the protein expression of HMGB1 and LC3. (G) MTT assay was used to detect the cell viability of HCs cells under different temperature stimulation after overexpression HMGB1 with or without the autophagy inhibitor Chloroquine (20 µM) for 24 h. Data are presented as mean ± SD; significance determined by Two-way ANOVA with Bonferroni’s multiple comparisons test (C, D, F and G). *p < 0.05

HMGB1 secreted by HCs promotes macrophage M2 polarization via the TREM1/JAK2/STAT3 axis

To explore the effect of HCs thermal injury on macrophages, HCs were co-cultured with KCs. We observed that at the temperature that induced thermal injury (47.5 °C), the levels of the pro-inflammatory factor TNF-α were significantly lower, whereas the levels of the anti-inflammatory factor IL-10 were significantly greater in the co-culture system than in the supernatant of KCs cells alone (Fig. 3A, B). These findings suggest that heat injury in HCs may promote macrophage polarization toward an anti-inflammatory phenotype. Subsequently, we investigated whether the regulatory effect of heat-injured HCs on macrophages was associated with HMGB1 secretion (Fig. 3C). We found that the proportion of M2 macrophages was increased after HMGB1 stimulation, and that the levels of the anti-inflammatory factor IL-10 were increased, whereas the levels of the pro-inflammatory factor IL-6 and IL-8 were decreased in the co-culture system treated with HMGB1 (Fig. 3D). In addition, macrophage polarization and inflammatory factor secretion was restored after TREM1 knockdown or adding TREM1 inhibitor (LP17), indicating that heat-injured HCs promote macrophage M2 polarization via the HMGB1/TREM1 pathway (Fig. 4A-D).

Fig. 3.

Fig. 3

Open in a new tab

HMGB1 secreted by HCs promotes macrophage M2 polarization. (A and B) ELISA was used to detect the levels of TNF-α and IL-10 in the supernatant of HCs (A) and co-cultured HCs with KCs (B). (C) The ratio of CD206 on HMGB1- treated KCs was assessed by Flow cytometry. (D) The concentration of IL-6, IL-8, and IL-10 in HMGB1-treated KCs was assessed by ELISA. Data are presented as mean ± SD; significance determined by Two-way ANOVA with Bonferroni’s multiple comparisons test (A, B and D) and Student’s unpaired t-test (C). *p < 0.05, **p < 0.01, ***p < 0.001

Fig. 4.

Fig. 4

Open in a new tab

Blockade of TREM1 recovers macrophage polarization and anti-inflammatory factors secretion induced by HMGB1. (A-D) KCs were transfected with shRNA-TREM1 or shRNA-NC for 72 h, and stimulated with HMGB1 (500 ng/ml) or LP17 (200 ng/ml) for 24 h. Fluorescence microscopy was used to analyze the transfection efficiency (A). CD206 was detected by flow cytometry (B). The levels of IL-6, IL-8, and IL-10 in the culture supernatant were determined by ELISA (C and D). Data are presented as mean ± SD; significance determined by One-way ANOVA with Tukey’s multiple comparisons test (B-D). *p < 0.05

The Janus kinase 2 (JAK2)/ signal transducer and activator of transcription 3 (STAT3) signaling pathway is pivotal in regulating macrophage inflammatory responses(Yan et al. 2024b; Zeng et al. 2024). Our findings demonstrated that co-cultured with heat-injured HCs or HMGB1 stimulation could induce the expression of p-JAK2 and p-STAT3 in KCs (Fig. 5A, B). Nevertheless, silencing TREM1 reversed the upregulation of p-JAK2 and p-STAT3 in KCs induced by heat-injured HCs. (Fig. 5A). Next, we explored the underlying mechanism by which TREM1 affects the JAK2/STAT3 signaling pathway, and found that TREM1 can interact with gp130 (Fig. 5C). gp130 is a key molecule that activates JAK2(Wu et al. 2016). In addition, the interaction between gp130 and JAK2 was stronger after HMGB1 treatment (Fig. 5D). Taken together, our results suggest that HMGB1 secreted by heat-injured HCs activated the TREM1/JAK2/STAT3 signaling pathway in KCs.

Fig. 5.

