Research ArticleExperimental Studies
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Combination of 3-(Trihydroxygermyl)propanoic Acid (THGP) and Lipopolysaccharide Promotes Macrophage Differentiation to M1, and Antitumor Activity

JUNYA AZUMI, TOMOYA TAKEDA, YASUHIRO SHIMADA, HISASHI ASO, HIROYUKI INAGAWA and TAKASHI NAKAMURA
Anticancer Research August 2025, 45 (8) 3425-3440; DOI: https://doi.org/10.21873/anticanres.17704

Abstract

Background/Aim: Macrophage polarization plays a critical role in cancer immunotherapy. This study aimed to evaluate the synergistic effects of 3-(trihydroxygermyl)propanoic acid (THGP), an organogermanium compound, and the immunostimulant lipopolysaccharide (LPS) derived from Pantoea agglomerans on M1 macrophage differentiation and antitumor activity.

Materials and Methods: RAW 264.7 cells were treated with THGP, LPS, or their combination for 1, 4, or 10 days. Morphological changes, M1 marker (CD80 and CD86) expression, cytokine production (IL-1β and IL-6), phagocytic activity, and cytotoxicity against B16-F10 melanoma cells were assessed using microscopy, qPCR, western blotting, immunofluorescence staining, and luciferase assays.

Results: After one day of treatment, LPS treatment, both alone or in combination with THGP, increased M1 marker expression. By day 4, both agents individually induced M1 differentiation; their combination had a synergistic effect on cytokine production and phagocytic activity. Antitumor effects were observed only with the combined treatment. After 10 days, single and combined treatments resulted in comparable phagocytic and antitumor activities.

Conclusion: The combination of THGP and LPS synergistically promotes M1 macrophage differentiation and enhances phagocytic and antitumor activities through distinct mechanisms. These findings suggest potential applications of this combination in cancer immunotherapy.

Keywords:

Introduction

Macrophages are crucial cells in the immune system that differ in response to environmental cues and perform diverse functions (1). These cells are primarily classified into M1 macrophages (proinflammatory) and M2 macrophages (anti-inflammatory) (2). M1 macrophages are induced by interferon-γ (IFN-γ) secreted from type 1 T helper (Th1) cells, cytotoxic T cells, and natural killer (NK) cells (3). These M1 macrophages possess high antigen-presenting and phagocytic abilities; secrete inflammatory cytokines such as tumor necrosis factor α (TNF-α), interleukin-1β (IL-1β), and IL-6; and contribute to the elimination of bacteria and viruses (4). In contrast, M2 macrophages are induced by IL-4 secreted from type 2 T helper (Th2) cells and regulatory T (Treg) cells. M2 macrophages secrete transforming growth factor-β (TGF-β) and IL-10, which participate in wound healing and immune tolerance (5). The M1/M2 macrophage ratio is typically maintained at homeostasis, and an imbalance can lead to various inflammatory diseases (6, 7).

Many well-known immunostimulatory substances are of bacterial or viral origin and are recognized via pathogen-associated molecular patterns (PAMPs) (8). These include lipopolysaccharides (LPSs), which are components present on the surface of bacterial cell membranes. Even in minute quantities, LPSs bind to Toll-like receptor 4 (TLR4), promoting the secretion of inflammatory cytokines such as IL-1β and IL-6 and triggering an inflammatory response (8, 9). However, the oral administration of LPS has been reported to protect against lethal paramyxovirus infection (10), increase phagocytic activity via TLR4, induce antitumor effects, and prevent diabetes (11). Consistent with these mechanisms of action of oral LPS administration, in vitro experiments have shown that macrophage stimulation at low concentrations can be observed. High concentrations of LPS induce the production of reactive oxygen species from microglia, causing inflammation in neuronal cells (12). However, repeated low-dose LPS stimulation increases the expression of neuroprotective factors such as neurotrophin 5 (Ntf5) and CC chemokine ligand 7 (Ccl7) in microglia (13). In a study using mouse macrophage-like RAW 264.7 cells, repeated stimulation with low-dose LPS (100 pg/ml to 1 ng/ml) was shown to enhance phagocytic activity against foreign substances (14).

