Midazolam exhibits antitumour and enhances the efficiency of Anti-PD-1 immunotherapy in hepatocellular carcinoma

Abstract

Background: Midazolam (MDZ) is an anaesthetic that is widely used for anxiolysis and sedation. More recently, MDZ has also been described to be related to the outcome of various types of carcinomas. However, how MDZ influences the progression of hepatocellular carcinoma (HCC) and its effects on the biological function and tumour immune microenvironment of this type of tumour remain unknown.

Methods: The effects of MDZ on the proliferation, invasion, and migration of HCC cell lines were examined in vitro using the Cell Counting Kit 8 (CCK8), 5-ethynyl-2'-deoxyuridine (EdU), Transwell, and wound healing assays. Additionally, western blotting was employed to confirm that PD-L1 was expressed. Chromatin immunoprecipitation-seq (ChIP-seq) analysis was used to pinpoint the transcriptional regulation regions of NF-κB and programmed death-ligand 1 (PD-L1). A C57BL/6 mouse model was used to produce subcutaneous HCC tumors in order to evaluate the in vivo performance of MDZ. Mass spectrometry was also used to assess changes in the tumour immunological microenvironment following MDZ injection.

Results: The HCC-LM3 and Hep-3B cell lines' proliferation, invasion, and migration were controlled by MDZ, according to the results of the CCK8, EdU, Transwell, and wound healing assays. PD-L1 expression was shown by ChIP-seq analysis to be boosted by NF-κB, and by Western blotting analysis, it was shown that MDZ downregulated the expression of NF-κB. Additionally, in vivo tests revealed that intraperitoneal MDZ injections reduced HCC tumor development and enhanced the effectiveness of anti-PD-1 therapy. The CD45⁺ immune cell proportions were higher in the MDZ group than in the PBS group, according to the mass spectrometry results. Injection of MDZ resulted in a decrease in the proportions of CD4⁺ T cells, CD8⁺ T cells, natural killer (NK) cells, monocytes, Tregs, and M2 macrophages and a rise in the proportion of dendritic cells. Additionally, the concentrations of the cytokines IFN-g and TNF-a were noticeably raised whereas the concentrations of the CD8⁺ T-cell fatigue markers ICOS, TIGIT, and TIM3 were noticeably lowered.

Conclusion: According to this study, MDZ inhibited the progression of HCC by inhibiting the NF-κB pathway and reducing the exhaustion of CD8⁺ T cells. In clinical practice, MDZ combined with anti-PD-1 therapy might contribute to synergistically improving the antitumor efficacy of HCC treatment.

Keywords: Hepatocellular carcinoma; Immune escape; MDZ; PD-1; PD-L1.

Fig. 1

MDZ inhibited proliferation and invasion in HCC cell lines. A, B The cell viability histograms of the HCC-LM3 and Hep-3B cell lines were drawn after treatment with midazolam at increasing concentrations (0, 50, 75, 100, 150, or 200 µM) based on CCK-8 assays. C, D EdU assays were performed to assess the proliferation of HCC-LM3 and Hep-3B cells cultured with 75 and 150 µM MDZ for 24 h. Then, statistical analysis was performed on the EdU( +) proliferating cells. Cells were treated with stroke-physiological saline solution (SPSS) as the control. E, F Transwell experiments were used to assess the invasion of HCC cells incubated with MDZ. The left panel shows HCC-LM3 and Hep-3B cell invasion after MDZ treatment. The right panel shows the counts of invading cells. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 2

MDZ inhibited migration in HCC cell lines. A–D Wound healing assays were used to assess the migration of HCC cells treated with MDZ for 24 h and 48 h. The left panel shows HCC-LM3 and Hep-3B cell migration after MDZ treatment. The right panel shows the cell migration rate. **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 3

MDZ downregulated PD-L1 expression in HCC cells through the NF-κB pathway. (A) The protein expression levels of PD-L1, P65, and p-P65 in HCC cells after MDZ treatment. B, C The panels show the protein band grey values. ***P < 0.001, ****P < 0.0001

Fig. 4

MDZ reduced tumour growth and increased the efficiency of the anti-PD-1 monoclonal antibody in a xenograft mouse model. A Procedures for establishing a subcutaneous tumour-bearing mouse model. PD-1 antibody was injected intraperitoneally twice a week 7 days after MDZ injection. Mice injected with PBS were the control. B Images of the subcutaneous tumours in the different groups (PBS, MDZ, α-PD-1, and α-PD-1 + MDZ). C, D The weights (C) and volumes (D) of the subcutaneous tumours in the indicated groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001

Fig. 5

Immunohistochemistry showed that MDZ affected tumour growth and enhanced anti-PD-1 monoclonal antibody treatment efficacy in a xenograft mouse model. A The morphology of subcutaneous tumours in the four groups was confirmed by HE staining, as shown in the upper panel. Moreover, the upper panel indicates the results immunohistochemical staining for Ki67, TUNEL, PD-L1, CD3, and CD8 expression in the indicated groups. B The lower panel illustrates the statistical analysis of the levels of these indicators in the four groups. *P < 0.05, **P < 0.01

Fig. 6

Mass cytometry was used to analyse the tumour immune microenvironment of subcutaneous HCC tumours after PBS/MDZ treatment. A The process of selecting CD45⁺ immune cells. B The proportions of CD45⁺ immune cells in different groups. C A total of 37 cell clusters were identified, and we defined the respective groups based on marker expression. D TSNE plot showing the distributions of 37 cell clusters. E The distribution of cell clusters in the indicated sample. F Histogram showing the number of the cell clusters in different groups by mass cytometry

Fig. 7

Mass cytometry revealed changes in marker expression after PBS/MDZ treatment. A–G TSNE plot showing the distribution of PD1, PD-L1, TIGIT, TIM3, ICOS, IFN-g and TNF-α expression in subcutaneous Hepa1-6 tumours in the PBS and MDZ groups. H The histogram shows the number of PD1⁺, CD8⁺PD1⁺, Treg⁺ PD1⁺, PD-L1⁺, MDSC⁺ PD-L1⁺, TIGIT, TIM3, ICOS, IFN-g⁺, and TNF-α⁺ cell clusters in the PBS and MDZ groups

Fig. 8

Schematic diagram of the changes in the tumour immune microenvironment of subcutaneous tumour-bearing mouse models after MDZ treatment. MDZ inhibited subcutaneous HCC tumour growth and enhanced anti-PD-1 monoclonal antibody immunity in HCC