Fig. 5

Open in a new tab

HMGB1 secreted by thermal injury HCs activates the TREM1/JAK2/STAT3 axis in macrophages. (A) KCs were transfected with shRNA-TREM1 or shRNA-NC for 72 h, and co-cultured with thermal injury HCs for 24 h. Western blot was used to detect the protein expression of TREM1, p-JAK2/JAK2 and p-STAT3/STAT3 in KCs. (B) KCs were treated with HMGB1 for 24 h, and Western blot was used to detect the protein expression of TREM1, p-JAK2/JAK2 and p-STAT3/STAT3. (C) Interaction of gp130 with TREM1 was analyzed by co-IP in KCs. (D) Interaction of gp130 with JAK2 was analyzed by co-IP in KCs treated with or without HMGB1. Data are presented as mean ± SD; significance determined by Two-way ANOVA with Bonferroni’s multiple comparisons test (A). *p < 0.05

Heat-injured HCs enhance the migration and invasion ability of HCC cells through activating macrophages

We found that co-cultured with heat-injured HCs and KCs enhanced the invasive ability of HepG2 cells but co-cultured with heat-injured KCs did not significantly affect the invasive ability of HepG2 (Fig. 6A). HMGB1-treated KCs promoted the migration and invasion of HepG2 cells, and highly expressed the metastasis-associated biomarkers (TGF-β and MMP-9) (Fig. 6B-D). Reciprocally, inhibition of TREM1 could significantly inhibit the expression TGF-β and MMP-9 in HMGB1-treated KCs, and decrease the migration and invasion of HepG2 cells (Fig. 6E-G). Together, HMGB1 secreted by heat-injured HCs promoted the pro-metastatic ability of KCs and enhanced the migration and invasion ability of HCC cells.

Fig. 6.

Fig. 6

Open in a new tab

HMGB1/TREM1 interaction in HCs and macrophages enhance the migration and invasion ability of HCC cells. (A) HepG2 cells were co-cultured with thermal injury HCs and KCs, and the invasion ability were detected by Transwell assay. (B and C) HepG2 were co-cultured with HMGB1-treated KCs, and the migration and invasion ability were detected by Transwell (B) and wound healing (C) assays. (D) The protein expression of VEGF, TGF-β and MMP-9 was detected by Western blot in HMGB1-treated KCs. (E-G) KCs were transfected with shRNA-TREM1 or shRNA-NC for 72 h, and stimulated by HMGB1 (500 ng/ml) or LP17 (200 ng/ml) for 24 h. HepG2 were co-cultured with KCs, and the migration and invasion ability were detected by Transwell (E) and wound healing (F) assays. The protein expression of VEGF, TGF-β and MMP-9 was detected by Western blot (G). Data are presented as mean ± SD; significance determined by One-way ANOVA with Tukey’s multiple comparisons test (A, E and F) and Student’s unpaired t-test (B and C). *p < 0.05, **p < 0.01

Discussion

Primary liver cancer is a highly malignant tumor that causes a substantial disease burden, with a global mortality rate as high as 8.2%(Jiang et al. 2023). RFA, as one of the three radical treatments for liver cancer, has gained widespread use in clinical practice owing to its advantages, such as its minimal invasiveness, rapid recovery, and cost-effectiveness. While the 5-year overall survival rate for early-stage liver cancer patients treated with RFA aligns closely with that of patients treated with surgical resection, a notable drawback is the elevated postoperative recurrence rate, a pivotal factor influencing clinical efficacy and prognosis(Shin et al. 2021). The diminished energy of RFA from the ablation center renders cancer cells in the energy center susceptible to destruction(Chu et al. 2014). However, as the energy decreases to a specific temperature, RFA fails to effectively eliminate tumor cells, contributing to increased invasion ability among residual tumor cells(Chu et al. 2014, Ruzzenente et al. 2004; Seki et al. 2001). In our study, we observed that HMGB1 expression was increased in HCs near the RFA necrotic area of the liver. Heat-injured HCs were identified as inducers of macrophage M2 polarization and facilitated liver cancer cell invasion in the TME through HMGB1 secretion. Silencing TREM1, the receptor for HMGB1 on macrophages, inhibited the phenotypic and biological changes in macrophages and liver cancer cells induced by the heat-injured HCs. Our findings provide potential theoretical support for the prevention and treatment of liver cancer recurrence after thermal ablation.

The TME comprises tumor cells, immune cells, including tumor-associated macrophages (TAMs), HCs, and various other cellular and non-cellular components, such as cytokines and chemokines. Intricate interactions occur among these elements. Recent studies have indicated significant inhibition of respiratory chain components in HCs following perfusion at 42℃, with irreversible uncoupling observed at 46℃(Žūkienė et al. 2017). In vitro studies revealed various degrees of tolerance to thermal stimulation among HCs, macrophages, and liver cancer cells, with HCs being the most susceptible. Notably, at 47.5℃, approximately half of the HCs died, whereas the activities of macrophages and HepG2 cells remained relatively unaffected. Consequently, it is hypothesized that HCs may serve as the primary instigators of TME alterations following thermal injury.

