
==== Front
J Pharm Anal
J Pharm Anal
Journal of Pharmaceutical Analysis
2095-1779
2214-0883
Xi'an Jiaotong University

S2095-1779(24)00030-3
10.1016/j.jpha.2024.01.010
100942
Original Article
Melatonin enhances the efficacy of anti-PD-L1 by improving hypoxia in residual tumors after insufficient radiofrequency ablation
Ren Yanqiao 1
Zhu Licheng 1
Guo Yusheng
Ma Jinqiang
Yang Lian
Zheng Chuansheng hqzcsxh@sina.com
∗∗
Dong Xiangjun dongxiangjun3100@163.com
∗
Department of Radiology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430022, China
∗ Corresponding author. dongxiangjun3100@163.com
∗∗ Corresponding author. hqzcsxh@sina.com
1 Both authors contributed equally to this work.

02 2 2024
8 2024
02 2 2024
14 8 1009421 10 2023
25 1 2024
29 1 2024
© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
The hypoxic microenvironment and inflammatory state of residual tumors caused by insufficient radiofrequency ablation (iRFA) are major reasons for rapid tumor progression and pose challenges for immunotherapy. We retrospectively analyzed the clinical data of patients with hepatocellular carcinoma (HCC) treated with RFA and observed that iRFA was associated with poor survival outcomes and progression-free survival. Using an orthotopic HCC mouse model and a colorectal liver metastasis model, we observed that treatment with melatonin after iRFA reduced tumor growth and metastasis and achieved the best outcomes when combined with anti-programmed death-ligand 1 (anti-PD-L1) therapy. In mechanism, melatonin inhibited the expression of epithelial-mesenchymal transitions, hypoxia-inducible factor (HIF)-1α, and PD-L1 in tumor cells after iRFA. Flow cytometry revealed that melatonin reduced the proportion of myeloid-derived suppressor cells and increased the proportion of CD8+ T cells. Transcriptomic analysis revealed an upregulation of immune-activated function-related genes in residual tumors. These findings demonstrated that melatonin can reverse hypoxia and iRFA-induced inflammation, thereby overcoming the immunosuppressive tumor microenvironment (TME) and enhancing the efficacy of immunotherapy.

Graphical abstract

Image 1

Highlights

• Melatonin can inhibit malignant degree in heated HCC under hypoxic conditions.

• Melatonin inhibits the expression of HIF-1α and regulates the TME.

• Melatonin combined with anti-PD-L1 antibody is effective in liver cancer.

Keywords

Insufficient radiofrequency ablation
Melatonin
Hypoxia
Immunotherapy
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pmc1 Introduction

Radiofrequency ablation (RFA), a minimally invasive treatment, is increasingly becoming a reliable therapeutic method for solid tumors, including hepatocellular carcinoma (HCC), renal cell carcinoma, and lung cancer [1,2]. The latest Barcelona Clinic Liver Cancer Guidelines recommend RFA for treating early-stage HCC [3]. In addition, RFA has led to favorable oncological outcomes in patients with unresectable liver metastasis of colorectal cancer (CRLM), comparable to those who meet the surgical conditions [[4], [5], [6]]. In particular, RFA is beneficial for patients with unresectable CRLM, patients with comorbidities preventing them from undergoing surgery, and patients with insufficient liver reserve [7]. Previous studies have indicated a 40% increase in the 5-year survival for small (<4 cm) isolated CRLM [8]. However, postoperative tumor recurrence and metastasis remain therapeutic challenges. Furthermore, residual tumors after RFA may be more malignant, invasive, and capable of distant metastasis, thereby severely affecting the prognosis of patients with liver cancer [9,10]. Hence, effectively eradicating residual and distant metastatic tumors after insufficient RFA (iRFA) and improving the efficacy of RFA for treating liver cancer are critical.

Tumors with a low mutation burden and few neoantigens are generally less immunogenic, with little response to immunotherapy. RFA not only reduces the tumor load but also promotes the release of neoantigens, thus effectively promoting lymphocyte infiltration of tumor tissues, improving the inhibition of the tumor immune microenvironment, and ultimately enhancing the immune response [11,12]. However, studies have shown that iRFA-induced immune responses are insufficient to prevent tumor recurrence and may impede the efficacy of anti-programmed death-ligand 1 (anti-PD-L1) antibodies [9,13]. Thus, understanding the immunological mechanisms underlying the prooncogenic effects of iRFA and using combined immunotherapies integrated with locoregional strategies may maximize the clinical response to RFA. After RFA, a band of inflammatory edema is formed between the normal liver tissue and area of tumor coagulative necrosis, causing blood stasis and thrombosis, exacerbating residual tumor hypoxia [14]. Hypoxia can lead to increased expression of tumor hypoxia-inducible factor (HIF)-1α, facilitating the development of various cancer cells by promoting tumor migration, angiogenesis, and metastasis. Meanwhile, HIF-1α can directly upregulate the expression of PD-L1 in myeloid-derived suppressor cells (MDSCs), dendritic cells, and cancer cells [15], ultimately leading to MDSC-mediated T cell suppression. Hence, improving the hypoxic immunosuppressive microenvironment of residual tumors after iRFA in combination with immunotherapy may enhance its therapeutic effects.

Studies have recently indicated that melatonin (PubChem CID: 896) can effectively inhibit HIF-1α expression and epithelial-mesenchymal transition (EMT) by blocking the HIF-1α/Snail/matrix metalloprotein-9 (MMP-9) signaling pathway, thereby reducing tumor invasion and metastasis [[16], [17], [18], [19], [20]]. In addition, melatonin has been reported to inhibit the proliferation of immunosuppressive cells, including MDSCs, regulatory T cells (Tregs), and tumor-associated macrophages, thereby increasing the number and function of CD8+ T lymphocytes [21,22]. Therefore, we hypothesized that melatonin administration after iRFA could significantly improve hypoxia in residual tumors and enhance the efficacy of immunotherapy. We tested this hypothesis by establishing various iRFA models and observing the effects of different treatments on residual and metastatic tumors using melatonin and anti-PD-L1 antibodies.

