Lidocaine Modulates Cytokine Production and Reprograms the Tumor Immune Microenvironment to Enhance Anti-Tumor Immune Responses in Gastric Cancer

Abstract

Lidocaine, a local anesthetic, has been shown to modulate immune responses. This study examines its effects on cytokine production in peripheral blood mononuclear cells (PBMCs) from healthy donors and tumor-infiltrating immune cells (TIICs) from gastric cancer patients. PBMCs from healthy donors and TIICs from gastric cancer patients were treated with lidocaine. Cytokine production was assessed using flow cytometry and cytokine assays, with a focus on IFN-γ, IL-12, IL-10, TGF-β, and IL-35 levels. Cytotoxicity against primary gastric cancer cells (PGCCs) was also evaluated. Lidocaine inhibited IFN-γ production in CD8⁺ PBMCs and IL-12 in CD14⁺ PBMCs while increasing anti-inflammatory cytokines (IL-10, TGF-β, IL-35) in CD4⁺CD25⁺ and CD14⁺ cells. In TIICs, lidocaine enhanced IFN-γ and IL-12 production in CD8⁺ and CD14⁺ cells while reducing IL-10, TGF-β, and IL-35 levels, promoting an M1-like phenotype in macrophages. Mechanistically, lidocaine enhanced IFN-γ production in sorted CD8⁺ TIICs through G-protein-coupled receptor (GPCR) signaling and increased IL-12 production in sorted CD14⁺ TIICs via the toll-like receptor 4 (TLR4) signaling pathway. Lidocaine also increased IFN-γ production and cytotoxicity in CD8⁺ TIICs via NF-κB activation. Importantly, lidocaine did not affect the viability of PBMCs, TIICs, or PGCCs at concentrations up to 1.5 mM. Lidocaine reprogrammed the tumor immune microenvironment, enhancing anti-tumor immune responses, suggesting its potential to modulate immune functions in gastric cancer.

Keywords: IFN-γ production; M1 macrophages; NF-κB activation; cytokine modulation; gastric cancer; gastrointestinal disease; immune response; inflammation; lidocaine; tumor-infiltrating immune cells.

Figure 1

Lidocaine reduced the secretion levels of IFN-γ by CD8⁺ PBMCs and IL-12 by CD14⁺ PBMCs and the effect of lidocaine on IFN-γ secretion by CD8⁺ TIICs and IL-12 secretion by CD14⁺ TIICs. The effects of lidocaine (from 0.25 mM to 1.5 mM) on PMA and PHA-stimulated IFN-γ secretion from CD8⁺ PBMCs (A) were investigated. The IFN-γ level in the supernatant was determined at 72 h by ELISA. Additionally, CD14⁺ PBMCs (B) were stimulated by PMA and PHA, then cultured in the absence or presence of graded concentrations of lidocaine. The IL-12 level in the supernatant was also determined at 72 h by ELISA. Sorted normal PBMCs (1 × 10⁵ cells/well) were cultured with lidocaine. Because PBMCs were from normal peripheral blood, there was no expression of IFN-γ and IL-12 spontaneity before pretreatment with PMA and PHA. NC: negative control (non-stimulated cells). The effects of lidocaine on IFN-γ secretion from CD8⁺ TIICs (1 × 10⁵ cells/well). (C) and IL-12 secretion from CD14⁺ TIICs (1 × 10⁵ cells/well) (D) were investigated. CD8⁺ and CD14⁺ TIICs were stimulated in the absence or presence of graded concentrations of lidocaine (from 0.25 mM to 1.5 mM). The levels of IFN-γ and IL-12 in the supernatant were determined at 72 h by ELISA. Cell viability was >95%, as assessed by trypan blue exclusion. Data are from distinct samples and are presented as the mean± SEM from three different experiments, each performed in duplicate. * p < 0.05, ** p < 0.01.