HMGB1, a non-histone chromatin-binding protein, plays pivotal roles in processes such as chromatin remodeling, transcription, DNA repair, and nucleosome assembly(Dulmovits et al. 2022; Wyganowska-Swiatkowska et al. 2020). Additionally, HMGB1 can be secreted into the extracellular space as damage-associated pattern molecules (DAMPs), which bind to receptors and participate in inflammatory responses, cell differentiation, angiogenesis, tumor metastasis, and drug resistance(Hernandez et al. 2018; Jube et al. 2012). HMGB1 is highly expressed in various solid tumors including colorectal cancer, liver cancer, breast cancer, pancreatic cancer, melanoma, and mesothelioma (Wang et al. 2020). In our study, we observed a significant increase in HMGB1 expression in HCs adjacent to the necrotic area following RFA, enhancing HCs tolerance to thermal injury. Studies have shown that HCs/liver cancer cells exhibit a slight autophagy, thereby safeguarding themselves from heat stress. We found that cytoplasmic HMGB1 in heat-stressed HCs could directly bind to the autophagy-related protein LC3, thereby activating autophagy to protect HCs from heat injury.

TAMs constitute a crucial component of the TME and are intricately linked to cancer onset and progression(Arneth 2019; Vitale et al. 2019). Macrophage polarization encompasses undifferentiated M0, classically activated M1, and selectively activated M2 states(Funes et al. 2018). Typically, M1 macrophages exhibit pro-inflammatory properties in acute inflammation-related conditions, whereas M2 macrophages demonstrate anti-inflammatory effects(Atri et al. 2018). Within the tumor milieu, M1 TAMs can eliminate tumor cells, whereas M2 TAMs tend to promote tumor growth. Notably, TAMs frequently skew toward the M2 phenotype, which is closely correlated with adverse prognoses across various cancers(Boutilier et al. 2021, Wu et al. 2020). Our investigation revealed that HMGB1 released from heat-injured HCs interacts with TREM1 receptors on macrophages to promote macrophage M2 polarization. Thus, while HMGB1 confers protection to heat-injured HCs, its effect on macrophages may contribute to the establishment of an immunosuppressive TME.

The enhanced invasive ability of liver cancer cells is a primary driver of tumor recurrence and postoperative cancer-related mortality(Liu et al. 2012). The cells within the liver, including HCs and macrophages, act as a fertile “soil,” furnishing liver cancer cells with abundant nutrients. Liver cancer cells constantly adapt to this milieu, promoting high invasion capacity and persistent recurrence and metastasis(Li et al. 2017, Ma et al. 2019). Remarkably, we observed that HMGB1 secretion by heat-damaged HCs fosters liver cancer cell invasion by modulating macrophage polarization. These findings underscore the pivotal role of HMGB1 in tumor recurrence following thermal ablation.

There are several limitations in our study. First, the HMGB1-TREM1 signaling pathway is only exploratory. We failed to block other HMGB1 signaling pathways and to clarify the pair of potential pathways involved. Second, most of the experimental results were obtained in vitro, and in vivo experiments are needed to achieve clinical translation in the future.

In summary, our research revealed that thermal injury to HCs following RFA induces HMGB1 expression and release. HMGB1 within the TME stimulates macrophage M2 polarization via the TREM1/JAK2/STAT3 signaling pathway, consequently bolstering liver cancer cell invasion, thereby contributing to a promising therapeutic strategy for recurrence after RFA by targeting the HMGB1/TREM1 axis.

Acknowledgements

We thank all members of our team for technical support suggestions.

Author contributions

X. L. conceived and supervised this project. B. X., C. L., G. H., J. L., and Q. L. performed experiments and analyzed the data. B. X., C. L., and J. G. wrote the final manuscript. Final approval of manuscript: All authors.

Funding

The work was supported by the Chongqing Science and Health Joint Medical Research Project (2023MSXM046).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethical approval

This study was subjected to review by the Ethics Committee of Chongqing Medical University and adhered to the Guidelines for Ethical Review of Laboratory Animal Welfare of the People’s Republic of China.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Bin Xiong and Chunming Li contributed equally to this work.