Poly-trans-[(2-carboxyethyl)germasesquioxane] (Ge-132) is a polymeric organogermanium compound that undergoes hydrolysis in aqueous or physiological media to yield the monomer 3-(trihydroxygermyl)propanoic acid (THGP) (15, 16). Ge-132 has various effects, including antioxidant effects (17, 18), bone metabolism-promoting effects (19, 20), and analgesic effects (21, 22). Additionally, Ge-132 activates NK cells and promotes the secretion of IFN-γ, enhancing the antitumor effects of macrophages (23–25). Because Ge-132 is completely hydrolyzed to THGP under physiological conditions, the biological effects ascribed to Ge-132 are understood to be mediated by THGP. Recent research has shown that long-term culture of RAW 264.7 cells in the presence of THGP induces the differentiation of macrophages into M1 macrophages via NF-κB. In addition, macrophages treated with THGP have shown strong antitumor effects on melanoma cells (26).

Therefore, THGP and LPS induce macrophage activation through different mechanisms, intracellular or cell surface mechanisms. Macrophage activation can be synergistically enhanced by engaging different receptors rather than relying on a single immunostimulatory substance. These include, for example, LPS and CpG DNA (27), zymosan and polyIC (28), and others. On the basis of these findings, we hypothesized that THGP, when combined with other immunostimulatory substances, could achieve a stronger antitumor effect.

In this study, we examined the characteristic changes related to M1-type macrophage activation by coculturing RAW 264.7 cells with low concentrations of LPS and THGP. These two substances act synergistically without interfering with each other’s functions, demonstrating that this combination has promising immunostimulatory effects.

Materials and Methods

Preparation of THGP. Ge-132 (Lot. 020X22A3) was manufactured by Asai Germanium Research Institute. Ge-132 was dissolved in ultrapure water to a concentration of 500 mM THGP. The solution was then neutralized to pH 7.10 using NaOH (Wako Pure Chemical Industries Ltd., Osaka, Japan). The solution was sterilized by filtration through a 0.2 μm membrane filter (Advantec Co., Ltd., Tokyo, Japan) and stored at −20°C.

Preparation of LPS. Lipopolysaccharide (LPS) derived from Pantoea agglomerans was obtained from Macrophi Inc. (Kagawa, Japan). LPS was prepared at a concentration of 1 μg/ml in sterile water and stored at 4°C. Immediately before use, the solution was sonicated for 15 s via an NS-300 ultrasonic cleaner (Nihonseiki Kaisha LTD., Tokyo, Japan).

Cell culture. RAW 264.7 cells (Riken BRC, Ibaraki, Japan) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) (Nissui Pharmaceutical Co., Ltd., Tokyo, Japan) supplemented with 10% fetal bovine serum (FBS). For maintenance culture, the cells were passaged at a 1:5 to 1:10 ratio when they reached subconfluence. For the experiments, RAW 264.7 cells were seeded at 1×106 cells/100 mm dish and cultured in medium containing 500 μM THGP and/or 1 ng/ml LPS, with passaging every three days. B16-F10/CMV-LUC#2 cells (B16-F10 cells) (JCRB Cell Bank, Osaka, Japan), which stably express the luciferase gene, were cultured in DMEM supplemented with 10% FBS. These cells were passaged at a 1:4 to 1:8 ratio following trypsinization (Nacalai Tesque Inc., Kyoto, Japan) when they reached subconfluence.

Morphological observation. RAW 264.7 cells were seeded at 1×106 cells/100 mm dish. After 24 h of culture, images of the cells were captured using a BZ-X810 microscope (Keyence Co., Osaka, Japan). The lengths of the dendritic processes were measured using Fiji software. The cells showing an amoeboid shape with extended dendritic processes were defined as spindle cells, and their percentage relative to the total cell count was calculated.