2 Materials and methods

2.1 Study design and patient selection

The clinical data of patients with HCC who underwent RFA at our medical center between January 2009 and December 2018 were analyzed. Insufficient ablation was defined as the presence of a recurrent tumor in or around the original ablation site within 4 months of RFA. Clinical data including age, sex, and tumor size were compared between the two groups. The inclusion criteria of this study were as follows: 1) patients older than 18 years were diagnosed with HCC; 2) a solitary HCC (≤5.0 cm) or multiple (up to three) HCC lesions (each ≤3.0 cm); 3) patients were not eligible for surgical resection or liver transplantation; 4) no vascular invasion or no extrahepatic metastasis; 5) Eastern Cooperative Oncology Group performance status 0; 6) Child-Pugh class A or B; and 7) blood platelet count >40 × 109/L. The exclusion criteria of this study were as follows: 1) before RFA treatment, patients received other treatments for HCC, such as hepatectomy, liver transplantation, and transarterial chemoembolization (TACE); 2) patients were accompanied by other malignancies; 3) patients were accompanied by severe cardiac and renal dysfunction; and 4) the clinical data of patients were incomplete or lost to follow-up. Abdominal contrast-enhanced computed tomography (CT) or magnetic resonance imaging (MRI) was performed every 6–8 weeks after the initial RFA in all patients to detect residual tumors and new metastases. Overall survival (OS) and progression-free survival (PFS) were assessed.

Ultrasound- or CT-guided RFA was performed following a previously reported standard treatment regimen [23]. A RITA 1500 generator (Angiodynamics, Inc., Latham, NY, USA) and a monopolar or multi-hook probe needle electrode (Angiodynamics, Inc.) were used. A monopolar electrode, measuring < 2 cm in diameter, was placed at the center of tumors. Alternatively, a multihook electrode was used.

The present retrospective study adhered to the principles outlines in the Declaration of Helsinki and received the approval from the Ethic Committee of the Union Hospital (Approval No.: UHCT-IEC-SOP-016-02-01). Written informed consent was obtained from patients prior to treatment.

2.2 Data from The Cancer Genome Atlas (TCGA) dataset

RNA-sequencing (RNA-seq) expression (level 3) profiles for HCC were downloaded from the TCGA dataset (https://portal.gdc.com). The two-gene correlation map was analyzed using the R software “ggstatsplot” package. Gene sets related to EMT markers (Vimentin, Cadherin 2, Forkhead box C2, SNAI1, SNAI2, TWIST1, Fibronectin 1, Integrin subunit beta 6, MMP-2, MMP-9, SRY-box transcription factor 10, GC vitamin D binding protein, Cadherin 1, Desmoplakin, and Occludin) were collected, and the enrichment scores for each sample on EMT markers were calculated based on single-sample gene set enrichment analysis. The correlation between HIF-1α and EMT markers scores/CD274 (PD-L1) was analyzed by Spearman correlation. All analysis methods and R packages were implemented in R, version 4.0.3. Values of P < 0.05 was considered to be statistically significant.

2.3 Cells and culture conditions

Human HCC cell lines (Huh7 and HCCLM3), mouse hepatoma cell lines (Hepa1–6), and mouse colon adenocarcinoma cell lines CT26 were purchased from the American Type Culture Collection (ATCC, Mansas, VA, USA). All cell lines were cultivated in Dulbecco's modified Eagle's medium (DMEM; Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS; Gibco) and 1% penicillin/streptomycin (Gibco) in a cell humidified incubator at 37 °C with 5% CO2, or in a hypoxic incubator (Thermo Fisher Scientific Inc., Waltham, MA, USA). In vitro sub-lethal heat stress model was established to simulate iRFA in vivo. Cells were first inoculated in the flasks for 24 h, following which the medium in the flasks, maintained at 37 °C, was replaced with the medium preheated at 47 °C. The flasks were immediately placed in a water bath at 47 °C for 5 min. After the thermal exposure, the flasks were placed in an incubator for subsequent experiments. In addition, CoCl2 (Sigma-Aldrich, St. Louis, MO, USA) at a concentration of 150 μM was added to mimic the hypoxia condition.

2.4 Western blot analysis

MMP-2, MMP-9, E-cadherin, N-cadherin, Vimentin, Snail, HIF-1α, and PD-L1 were detected in Huh7 and HCCLM3 cells through Western blot analysis. After exposure to heat, cells were categorized into normoxic and hypoxic groups. Cells were cultured in normoxic (21% O2) and hypoxic (150 μM CoCl2) conditions for 12 and 24 h, respectively, with or without melatonin (0, 0.5, 1, and 2 mM) in the culture medium. The cells were lysed on ice using cell lysis buffer (pH 8.0) and phosphatase inhibitors (Sigma-Aldrich) as needed, boiled for 10 min under reducing conditions, and frozen at −20 °C until use. Approximately 20 μg of protein per well was loaded onto gels and transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Targeted protein levels were assessed through Western blot analysis using primary antibodies (Proteintech, Wuhan, China), followed by incubation with the corresponding peroxidase-conjugated secondary antibodies and Immobilion Western Chemilum HRP Substrate (Millipore).

2.5 Real-time polymerase chain reaction (RT-PCR)

Experimental grouping and cell intervention followed the same protocol as described for Western blotting experiment. Total RNA was extracted from Huh7 and HCCLM3 cells using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions, and then subjected to complementary DNA (cDNA) synthesis by reverse transcription using a SuperScript III kit (Invitrogen, Waltham, MA, USA). All the primers used are listed in Table S1. The results were normalized according to the expression of the internal control, β-actin messenger RNA (mRNA).

2.6 Cell viability assays

Experimental grouping and cell intervention followed the same protocol as described for western blotting experiment. Each well of a 96-well plate was inoculated with 10,000 cells and treated with a complete medium for the Cell Counting Kit-8 (CCK-8) assay. Optical density was measured at 450 nm after 12 and 24 h using a microplate reader (Allsheng, Hangzhou, China).