Figure 2

Lidocaine induced the secretion of IL-10, TGF-β, and IL-35 by CD4⁺CD25⁺ and CD14⁺ PBMCs and inhibited the secretion of IL-10, TGF-β, and IL-35 by CD4⁺CD25⁺ and CD14⁺ TIICs. The effect of lidocaine on the secretion of IL-10 (A,B), TGF-β (C,D), and IL-35 (E,F) from CD4⁺CD25⁺ and CD14⁺ PBMCs and tumor-infiltrating immune cells (TIICs) was investigated. CD4⁺CD25⁺ and CD14⁺ PBMCs or TIICs were cultured in the absence or presence of graded concentrations of lidocaine (from 0.25 mM to 1.5 mM). The levels of IL-10, TGF-β, and IL-35 in the supernatant were determined at 72 h by ELISA. Cell viability was >95%, as assessed by trypan blue exclusion. Data are presented as the mean ± SEM from three independent experiments, each performed in duplicate, using distinct samples (PBMCs) or three different donors (TIICs). * p < 0.05, ** p < 0.01. The values marked as 0 without lidocaine represent the basal levels of IL-10, TGF-β, and IL-35 in CD4⁺CD25⁺PBMCs and CD14⁺PBMCs.

Figure 3

Lidocaine may modulate macrophage polarization, promoting a pro-inflammatory and potentially anti-tumor immune environment. Panels (A,B) illustrate the expression profiles of CD14⁺ tumor-infiltrating immune cells (TIICs). (A) The expression of CD163, a marker for M2 macrophages, is shown prior to treatment (Mock). (B) After a 3-day treatment with lidocaine, CD14⁺ TIICs exhibited increased expression of CD40, a hallmark marker for M1 macrophage polarization. Cell viability was >95%, as assessed by trypan blue exclusion. Results obtained from three different donors are shown. Data are from distinct samples and are presented as the mean ± SEM., ** p < 0.01.

Figure 4

Schematic diagram showing lidocaine-modulated immunosuppression of normal PBMCs and anti-tumor effect of gastric TIICs. This diagram illustrates the dual effects of lidocaine on immune cells: 1. PBMCs: Demonstrates how lidocaine modulates immunosuppression in normal PBMCs, highlighting changes in cytokine secretion. 2. TIICs: Illustrates the anti-tumor effect of lidocaine on gastric TIICs, focusing on its impact on cytokine secretion.

Figure 5

TLR4 inhibition reduced IL-12 levels in lidocaine-treated CD14⁺ TIICs, while GPCR inhibition decreased the levels of IFN-γ in lidocaine-treated CD8⁺ TIICs. (A) TLR4 inhibition was achieved using 0.5 μg/mL resatorvid (MedChemExpress, Monmouth Junction, NJ, USA). CD14⁺ TIICs were incubated with or without 0.5 μg/mL resatorvid for 24 h, followed by culture in the presence of 1.5 mM lidocaine. (B) GPCR inhibition was performed using 500 nM paroxetine (MCE). CD8⁺ TIICs were pre-incubated with paroxetine for 45 min before stimulation with 1.5 mM lidocaine for 72 h. Cytokine levels of IL-12 and IFN-γ in the supernatant were measured at 72 h by ELISA. Cell viability remained > 95%, as determined by trypan blue exclusion. Data are presented as the mean ± SEM from three independent experiments, each performed in duplicate, using TIICs from three different donors. * p < 0.05, ** p < 0.01. Paroxetine (MedChemExpress) is a direct GPCR inhibitor; resatorvid (MedChemExpress) is a TLR4 inhibitor [28].

Figure 6

Lidocaine inhibited IL-35, IL-10, and TGF-β production by CD4⁺CD25⁺Foxp3⁺ and CD4⁺CD25⁺CD127⁻ TIICs. Sorted CD4⁺CD25⁺TIICs were treated with 1.5 mM lidocaine for 72 h followed by staining with anti-IL-35, anti-IL-10, anti-TGF-β, and anti-Foxp3 antibodies for flow cytometry analysis. Isotype controls were used to distinguish between positive and negative cells for IL-35, IL-10, TGF-β, and Foxp3. Typical flow cytometry dot plot analysis revealed the percentage of (A,B) CD35⁺Foxp3⁺CD4⁺CD25⁺ TIICs, (C,D) IL-10⁺Foxp3⁺CD4⁺CD25⁺ TIICs, and (E,F) TGF-β⁺Foxp3⁺CD4⁺CD25⁺ TIICs treated with lidocaine (1.5 mM). (G,H) The expression levels of CD127 in lidocaine-treated IL-35⁺CD4⁺CD25⁺, IL-10⁺CD4⁺CD25⁺, and TGF-β⁺CD4⁺CD25⁺ cells were analyzed using flow cytometry. The expression levels are shown on histograms. Isotype controls were used to distinguish between positive and negative cells for CD127. Cell viability was >95%, as assessed by trypan blue. RMFI: relative mean fluorescence intensity. Data are from distinct samples and presented as the mean± SEM in three different experiments, each performed in duplicate. * p < 0.05, ** p < 0.01.