Western blot analysis. RAW 264.7 cells were seeded at 1×106 cells/60 mm dish. After 24 h, proteins were extracted with RIPA buffer. Protein concentrations were measured using the Bradford method (Bio-Rad Laboratories Inc., Hercules, CA, USA). A total of 10 μg of protein from each sample was separated using polyacrylamide gel electrophoresis. The following primary antibodies were used: anti-B7-2 (CD86) antibody (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), anti-SIRP-α antibody (Abcam Ltd., Cambridge, UK), and anti-β-actin antibody (Abcam). The primary antibodies were diluted according to the manufacturer’s recommendations. Secondary antibody reactions were performed using corresponding secondary antibodies (Abcam). Five percent skim milk in TBS-T (Morinaga Milk Industry Co., Ltd., Tokyo, Japan) was used for blocking and antibody dilution. After the secondary antibody reaction, the membranes were imaged using a ChemiDocTM XRS Plus system (Bio-Rad) and analyzed using Image Lab software (Bio-Rad). β-actin was used as an internal standard for protein expression quantification.

Real-time RT–PCR. RAW 264.7 cells were seeded at 2×105 cells/well in a 6-well plate. After 24 h, RNA was extracted using ISOGEN II (Nippon Gene Co. Ltd., Tokyo, Japan) following the manufacturer’s protocol. One microgram of extracted RNA was used as a template for reverse transcription using SuperScript III (Invitrogen, Life Technologies Corp., Carlsbad, CA, USA) (50°C for 1 hour, 95°C for 5 min). PCR was performed using TB Green Premix Ex Taq II (Tli RNaseH Plus) (Takara Bio Inc., Otsu, Japan) (95°C for 5 s, 60°C for 30 s, 40 cycles). Rps18 was used as an internal standard for expression normalization. The sequences of the primers used are shown in Table I.

View this table:
Table I.

Primer list.

Immunofluorescence. RAW 264.7 cells were seeded at 5×104 cells/well in 8-well glass slides (Matsunami Glass Ind., Ltd., Osaka, Japan). After 24 h of culture, the cells were fixed with 4% paraformaldehyde in PBS (Wako Pure Chemical Industries Ltd.). The cell membranes were permeabilized with 0.2% Triton X-100 (Nacalai Tesque, Inc., Kyoto, Japan) for 10 min at room temperature, followed by blocking with 1.5% BSA in PBS-T for 30 min at room temperature. Primary antibody reactions were performed overnight at 4°C, and secondary antibody reactions were performed for 1 h at room temperature. The primary antibodies used were anti-CD86 (Santa Cruz) and anti-CD206 (Abcam), and the secondary antibodies used were a goat anti-mouse IgG H&L antibody (Texas Red, Abcam) and a goat anti-rabbit IgG H&L antibody (FITC) (Abcam). Nuclei were stained with DAPI (Dojindo Laboratories, Kumamoto, Japan). Observations were made using a fluorescence microscope (BZ-X810, Keyence Corporation). The primary and secondary antibodies were diluted to the recommended concentrations with blocking buffer. The captured images were analyzed using a BZ-X800 analyzer (Keyence). The cell populations with high CD86 expression and low CD206 expression were defined as M1 macrophages, and their percentage relative to the total cell count (DAPI-stained) was calculated.

Phagocytosis assay. RAW 264.7 cells were seeded at 5×104 cells/well on 8-well glass slides. After 24 h, the cells were washed once with PBS (−). The cells were subsequently incubated for 30 min with a phagocytosis assay kit (Cayman Chemical Company, Ann Arbor, MI, USA) (1:200) and Hoechst 33452 (1:100) (Dojindo Laboratories). After being washed twice with PBS (−), the cells were fixed with 4% paraformaldehyde in PBS (Wako) and mounted with ProLong™ Glass Antifade Mountant (Invitrogen). Fluorescence observations were made via a BZ-X810 microscope. Phagocytic activity was calculated by dividing the total fluorescence intensity of beads per field of view by the total cell count.