2.7 Migration assays

The migratory ability of HCC cells was evaluated using a transwell assay (Corning Inc., Corning, NY, USA). First, HCC cells (1 × 105 cells/mL) in serum-free medium were seeded into the transwell chambers. Subsequently, 600 μL of DMEM supplemented with 15% FBS was added to the lower compartments of the transwell chambers. After 24 h, the cells on the lower surface of the upper chambers were fixed in 4% paraformaldehyde and stained with 0.25% crystal violet for 30 min.

2.8 Colony formation

HCC cells were collected and seeded in six-well plates at a density of 1.0 × 103 cells/well, followed by incubation for 14 days. The colonies were fixed with methanol, stained with 0.1% crystal violet, and subsequently counted.

2.9 In vivo tumor models and therapy

All experimental animals adhered to the Animal Welfare Act regarding the use of laboratory animals. All animal experiments were approved by the Ethics Committee of Tongji Medical College, Huazhong University of Science and Technology (IACUC No.: 3041). C57BL/6 mice (male, 6–8 weeks old, weighing 19–22 g) were purchased from the Animal Center of Tongji Medical College, kept under optimal light, temperature, and humidity conditions, and provided with sufficient food and water.

2.10 Model 1: orthotopic syngeneic Hepa1–6 HCC mouse model

In this model, Hepa1–6 tumors extracted from carrier mice were cut into 1 mm3 blocks under sterile conditions and then placed in physiological saline on ice. The recipient mouse's left liver lobe was then fully exposed via a midline abdominal incision, and a small fragment of the Hepa1–6 tumor was inoculated into the left liver lobe. A small piece of gelatin sponge was placed in the liver wound, and the abdominal muscle and skin layers were sutured. RFA was initiated when the maximum tumor diameter reached 0.6–0.8 cm. A midline abdominal incision was made to fully expose the tumor in the left lobe of the liver. RFA was performed under direct vision using a 460-kHz RF generator (Angiodynamics, Inc.) and a 16-gauge monopolar needle electrode (Angiodynamics, Inc.). The electrode was eccentrically inserted into the tumor for ablation to ensure iRFA. The ablation power was set to 5 W and maintained for 30 s. An infrared camera (MAG64AI; Magnity Electronics Co., Ltd., Shanghai, China) was used to observe the dynamic temperature changes in the ablation zone. A day after ablation, diffusion-weighted imaging was performed to examine the restricted diffusion in the ablation site. Hematoxylin and eosin (H&E) staining was performed to confirm necrosis and residual tumor areas. The experimental mice were categorized into the following five groups (n = 6 mice per group): 1) untreated, 2) iRFA, 3) iRFA + melatonin (iRFA + M), 4) iRFA + anti-PD-L1 antibody (iRFA + P), and 5) iRFA + melatonin + anti-PD-L1 antibody (iRFA + M + P). Melatonin (10 mg/kg, 100 μL) was administered on the same day as with iRFA, with an intraperitoneal injection once a day. Anti-PD-L1 antibody (10 mg/kg, 100 μL; Clone: 10F.9G2, rat IgG2b; BioXCell, West Lebanon, NH, USA) was administered on the same day as iRFA, and intraperitoneal injection was administered every three days. The tumor volume in each mouse was assessed using a 3.0-T MRI a day before and immediately after iRFA and in every five days after iRFA. After 21 days of treatment, the mice in all five groups were euthanized and liver tumor tissues were harvested for further analysis. Survival analysis was performed as an independent experiment with 10 mice in each group.

2.11 Model 2: intrasplenic injection of CT26 cells to establish liver metastasis model

Luciferase (Luc)-CT26 cells were exposed to 47 °C for 5 min and incubated for 1 h. Subsequently, C57BL/6 mice received 100 μL (5 × 106 cells) of the cell suspension through intrasplenic injection. After 3 days of the injection, the mice were randomly categorized into the following four groups (n = 6 mice per group): 1) iRFA, 2) iRFA + M, 3) iRFA + P, and 4) iRFA + M + P. Meanwhile, the experimental intervention was initiated; the dose and regimen of melatonin and anti-PD-L1 antibody were the same as those used in Model 1. The entire intervention cycle lasted 14 days. The untreated mice (n = 6) received 100 μL (5 × 106 cells) of the Luc-CT26 cell suspension without thermal exposure through intrasplenic injection. At 3, 10, and 17 days after cell injection, small-animal live imaging was performed to detect the metastatic progression of Luc-CT26 cells in the liver. After 17 days of cell injection, the body weights of the mice in all five groups were measured, following which the mice were euthanized, and their livers were removed and weighed. In addition, survival analysis was performed as an independent experiment on the indicated days (n = 10 mice per group).

2.12 Histological analysis

The 4-μm paraffin-embedded tissue sections were prepared for H&E staining, immunohistochemistry (IHC), and immunofluorescence. Paraffin was removed from tissue sections, followed by antigen retrieval. IHC for target proteins was performed using different antibodies including anti-HIF-1α (1:50; Proteintech), anti-CD4 (1:16000, Abcam, Boston, MA, USA), anti-CD8 (1:16000; Proteintech), anti-MMP-2 (1:2000; Proteintech), and anti-MMP-9 (1:2000; Proteintech). For immunofluorescence, the primary antibodies used were anti-CD8 (1:16000; Proteintech) and anti-Ki67 (1:200; Abcam). A terminal-deoxynucleoitidyl transferase dUTP nick-end labeling (TUNEL) apoptosis assay kit (Yeasen, Wuhan, China) was used to evaluate cell apoptosis following the manufacturer's instructions. The nuclei of the tumor cells were stained with 4′,6-diamidino-2-phenylindole (DAPI). Hypoxyprobe-1 (60 mg/kg; HypoxyprobeTM-1 Plus Kit; Hypoxyprobe, Inc. Burlington, MA, USA) solution was injected into the caudal vein to assess tumor hypoxia. Tumor tissues were removed after 1 h, sectioned, and stained with the Hypoxyprobe Plus Kit following the manufacturer's instructions. Six to eight independent microscopic fields (magnification, ×200) per specimen were used for quantitative IHC and immunofluorescence analyses. Image analysis was performed using Image-Pro Plus software (version 6.0; Media Cybernetics, Warrendale, PA, USA).