Figure 7

Lidocaine enhanced anti-tumor immunity by reducing PD-1 and increasing IFN-γ expression on CD8⁺ TIICs through the NF-κb signaling pathway. Gastric CD8⁺ TIICs treated with lidocaine (1.5 mM) were analyzed by flow cytometry. CD8⁺ TIICs were stimulated with ionomycin and PMA to enhance IFN-γ production as a positive control. Single-cell suspensions obtained from sorted CD8⁺ TIICs were stained to detect IFN-γ (A,B) and PD-1 (C–E). We gated on CD8⁺ TIICs, as described in our previously published paper [24]. Analysis of IFN-γ production and PD-1 expression by lidocaine-treated CD8⁺TIICs with the NF-κB inhibitor. CD8⁺TIICs were incubated for 1 h with or without BAY11-7082 (10 μM) and then treated with lidocaine for 72 h. IFN-γ and PD-1 was measured by flow cytometry. All flow cytometry analyses were gated on total live cells. Cell viability was >95%, as assessed by trypan blue exclusion. Data are from distinct samples and presented as the mean ± SEM. * p < 0.05, ** p < 0.01; n ≥ 3. MFI: mean fluorescence intensity.

Figure 8

Apoptotic effects of lidocaine on TIICs, PBMCs, PGCCs, and CD8⁺TIICs co-cultured with PGCCs assessed by flow cytometry. Concentrations of 5 mM and 10 mM lidocaine induced apoptosis of TIICs and PBMCs, but concentrations of 0.5 and 1.5 mM lidocaine did not induce PGCCs apoptosis. TIICs (A), PBMCs (C), and PGCCs (E) were assessed by flow cytometric analysis using propidium iodide-stained cells. Firstly, 10⁴ cells were incubated in 96-well plates in the presence or absence of the indicated concentrations of lidocaine. After 72 h treatment, cells were washed with PBS and fixed with 70% ethanol for 1 h on ice. Pelleted cells were incubated with RNaseA (0.1 mg/mL) and propidium iodide (40 μg/mL) for 1 h with shaking and protected from light. The percentage of subG1 population was determined by flow cytometry. (G) Flow cytometry assessment of cell death of lidocaine (1.5 m M)-treated CD8⁺TIICs, -treated PGCCs, or -treated CD8⁺TIICs were co-cultured with PGGCs. We gated on CD8⁺ TIICs and GRN⁺ PGGCs, as described in our previously published paper [24]. (I) CD8⁺TIICs-, PGCCs-, and CD8⁺TIICs-treated with lidocaine at a concentration of 1.5 mM were co-cultured with PGGCs at the 5:1 E:T ratios. Target cell cytotoxicity was determined at 2 h by a DELFIA EuTDA assay. MFI: mean fluorescence intensity. Data are representative of three independent experiments; n >= 3. (B,D,F,H,I) Data are from distinct samples and presented as the mean ± SEM in three different experiments, each performed in duplicate. ** p < 0.01.

Figure 9

Schematic diagram illustrating the lidocaine-mediated anti-tumoral mechanism through immunogenic cell death targeting PGCCs. Lidocaine inhibited the production of IL-35, IL-10, and TGF-β by CD4⁺CD25⁺Foxp3⁺ tumor-infiltrating immune cells (TIICs). Additionally, lidocaine enhanced anti-tumor immunity by reducing PD-1 expression and increasing IFN-γ expression on CD8⁺ TIICs via the NF-κB signaling pathway. The lidocaine-treated CD8⁺ TIICs subsequently promoted the immunogenic cell death of primary gastric cancer cells (PGCCs). Long→: treated. Short→: linked. ┬: inhibition.