Evaluation of the cytotoxicity against B16-F10 cells. RAW 264.7 cells and B16-F10/CMV-LUC#2 cells were seeded simultaneously at 5.0×103 cells and 2.5×103 cells/96-well, respectively. After 48 h, the culture supernatant was removed, and 100 μl of ONE-Glo™ Luciferase Assay System (Promega Corp., Fitchburg, WI, USA) and 100 μl of DMEM were added to each 96-well plate. Luminescence intensity was measured via an ARVO X3 plate reader (PerkinElmer, Inc., Waltham, MA, USA).

Statistical analysis. Statistical analysis was performed using the Excel statistical software “Statcel 4” (OMS Publishing Co., Ltd., Tokyo, Japan). One-way analysis of variance (ANOVA) and Tukey–Kramer’s multiple comparison test were used for comparisons among three or more groups. p-Values less than 0.05 were considered statistically significant. All the data are presented as the means±standard deviations.

Results

RAW 264.7 cells exhibit morphological characteristics of M1 macrophages upon THGP and LPS stimulation. RAW 264.7 cells were cultured for 4 days in media containing either 500 μM THGP or 1 ng/ml LPS. The cells were passaged every 3 days and used for experiments on days 1 and 4 (Figure 1A). We first examined the morphological changes in RAW 264.7 cells stimulated with 500 μM THGP and 1 ng/ml LPS for 1 day (Figure 1B, D, and F) or 4 days (Figure 1C, E, and G). THGP stimulation alone did not affect the proportion of spindle-shaped cells but affected the length of elongated dendritic processes on both day 1 and day 4 (Figure 1D and E). LPS stimulation alone increased the proportion of spindle-shaped cells without affecting the length of the dendritic processes (Figure 1F and G). When THGP and LPS were combined, a mixture of morphological features, such as an amoeboid shape and extended dendritic processes was observed (Figure 1D-G). These results suggest that the combination of THGP and LPS may promote M1 macrophage differentiation more effectively than either stimulus alone.

Figure 1.
Figure 1.

Morphological changes induced by THGP and LPS stimulation. (A) Schematic illustration of the experimental design. (B) Representative bright-field images of cells treated with THGP and LPS, alone or in combination, for one day. (C) Representative bright-field images of cells treated with THGP and LPS, alone or in combination, for four days. (D, E) Quantification of dendritic process length after one day (D) and four days (E) of treatment. (F, G) Quantification of spindle cells after one day (f) and four days (g) of treatment. Scale bar: 20 μm. N=6, **p<0.01, *p<0.05.

RAW 264.7 cells differentiate into M1 macrophages upon THGP and LPS stimulation. To investigate the polarization of macrophages in response to THGP and LPS stimulation, we examined the gene expression of the M1 macrophage markers CD80 (Figure 2A) and CD86 (Figure 2B). On day 1, LPS alone affected the expression of the M1 marker CD80. However, the combination of THGP and LPS significantly increased the expression of both CD80 and CD86. On day 4, both THGP and LPS alone significantly increased the expression of CD80 and CD86. Notably, on day 4, the expression levels of both markers were greater than those in the single treatment groups, suggesting a synergistic effect. Additionally, the expression of CD80 and CD86 on day 4 was increased 4-5-fold compared with that on day 1 following stimulation with THGP and LPS. Next, we examined CD86 protein expression. On day 4, THGP alone, LPS alone, or their combination increased CD86 expression (Figure 2C). However, no synergistic effect was observed with the combination treatment. These results suggest the possible involvement of a time lag between gene and protein expression or posttranslational modifications. Furthermore, we performed immunofluorescence staining for the M1 macrophage marker CD86 (red) and the M2 macrophage marker CD206 (green) in RAW 264.7 cells cultured with THGP and LPS for 4 days. In the control group, the majority of cells expressed both red and green fluorescence. However, treatment with THGP or LPS alone or in combination increased the proportion of cells showing red fluorescence (Figure 2D). When M1 macrophages were defined as CD86high CD206low cells, the proportion of M1 macrophages in the control group was 19%. THGP treatment alone doubled this proportion to 38% (Figure 2E). Compared with the control, LPS treatment alone or in combination with THGP increased the proportion of M1 macrophages to approximately 70%, a 3.7-fold increase. These results demonstrated that 4 days of stimulation with THGP and LPS induced the differentiation of RAW 264.7 cells into M1 macrophages.