2.13 Flow cytometry

For cell-surface analysis, cells were stained with anti-mouse fixable viability stain (564997), CD3 (563024), CD45 (557659), CD11b (563015), CD4 (566407), CD8a (563234), F4/80 (565411), and Gr-1 (553126) in the suggested antibody concentrations and incubated at 4 °C for 30 min. These were fixed and permeabilized for Foxp3 (563101) staining. All antibodies for flow cytometry were obtained from BD Pharmingen™ (Franklin Lake, NJ, USA). The FlowJo software was used to analyze the data (version 10).

2.14 RNA-seq analysis

In Model 1, total RNA was extracted from the untreated, iRFA, and iRFA + M groups after 21 days of treatment. The quality of the purified RNA was assessed using the Agilent Bioanalyzer 2100 system (Agilent Technologies, Santa Clara, CA, USA). RNA-seq was performed using NovaSeq X Plus (Majorbio Bio-pharm Technology Co., Ltd., Shanghai, China) and the data were analyzed using the Majorbio Cloud Platform (Majorbio Bio-pharm Technology Co., Ltd.). The data are presented as mean displayed at the center of the heatmaps. The fold change was calculated and converted to log2. A heat map of immune-activated function-related genes was obtained from a previous study [24]. Microenvironment cell populations-counter (MCPcounter), epigenetic profile inference from cell-free DNA (CfDNA) (EPIC), and cell-type identification by estimating relative subsets of RNA transcripts (CIBERSORT-ABS) algorithms were applied to quantify the relative proportions of infiltrating immune cells.

2.15 Enzyme-linked immunosorbent assay (ELISA) and biochemical analysis

Blood serum samples were collected from the mice of Model 1 group before euthanasia to measure cytokine levels, including tumor necrosis factor (TNF)-α, interferon-gamma (IFN-γ), interleukin (IL)-12, IL-10, IL-4, and IL-2, and analyzed using ELISA kits (Wuhan USCN Business Co., Ltd., Wuhan, China). In addition, the heart, liver, and kidney functions, encompassing plasma creatine kinase, alanine aminotransferase, aspartate aminotransferase, total bilirubin, blood urea nitrogen, and serum creatinine levels, were evaluated using a biochemical autoanalyzer (Model DXC 8000; Beckman Coulter Diagnostics, Brea, CA, USA).

2.16 Statistical analysis

All analyses were performed using GraphPad Prism (version 8). Quantitative data are presented as mean ± standard deviation (SD), and discrete variables are presented as proportions. The t-test and Chi-square test were used to compare the data between the groups. OS and PFS were analyzed using the Kaplan-Meier method and compared using the log-rank Mantel-Cox test. Variables with a P < 0.1 at univariate analysis were entered into the multivariate analysis. A Cox proportional hazard regression model was used to analyze potential prognostic factors affecting OS and PFS. The values of P < 0.05 was considered to be statistically significant.

3 Results

3.1 iRFA is related to poor prognosis in patients with HCC

A retrospective case-control study was conducted including patients with HCC to determine the association between residual tumors and rapid disease progression after iRFA. From January 2009 to December 2018, 21 patients in the iRFA group had received prior treatments before undergoing RFA treatment. Specifically, 11 patients had undergone liver resection, 1 patient had undergone liver transplantation, and 9 patients had received TACE before the RFA treatment. Meanwhile, in the complete ablation group, 283 patients were excluded from the analysis. This group included 162 patients who had undergone liver resection, 13 patients who had received liver transplantation, and 108 patients who had undergone TACE. Finally, 80 patients were enrolled in this study, including 28 patients in the iRFA group and 52 in the complete ablation group; Details have been presented in a flow chart in Fig. S1. Detailed clinicopathological characteristics of the 80 patients are summarized in Table S2. Typical CT images of local residual tumors are illustrated in Fig. 1A. The median OS was 25 months (95% confidence interval (CI): 19.8–30.2 months) and 78 months (95% CI: 40.4–115.6 months) in the iRFA and complete ablation group, respectively (P < 0.001) (Fig. 1B). Additionally, the median PFS in both groups was 9 months (95% CI: 5.1–12.9 months) and 29 months (95% CI: 12.5–45.5 months), respectively (P < 0.001) (Fig. 1C). The univariate and multivariate analyses (Tables S3 and S4) revealed iRFA as an independent risk factor for OS.Fig. 1 Imaging and survival curves of two groups of patients. (A) Typical computed tomography (CT) images of a patient with local residual tumor (white arrow: tumor before radiofrequency ablation (RFA); green arrow: low density ablation zone three months after ablation; and red arrow: residual tumor). (B, C) Kaplan-Meier curves of overall survival (B) and progression-free survival (C) of patients in insufficient RFA (iRFA) and complete ablation group. OS: overall survival; PFS: progression-free survival.