References

  1. Arneth B (2019) Tumor Microenvironment. Med (Kaunas) 56:15. 10.3390/medicina56010015 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Atri C, Guerfali FZ, Laouini D (2018) Role of human macrophage polarization in inflammation during infectious diseases. Int J Mol Sci 19:1801. 10.3390/ijms19061801 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Biguetti CC, Cavalla F, Silveira EV, Tabanez AP, Francisconi CF, Taga R, Campanelli AP, Trombone APF, Rodrigues DC, Garlet GP (2019) HGMB1 and RAGE as essential components of Ti Osseointegration process in mice. Front Immunol 10:709. 10.3389/fimmu.2019.00709 [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Bosco MC, Raggi F, Varesio L (2016) Therapeutic potential of Targeting TREM-1 in Inflammatory diseases and Cancer. Curr Pharm Des 22:6209–6233. 10.2174/1381612822666160826110539 [DOI] [PubMed] [Google Scholar]
  5. Boutilier AJ, Elsawa SF (2021) Macrophage polarization States in the Tumor Microenvironment. Int J Mol Sci 22:6995. 10.3390/ijms22136995 [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Bruni D, Angell HK, Galon J (2020) The immune contexture and immunoscore in cancer prognosis and therapeutic efficacy. Nat Rev Cancer 20:662–680. 10.1038/s41568-020-0285-7 [DOI] [PubMed] [Google Scholar]
  7. Chu KF, Dupuy DE (2014) Thermal ablation of tumours: biological mechanisms and advances in therapy. Nat Rev Cancer 14:199–208. 10.1038/nrc3672 [DOI] [PubMed] [Google Scholar]
  8. Dulmovits BM, Tang Y, Papoin J, He M, Li J, Yang H, Addorisio ME, Kennedy L, Khan M, Brindley E, Ashley RJ, Ackert-Bicknell C, Hale J, Kurita R, Nakamura Y, Diamond B, Barnes BJ, Hermine O, Gallagher PG, Steiner LA, Lipton JM, Taylor N, Mohandas N, Andersson U, Al-Abed Y, Tracey KJ, Blanc L (2022) HMGB1-mediated restriction of EPO signaling contributes to anemia of inflammation. Blood 139:3181–3193. 10.1182/blood.2021012048 [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Funes SC, Rios M, Escobar-Vera J, Kalergis AM (2018) Implications of macrophage polarization in autoimmunity. Immunology 154:186–195. 10.1111/imm.12910 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hernandez C, Huebener P, Pradere JP, Antoine DJ, Friedman RA, Schwabe RF (2018) HMGB1 links chronic liver injury to progenitor responses and hepatocarcinogenesis. J Clin Invest 128:2436–2451. 10.1172/jci91786 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Jiang J, Chen S, Li K, Zhang C, Tan Y, Deng Q, Chai Y, Wang X, Chen G, Feng K, Zhang L, Xie CM, Ma K (2019) Targeting autophagy enhances heat stress-induced apoptosis via the ATP-AMPK-mTOR axis for hepatocellular carcinoma. Int J Hyperth 36:499–510. 10.1080/02656736.2019.1600052 [DOI] [PubMed] [Google Scholar]
  12. Jiang C, Cai YQ, Yang JJ, Ma CY, Chen JX, Huang L, Xiang Z, Wu J (2023) Radiomics in the diagnosis and treatment of hepatocellular carcinoma. Hepatobiliary Pancreat Dis Int 22:346–351. 10.1016/j.hbpd.2023.03.010 [DOI] [PubMed] [Google Scholar]
  13. Jube S, Rivera ZS, Bianchi ME, Powers A, Wang E, Pagano I, Pass HI, Gaudino G, Carbone M, Yang H (2012) Cancer cell secretion of the DAMP protein HMGB1 supports progression in malignant mesothelioma. Cancer Res 72:3290–3301. 10.1158/0008-5472.Can-11-3481 [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Klune JR, Dhupar R, Cardinal J, Billiar TR, Tsung A (2008) HMGB1: endogenous danger signaling. Mol Med 14:476–484. 10.2119/2008-00034.Klune [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Li HM, Ye ZH (2017) Microenvironment of liver regeneration in liver cancer. Chin J Integr Med 23:555–560. 10.1007/s11655-017-2806-0 [DOI] [PubMed] [Google Scholar]