Fig. 1

3.2 Melatonin ameliorates hypoxia and reduces the invasion and metastasis

Initially, we examined 371 patients with HCC from the TCGA cohort. The correlation maps indicated a significant positive correlation between HIF-1α expression and EMT markers’ enrichment scores (P < 0.001, Fig. 2A) and the expression of CD274 (PD-L1) (P < 0.001, Fig. 2B). After incomplete ablation, the HCCLM3 and Huh7 cells cultured with CoCl2 to mimic the hypoxic microenvironment demonstrated that melatonin dose-dependently decreased the level of HIF-1α. Meanwhile, the expression of EMT markers, including N-cadherin, Vimentin, Snail, MMP-2, and MMP-9, decreased, and the expression of E-cadherin was upregulated. Additionally, PD-L1 expression decreased after melatonin treatment (Fig. 2C). Notably, HIF-1α was absent under normoxic conditions, and melatonin did not affect the expression of EMT markers or PD-L1 (Fig. 2D).Fig. 2 Melatonin improves hypoxia and reduces invasion and metastasis of incomplete ablated cells. (A, B) The expression correlation of hypoxia-inducible factor (HIF)-1α and epithelial-mesenchymal transition (EMT) markers (A)/CD274 (B) in 371 hepatocellular carcinoma (HCC) patients from The Cancer Genome Atlas (TCGA) cohort. (C, D) Western blotting was used to detect the expression of HIF-1α, N-cadherin, E-cadherin, Vimentin, Snail, matrix metalloprotein (MMP)-2, MMP-9, programmed death-ligand 1 (PD-L1), and β-actin in heated HCCLM3 and Huh7 cells treated with melatonin for different concentrations (0, 0.5, 1, and 2 mM) for 12 or 24 h in hypoxia (C) or in normoxia (D) (n = 3). (E, F) The relative messenger RNA (mRNA) expression level of Slug, Snail, and Twist in heated HCCLM3 (E) and Huh7 (F) cells treated with melatonin for different concentrations (0, 0.5, 1.0, and 2.0 mM) for 24 h in hypoxia (n = 3). (G, H) Migration of heated HCCLM3 and Huh7 cells treated with melatonin for different concentrations (0, 0.5, 1.0, and 2.0 mM) and quantitative analysis charts in hypoxia (G) or in normoxia (H) (n = 3). (I, J) Colony forming ability of heated HCCLM3 and Huh7 cells treated with melatonin for different concentrations (0, 0.5, 1.0, and 2.0 mM) and quantitative analysis charts in hypoxia (I) or in normoxia (J) (n = 3). (K, L) Cytotoxicity assays of heated HCCLM3 (K) and Huh7 (L) cells treated with melatonin for different concentrations (0, 0.5, 1.0, and 2.0 mM) in hypoxia or in normoxia. Data are presented as mean ± standard deviation (SD). The P values are calculated using two-tailed unpaired t-test. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. ns: no significance. TPM: transcripts per million.

Fig. 2

Subsequently, the mRNA expression of EMT-related transcription factors (Snail, Slug, and Twist) was assessed using quantitative PCR (qPCR). Melatonin reduced the relative mRNA expression of Slug, Snail, and Twist under hypoxic (Figs. 2E, 2F, S2A, and S2B) and normoxic conditions (Figs. S2C−F). Transwell migration assay indicated decreased migratory ability of heat-treated HCCLM3 and Huh7 cells under hypoxic conditions (Fig. 2G) but had no effect under normoxic conditions (Fig. 2H). These results suggest that melatonin inhibits EMT in cells after iRFA, especially in HCC cells under hypoxic conditions. Colony formation assays revealed that melatonin dose-dependently inhibited cell proliferation under hypoxic (Fig. 2I) and normoxic conditions (Fig. 2J). Finally, cytotoxicity assays revealed that although melatonin significantly killed tumor cells under normoxia, its tumor-killing ability was significantly improved under hypoxia (Figs. 2K and L).

3.3 Combining melatonin with anti-PD-L1 antibody induces optimal anti-tumor effects

An iRFA model was created to investigate the efficacy of melatonin combined with an anti-PD-L1 antibody for treating residual HCC. Residual tumors were treated with melatonin or anti-PD-L1 antibody, alone or in combination, and T2-weighted MRI was used to observe the growth of the tumor longitudinally (Fig. 3A). The needle was inserted at the edge of the tumor and a lower power was used for ablation. At the same time, an infrared camera was used to detect the temperature of the ablation site (Fig. 3B). A day after ablation, diffusion-weighted images showed restricted diffusion in the ablation site, and H&E staining confirmed the presence of residual tumors (Figs. 3C and D). Initially, we observed a decrease in tumor volume after iRFA, which was followed by a gradual increase in tumor volume in the iRFA group than in the untreated group (Figs. 3E and F). Notably, the combination of melatonin and anti-PD-L1 antibody significantly inhibited tumor growth, demonstrating superior antitumor efficacy than that of the monotherapy (Figs. 3G and H). In addition, melatonin in combination with an anti-PD-L1 antibody significantly prolonged the OS of mice bearing residual Hepa1–6 tumors, an effect not observed in any of the monotherapy arms (Fig. 3I).Fig. 3 Melatonin combined with anti-programmed death-ligand 1 (PD-L1) antibody can significantly inhibit residual tumor growth after radiofrequency ablation (RFA). (A) Diagram for the experimental design. C57BL/6 mice were inoculated with Hepa1–6 cells, treated with insufficient RFA (iRFA) when tumor grown to about 0.6–0.8 cm. Melatonin and/or anti-PD-L1 antibody were administered to mice after iRFA treatment. Group 1: untreated; group 2: iRFA; group 3: iRFA + melatonin (iRFA + M); group 4: iRFA + anti-PD-L1 antibody (iRFA + P); group 5: iRFA + melatonin + anti-PD-L1 antibody (iRFA + M + P). (B) The ablation needle was inserted at the edge of the tumor, and an infrared camera was used to record the temperature in the ablation area during RFA. (C) T2-weighted (left panel) and diffusion weighted imaging (DWI) (right panel) magnetic resonance (MR) were used to detect the residual tumors. (D) Histological examination of residual tumors by hematoxylin and eosin (H&E) staining, which was classified into three regions: tumor necrosis area (region a), residual tumor area (region b), and normal liver area (region c). (E, F) T2-weighted MR was used to detect of the growth of liver tumors (E), and the tumor growth curves were plotted using the tumor volume in the six time points (F). (G, H) The mice were sacrificed at the end of the treatments (G), and liver/body weight ratio was recorded (H). (I) Kaplan-Meier survival curves for mice in different treatment groups. (J) Confocal fluorescence microscopy images of 4′,6-diamidino-2-phenylindole (DAPI) (blue color) and terminal-deoxynucleoitidyl transferase dUTP nick-end labeling (TUNEL) (green color), and quantitative analysis of tumor cell apoptosis (lower right). (K) Confocal fluorescence microscopy images of DAPI (blue color) and Ki67 (red color), and quantitative analysis of tumor cell proliferation (lower right). (L) Body weight curves from the mice in the different treatment groups. (M) Representative H&E staining images of lung, heart, liver, spleen, and kidney are shown in the untreated group and iRFA + M + P group. Data are presented as mean ± standard deviation (SD). The P values are calculated using two-tailed unpaired t-test or log-rank test. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. ns: no significance.

Fig. 3

TUNEL and Ki67 immunohistochemical staining were performed on tumor tissue sections to further elucidate the status of tumor cells in terms of apoptosis and proliferation. As illustrated in Fig. 3J, the green fluorescence of the TUNEL-positive cell nuclei was sequentially enhanced in the following order: iRFA + M + P > iRFA + P > iRFA + M > untreated > iRFA groups. This observation indicates that melatonin combined with an anti-PD-L1 antibody has the most potent triggering effect on tumor cell apoptosis. Furthermore, the decreased expression of Ki67 in the iRFA + M + P group also indicated a noticeable decline in tumor cell proliferation compared to the other four groups (Fig. 3K). Hence, this combination of melatonin and anti-PD-1 antibody is suggested as the best therapeutic effect in orthotopic syngeneic Hepa1–6 HCC residual tumors.

To evaluate drug efficacy, it is important to focus on the side effects of treatment. The changes in body weight during treatment fluctuated with an increasing trend, but showed no significant difference (Fig. 3L). In addition, the heart, liver, and renal functions in each group were evaluated, as shown in Fig. S3, demonstrating no significant differences in liver or renal function between the groups (P > 0.05). In addition, H&E staining of the lungs, heart, liver, spleen, and kidneys revealed no pathological abnormalities in the untreated and iRFA + M + P groups (Fig. 3M).

3.4 Melatonin ameliorates hypoxia and immunosuppressive microenvironment in residual tumors

To understand the molecular mechanisms underlying melatonin therapy for residual tumors, we performed RNA-seq analysis using Hepa1–6 tumors from the untreated, iRFA, and iRFA + M groups after 21 days of treatment. Reactome enrichment analysis of differentially expressed genes revealed several top enriched terms, including immune system, innate immune system, immunoregulatory interactions between lymphoid and non-lymphoid cells, adaptive immune system, cytokine signaling in the immune system, and neutrophil degranulation (Figs. 4A and B). The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation revealed a downregulation of 87 genes, with 2 genes specifically downregulated in the second category of the immune system when comparing iRFA and untreated groups (Fig. 4C). Notably, the number of downregulated genes decreased to 38 when comparing the iRFA and Irfa + M groups (Fig. 4D). A heatmap of immune-activated function-related genes demonstrated that iRFA contributed to the repression of these genes. However, this inhibitory effect was reversed by melatonin administration (Fig. 4E).Fig. 4 Bioinformatics analysis and hypoxia degree analysis of tumor tissue in different groups of mice. (A, B) Pathway enrichment analysis was performed based on the Reactome Pathway Database in comparing insufficient radiofrequency ablation (iRFA) group with untreated group (A) or iRFA + melatonin (M) group (B). (C, D) Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation of the transcriptome in comparing iRFA group with untreated group (C) or iRFA + M group (D). (E) Heatmap of immune-activated function related genes in three groups. (F) The MCPconter method estimated the abundance of immune and stromal cell populations was estimated from gene expression data. (G) Representative immunofluorescence for hypoxyprobe-1 (green), CD8 (red), and 4′,6-diamidino-2-phenylindole (DAPI) counterstaining in Hepa1–6 hepatocellular carcinoma (HCC) tissues. Quantification analysis of hypoxyprobe+ areas and CD8+ areas (lower right). Data are presented as mean ± standard deviation (SD). The P values are calculated using two-tailed unpaired t-test. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. ns: no significance. FCGR: Fc gamma receptor; TCR: T-cell receptor; ROS: reactive oxygen species; RNS: reactive nitrogen species; BCR: B-cell receptor; FCERI: Fc epsilon receptor I; MAPK: mitogen-activated protein kinase; iRFA + P: iRFA + anti-programmed death-ligand 1 (anti-PD-L1) antibody (P).

Fig. 4

The abundance of immune and stromal cell populations was estimated from gene expression data using the MCPcounter method. Results indicated that melatonin recovered the downregulation of T cells, CD8+ T cells, and B cells and iRFA-induced upregulation of monocytes, mast cells, neutrophils, and endothelial cells (Fig. 4F). Additionally, other two immune infiltration algorithms (EPIC and CIBERSORT-ABS) yielded similar results (Fig. S4).

These findings suggest that iRFA elicits a complex immune response in residual tumors that ultimately promotes immunosuppression in the tumor microenvironment (TME) and melatonin ultimately ameliorates this immunosuppressive microenvironment.

Hypoxyprobe-1 was used to further investigate the effects of iRFA and melatonin on the residual TME. We observed that residual tumors in the iRFA group exhibited the most pronounced degree of hypoxia, whereas that the iRFA and iRFA + M + P groups exhibited significantly improved degrees of hypoxia (Fig. 4G). Interestingly, CD8 expression was primarily observed in areas with mild hypoxia. Furthermore, immunohistochemical analysis confirmed significantly higher expression of HIF-1α in the iRFA group than in other groups. The positive rate of CD8+ T cells in the iRFA + M group was higher than that in the iRFA group, and it was further increased in the iRFA + M + P group (Fig. S5).