  16. Li A, Ji B, Yang Y, Ye B, Zhu Q, Hu X, Liu Y, Zhou P, Liu J, Gao R, Zhou Q, Kang B, Jiang Y (2023) Single-cell RNA sequencing highlights the role of PVR/PVRL2 in the immunosuppressive tumour microenvironment in hepatocellular carcinoma. Front Immunol 14:1164448. 10.3389/fimmu.2023.1164448 [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Liu Y, Tong Z, Li T, Chen Q, Zhuo L, Li W, Wu RC, Yu C (2012) Hepatitis B virus X protein stabilizes amplified in breast cancer 1 protein and cooperates with it to promote human hepatocellular carcinoma cell invasiveness. Hepatology 56:1015–1024. 10.1002/hep.25751 [DOI] [PubMed] [Google Scholar]
  18. Liu T, Zhao H, Wang Y, Qu P, Wang Y, Wu X, Zhao T, Yang L, Mao H, Peng L, Zhan Y, Li P (2024) Serum high mobility group box 1 as a potential biomarker for the progression of kidney disease in patients with type 2 diabetes. Front Immunol 15:1334109. 10.3389/fimmu.2024.1334109 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Luo YD, Fang L, Yu HQ, Zhang J, Lin XT, Liu XY, Wu D, Li GX, Huang D, Zhang YJ, Chen S, Jiang Y, Shuai L, He Y, Zhang LD, Bie P, Xie CM (2021) p53 haploinsufficiency and increased mTOR signalling define a subset of aggressive hepatocellular carcinoma. J Hepatol 74:96–108. 10.1016/j.jhep.2020.07.036 [DOI] [PubMed] [Google Scholar]
  20. Ma L, Hernandez MO, Zhao Y, Mehta M, Tran B, Kelly M, Rae Z, Hernandez JM, Davis JL, Martin SP, Kleiner DE, Hewitt SM, Ylaya K, Wood BJ, Greten TF, Wang XW (2019) Tumor Cell Biodiversity drives Microenvironmental Reprogramming in Liver Cancer. Cancer Cell 36:418–430e6. 10.1016/j.ccell.2019.08.007 [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Na YR, Kim SW, Seok SH (2023) A new era of macrophage-based cell therapy. Exp Mol Med 55:1945–1954. 10.1038/s12276-023-01068-z [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Otoo A, Czika A, Lamptey J, Yang JP, Feng Q, Wang MJ, Wang YX, Ding YB (2023) Emodin improves glucose metabolism and ovarian function in PCOS mice via the HMGB1/TLR4/NF-κB molecular pathway. Reproduction 166:323–336. 10.1530/rep-22-0449 [DOI] [PubMed] [Google Scholar]
  23. Ruzzenente A, Manzoni GD, Molfetta M, Pachera S, Genco B, Donataccio M, Guglielmi A (2004) Rapid progression of hepatocellular carcinoma after Radiofrequency ablation. World J Gastroenterol 10:1137–1140. 10.3748/wjg.v10.i8.1137 [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Seki T, Tamai T, Ikeda K, Imamura M, Nishimura A, Yamashiki N, Nakagawa T, Inoue K (2001) Rapid progression of hepatocellular carcinoma after transcatheter arterial chemoembolization and percutaneous radiofrequency ablation in the primary tumour region. Eur J Gastroenterol Hepatol 13:291–294. 10.1097/00042737-200103000-00014 [DOI] [PubMed] [Google Scholar]
  25. Shin SW, Ahn KS, Kim SW, Kim TS, Kim YH, Kang KJ (2021) Liver resection Versus local ablation therapies for Hepatocellular Carcinoma within the Milan Criteria: a systematic review and Meta-analysis. Ann Surg 273:656–666. 10.1097/sla.0000000000004350 [DOI] [PubMed] [Google Scholar]
  26. Visioni A, Skitzki J (2016) Technical considerations for the generation of Adoptively Transferred T cells in Cancer Immunotherapy. Cancers (Basel) 8:86. 10.3390/cancers8090086 [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Vitale I, Manic G, Coussens LM, Kroemer G, Galluzzi L (2019) Macrophages and metabolism in the Tumor Microenvironment. Cell Metab 30:36–50. 10.1016/j.cmet.2019.06.001 [DOI] [PubMed] [Google Scholar]