3.5 Myeloid cells increase in residual tumor and spleen

To further investigate changes in the immune microenvironment, we initially analyzed the infiltrating immune cells in residual Hepa1–6 tumors in each group using multicolor flow cytometry. The t-Stochastic neighbor embedding (t-SNE) analysis included several immune cell subsets (MDSCs, macrophages, CD8+ T cells, CD4+ T cells, and Tregs) (Fig. 5A). We observed that the proportion of CD11b+ myeloid cells increased significantly in the iRFA group than in the untreated group, and this increase was reversed by melatonin, anti-PD-L1 antibody, or their combination (Fig. 5B). Consistent with previous reports, iRFA increased the percentage of MDSCs (CD11b+Gr-1+) in CD45+ cells [9], and this high level was maintained for 21 days following iRFA. However, melatonin, anti-PD-L1 antibody, or their combination significantly decreased the proportion of MDSCs (Fig. 5C). Notably, the five groups did not show significantly altered numbers of macrophages (CD11b+F4/80+) (Fig. S6A). Additionally, we observed that melatonin or the two-drug combination reversed the downregulation of CD8+ T cells in the iRFA group (Fig. 5D) and decreased the proportion of Tregs (Fig. 5E). We measured the immune cell composition in the spleens of the five groups and observed that iRFA treatment increased the percentage of CD11b+ myeloid cells in the spleen (Fig. S6B). This could be attributed to a significant increase in the number and proportion of MDSC (Fig. 5F), which was reduced by melatonin, anti-PD-L1 antibody, or their combination. In addition, no significant difference in the proportions of macrophages, CD8+ T cells, or Tregs was observed among the five groups (Figs. S6C−F). We also evaluated the expression levels of activated CD8+ T cell biomarkers, including IFN-γ and TNF-α. The ELISA results revealed that melatonin increased the levels of serum IFN-γ and TNF-α, with the two-drug combination exhibiting the highest level (Fig. 5G). Additionally, serum IL-12 and IL-2 levels, which mediate the activation of T cells, significantly increased after the addition of melatonin (Fig. 5H). Moreover, melatonin, anti-PD-L1 antibody, or their combination significantly reduced the levels of immunosuppressive cytokines (IL-4 and IL-10) (Fig. 5I).Fig. 5 Flow cytometry analysis of tumor and spleen tissue and serological analysis of mice. (A) Multi-color flow cytometry data from cells sorted from untreated tumors and residual tumors treated with melatonin, anti-programmed death-ligand 1 antibody (anti-PD-L1), or two-drug combination. Tumors in the insufficient radiofrequency ablation (iRFA) group served as the control. (B) Flow cytometry analysis and quantification of myeloid cells (CD45+CD11b+) from tumors of five groups (n = 4). (C) Flow cytometry analysis and quantification of myeloid-derived suppressor cells (MDSCs) (CD11b+Gr-1+) from tumors of five groups (n = 4). (D) Flow cytometry analysis and quantification of CD8+ T cells from tumors of five groups (n = 4). (E) Flow cytometry analysis and quantification of regulatory T cells (Tregs) (CD4+ Foxp3+) from tumors of five groups (n = 4). (F) Representative histograms of myeloid cells (CD45+CD11b+) from spleens of five groups. (G–I) Concentration of serum tumor necrosis factor (TNF)-α and interferon-gamma (IFN-γ) (G), interleukin (IL)-2 and IL-12 (H), as well as IL-4 and IL-10 (I) were determined by enzyme-linked immunosorbent assay (ELISA) (n = 4). Data are presented as mean ± standard deviation (SD). The P values are calculated using two-tailed unpaired t-test. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. ns: no significance. iRFA + M: iRFA + melatonin (M); iRFA + P: iRFA + anti-PD-L1 antibody (P); t-SNE: t-Stochastic neighbor embedding.

Fig. 5

3.6 Melatonin combined with anti-PD-L1 antibody can significantly inhibit liver metastasis

The orthotopic Hepa1–6 HCC model demonstrated that the expression levels of MMP-2 and MMP-9 in the iRFA group were significantly higher than those in the other groups. However, the expression of MMP-2 and MMP-9 decreased after treatment with melatonin or anti-PD-L1 antibody (Fig. S7). To further study the effects of melatonin combined with an anti-PD-L1 antibody on tumor metastasis, CT26 cells, a murine colon cancer cell line sensitive to checkpoint blockade [25], were injected through the mouse spleen (Fig. 6A).Fig. 6 Melatonin combined with anti-programmed death-ligand 1 antibody (anti-PD-L1) can significantly inhibit liver metastasis of colon cancer. (A) Diagram for the experimental design. Intrasplenic injection CT26 cells was performed to establish liver metastasis model and observe the efficacy of different treatments on inhibiting tumor metastasis. Group 1: untreated; group 2: insufficient radiofrequency ablation (iRFA); group 3: iRFA + melatonin (iRFA + M); group 4: iRFA + anti-PD-L1 antibody (iRFA + P); group 5: iRFA + melatonin + anti-PD-L1 antibody (iRFA + M + P). (B) The tumor metastasis in the liver was detected by small animal live imaging. The mice were sacrificed at the end of the treatments. (C) Liver/body weight ratio was recorded. (D) Representative gross images and hematoxylin and eosin (H&E) staining of tumors in different groups. The yellow arrows indicate hepatic metastatic nodules. (E) Kaplan-Meier survival curves for mice in different groups. (F) Flow cytometry analysis and quantification of CD8+ T cells from tumors of five groups (n = 4). The P values are calculated using two-tailed unpaired t-test or log-rank test. ∗P < 0.05, ∗∗P < 0.01, and ∗∗∗P < 0.001. ns: no significance.

Fig. 6

Compared to iRFA, melatonin and anti-PD-L1 antibodies significantly reduced metastatic liver tumors, as demonstrated by bioluminescence imaging, liver weight loss, number of visible tumors, and histological micrometastases in the liver (Figs. 6B−D). In addition, an independent mouse cohort that underwent the same tumor inoculation and treatment was monitored to evaluate the effect of melatonin combined with an anti-PD-L1 antibody on the survival of tumor-bearing mice. The results indicated that the combination treatment markedly prolonged the survival of mice (Fig. 6E). Finally, we validated the changes in the proportion of T cells in the CRLM after different treatments using flow cytometry (Fig. 6F). The combination therapy group exhibited a significant increase in the infiltration of CD8+ T cells.