  28. Wang S, Zhang Y (2020) HMGB1 in inflammation and cancer. J Hematol Oncol 13:116. 10.1186/s13045-020-00950-x [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Wang X, Xiang L, Li H, Chen P, Feng Y, Zhang J, Yang N, Li F, Wang Y, Zhang Q, Li F, Cao F (2015) The role of HMGB1 signaling pathway in the Development and Progression of Hepatocellular Carcinoma: a review. Int J Mol Sci 16:22527–22540. 10.3390/ijms160922527 [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Wei H, Li W, Yang M, Fang Q, Nian J, Huang Y, Wei Q, Huang Z, Liu G, Xu Z, Hu A, Pu J (2024) METTL3/16-mediated m(6)a modification of ZNNT1 promotes hepatocellular carcinoma progression by activating ZNNT1/osteopontin/S100A9 positive feedback loop-mediated crosstalk between macrophages and tumour cells. Clin Immunol 261:109924. 10.1016/j.clim.2024.109924 [DOI] [PubMed] [Google Scholar]
  31. Wu J, Guo W, Lin SZ, Wang ZJ, Kan JT, Chen SY, Zhu YZ (2016) Gp130-mediated STAT3 activation by S-propargyl-cysteine, an endogenous hydrogen sulfide initiator, prevents doxorubicin-induced cardiotoxicity. Cell Death Dis 7:e2339. 10.1038/cddis.2016.209 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Wu K, Lin K, Li X, Yuan X, Xu P, Ni P, Xu D (2020) Redefining Tumor-Associated macrophage subpopulations and functions in the Tumor Microenvironment. Front Immunol 11:1731. 10.3389/fimmu.2020.01731 [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Wyganowska-Swiatkowska M, Nohawica M, Grocholewicz K, Nowak G (2020) Influence of Herbal Medicines on HMGB1 release, SARS-CoV-2 viral attachment, Acute Respiratory failure, and Sepsis. A literature review. Int J Mol Sci 21:4639. 10.3390/ijms21134639 [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Xu Z, Ma W, Wang J, Chen H, Li H, Yin Z, Hao J, Chen K (2024) Nuclear HMGB1 is critical for CD8 T cell IFN-γ production and anti-tumor immunity. Cell Rep 43:114591. 10.1016/j.celrep.2024.114591 [DOI] [PubMed] [Google Scholar]
  35. Yan Y, Zhang LB, Ma R, Wang MN, He J, Wang PP, Tao QW, Xu Y (2024) Jolkinolide B ameliorates rheumatoid arthritis by regulating the JAK2/STAT3 signaling pathway. Phytomedicine 124:155311. 10.1016/j.phymed.2023.155311 [DOI] [PubMed] [Google Scholar]
  36. Yan L, Wang J, Cai X, Liou YC, Shen HM, Hao J, Huang C, Luo G, He W (2024a) Macrophage plasticity: signaling pathways, tissue repair, and regeneration. MedComm (2020) 5:e658. 10.1002/mco2.658 [DOI] [PMC free article] [PubMed]
  37. Yao W, Tao R, Wang K, Ding X (2024) Icariin attenuates vascular endothelial dysfunction by inhibiting inflammation through GPER/Sirt1/HMGB1 signaling pathway in type 1 diabetic rats. Chin J Nat Med 22:293–306. 10.1016/s1875-5364(24)60618-7 [DOI] [PubMed] [Google Scholar]
  38. Zeng J, Lin Z, Tang J, Chen X, Huang K, Gan F (2024) The role of JAK2/STAT3 pathway in non-cytotoxic concentrations of DON-induced aggravation of inflammatory response in IL-10 deficient RAW264.7 cells. Food Chem Toxicol 186:114557. 10.1016/j.fct.2024.114557 [DOI] [PubMed] [Google Scholar]
  39. Zhang S, Huang Y, Pi S, Chen H, Ye F, Wu C, Li L, Ye Q, Lin Y, Su Z (2023) Autophagy-amplifying nanoparticles evoke immunogenic cell death combined with anti-PD-1/PD-L1 for residual tumors immunotherapy after RFA. J Nanobiotechnol 21:360. 10.1186/s12951-023-02067-y [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Zhao Y, Bai J, Wang X, Zhang Y, Yan X, Qi J, Xia X, Feng Y, Duan B (2024) Threatment strategies for recurrent Hepatocellular Carcinoma patients: ablation and its combination patterns. J Cancer 15:2193–2205. 10.7150/jca.93885 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Žūkienė R, Naučienė Z, Šilkūnienė G, Vanagas T, Gulbinas A, Zimkus A, Mildažienė V (2017) Contribution of mitochondria to injury of hepatocytes and liver tissue by hyperthermia. Med (Kaunas) 53:40–49. 10.1016/j.medici.2017.01.001 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

No datasets were generated or analysed during the current study.

Articles from Journal of Cancer Research and Clinical Oncology are provided here courtesy of Springer

RESOURCES