4 Discussion

Identifying optimal combinatorial strategies for anti-PD-1/PD-L1-based immunotherapy is a hot topic in the research on solid tumors, such as HCC, after ablation [13,26]. The present study demonstrated that combining melatonin with an anti-PD-L1 antibody enhances the efficacy of RFA in tumors, offering a promising strategy to extend the benefits of RFA therapy to a larger population of patients with HCC. Moreover, melatonin could improve hypoxia and immunosuppressive microenvironment, ultimately enhancing the efficacy of anti-PD-L1 antibody in the treatment of residual tumors after iRFA.

This retrospective analysis revealed that iRFA significantly shortened the median OS and PFS of patients with HCC, with local residual tumors identified as an independent prognostic factor affecting OS. We established an iRFA model and used RNA-seq to detect the gene expression profiles of tumor tissues after iRFA and compared the iRFA group with the untreated and iRFA + M groups. Consistent with the findings of Shi et al. [9], the present study demonstrated significant expression of genes in the immune process after iRFA. Therefore, elucidating changes in the tumor immune microenvironment after iRFA plays a vital role in treating residual tumors. Herein, we focused on changes in TME. Unraveling this puzzle may lead to the discovery of new biological biomarkers that will facilitate better treatment selection.

Hypoxia, a characteristic of tumor tissue, is considered as a barrier to antitumor therapy [[27], [28], [29], [30]]. It is further aggravated in the residual tumor tissue after iRFA, as confirmed in the present study. Wu et al. [31] reported that the hypoxic microenvironment in HCC induces tumor-associated macrophages to overexpress triggering receptor expressed on myeloid cells-1 (TREM-1), thereby promoting the apoptosis of CD8+ T cells. The present study indicated that residual tumors in the iRFA group exhibited increased hypoxia than that of the other groups, accompanied by a significant increase in MDSCs. Furthermore, iRFA could activate the EMT and cause the morphological transformation of residual HCC cells [32,33]. Tong et al. [10] revealed that either improving the hypoxic microenvironment or silencing HIF-1α signaling reduces the invasiveness and metastasis of residual tumor cells and reverses EMT to varying degrees. EMT has long been considered as an essential process in the progression from primary tumors to distant metastases [34]. Hence, a hypoxic microenvironment after iRFA can accelerate the progression of residual tumors and distant metastasis. The retrospective analysis results of this study underscore the significant influence of iRFA on the survival and prognosis of patients with HCC.

Melatonin, an endogenously produced molecule, plays a vital role in the circadian rhythm and cellular redox status [[35], [36], [37]]. Recent reports have indicated its inhibitory effect on the growth of certain cancers [[38], [39], [40]]. In addition, Li et al. [41] proposed that melatonin could enhance RFA-stimulated natural killer (NK) cell activity and exhibit a synergistic antitumor effect with RFA. However, the effect of melatonin on hypoxia in residual tumors after iRFA has not been reported. The present study demonstrated that melatonin could markedly decrease the proliferation, invasion, metastasis, and HIF-1α expression of sublethal heat-stressed cells under hypoxia. To determine the extent to which melatonin improved hypoxia in residual tumor tissues, mice were injected with pimonidazole prior to euthanasia, which is the gold standard for assessing hypoxia [42]. The results indicated that melatonin can significantly improve hypoxia in residual tumors after iRFA.

Although anti-PD-1/PD-L1 based immunotherapy has shown remarkable and durable clinical responses in certain patients with advanced HCC, many patients fail to benefit from monotherapy [43]. Recent studies have shown that anti-PD-1/PD-L1 antibody resistance can be attributed to poor production of immunogenic neoantigens, ineffective antigen processing, severe deficiency of infiltrating lymphocytes, and enrichment of immunosuppressive components [[44], [45], [46]]. Accordingly, enhancing or blocking these mechanisms using combination strategies may enhance the efficacy of anti-PD-L1 antibodies in the treatment of HCC. The current study demonstrated that melatonin improved the hypoxic immunosuppressive microenvironment, promoted intratumoral CD8+ T lymphocyte infiltration, and reduced MDSCs, which are key mediators in improving the treatment of residual tumors with anti-PD-L1 antibodies. Furthermore, we established liver and lung metastasis models and evaluated the inhibitory effects of melatonin in combination with an anti-PD-L1 antibody on tumor metastasis. Our study strongly supports the possibility that melatonin enhances the efficacy of anti-PD-L1 antibodies in treating residual tumors after iRFA and effectively inhibits liver and lung metastasis.

5 Conclusion

In conclusion, the present findings establish a clear role for melatonin in improving the hypoxic immunosuppressive microenvironment of tumors, reducing tumor progression, and enhancing the efficacy of anti-PD-L1 antibodies in treating residual tumors. Elucidating this potential mechanism further will provide a solid scientific basis for targeting the hypoxic microenvironment to prevent HCC recurrence and metastasis in clinical RFA practice.

CRediT author statement

Yanqiao Ren: Conceptualization, Methodology, Software, Data curation, Writing - Original draft preparation; Licheng Zhu: Conceptualization, Methodology, Software; Yusheng Guo: Data curation, Writing - Original draft preparation; Jinqiang Ma: Visualization, Investigation; Lian Yang: Supervision; Chuansheng Zheng: Software, Validation; Xiangjun Dong: Writing - Reviewing and Editing.

Declaration of competing interest

The authors declare that there are no conflicts of interests.

Appendix A Supplementary data

The following is the Supplementary data to this article.Multimedia component 1

Multimedia component 1

Acknowledgments

We thank Mr. Meng Xiong from Magnity Electronics Co., Ltd. (Shanghai, China) for providing infrared imaging system. This work was supported by grant from the 10.13039/501100001809 National Nature Science Foundation of China (Grant Nos.: 82102168 and 81873919 ).

Appendix A Supplementary data to this article can be found online at https://doi.org/10.1016/j.jpha.2024.01.010.
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