Figure 2.
Figure 2.

M1 macrophage differentiation induced by THGP and LPS stimulation. (A, B) qPCR analysis of CD80 (A) and CD86 (B) expression after one or four days of stimulation with THGP and LPS, alone or in combination. (C) Western blot analysis of CD86 expression after four days of stimulation with THGP and LPS, alone or in combination. (D) Representative immunofluorescence images after four days of stimulation with THGP and LPS, alone or in combination. Red: CD86; green: CD206; blue: DAPI. (E) Quantification of M1 macrophages (CD86high and CD206low) from the images in (D). Means with different letters (a-b or A-D) are significantly different (p<0.05). Scale bar: 20 μm. N=6, **p<0.01.

THGP and LPS stimulation alters the gene expression of cytokines secreted by M1 macrophages in RAW 264.7 cells. We examined the gene expression of the cytokines IL-1β and IL-6, which are highly secreted by M1 macrophages. On day 1, compared with the control, stimulation with LPS alone or in combination with THGP significantly up-regulated the expression of both IL-1β and IL-6 (Figure 3A and B). Notably, the combination of THGP and LPS had a synergistic effect, further enhancing this up-regulation. Furthermore, temporal analysis revealed that the expression levels of IL-1β and IL-6 were greater on day 4 than on day 1 across all treatment groups (THGP alone, LPS alone, or the combination). This trend was consistent for both cytokines.

Figure 3.
Figure 3.

Changes in the expression of cytokine genes secreted by M1 macrophages stimulated with THGP and LPS. We evaluated the expression of IL-1β (A) and IL-6 (B) via qPCR after 1 or 4 days of stimulation with THGP and LPS alone or in combination. Means with different letters (a-b or A-D) are significantly different (p<0.05). N=6, **p<0.01.

These findings indicate that individual stimulation with either THGP or LPS induces the gene expression of M1 macrophage-associated cytokines. Moreover, combined treatment with THGP and LPS had a synergistic effect on cytokine gene expression, suggesting a potential increase in M1 macrophage polarization.

THGP and LPS stimulation enhances the phagocytic activity of RAW 264.7 cells against foreign substances. The phagocytic activity of macrophages against foreign substances is increased after M1 polarization. Therefore, we examined the phagocytic activity of the macrophages against latex rabbit-IgG beads after stimulation with THGP, LPS or their combination. While LPS treatment alone increased phagocytic activity after four days of stimulation, THGP treatment alone did not significantly change phagocytic activity (Figure 4A and B). However, combined treatment with THGP and LPS enhanced phagocytic activity beyond that observed with LPS alone, confirming a synergistic effect.

Figure 4.
Figure 4.

Evaluation of the phagocytic activity of macrophages against foreign substances following THGP and LPS stimulation. (A) We assessed the phagocytic activity against foreign substances via a phagocytosis assay after 4 days of stimulation with THGP and LPS alone or in combination. Blue (Hoechst 33342) represents cell nuclei, whereas green (FITC) indicates phagocytosed beads. (B) The graph shows quantified values from the images in (a). Scale bar: 20 μm. N=6, **p<0.01, *p<0.05.

THGP and LPS stimulation enhances the antitumor effect on RAW 264.7 cells. Cancer cells escape immune responses through the binding of SIRP-α expressed on macrophages to CD47 expressed on cancer cells, known as the “do not eat me signal” (29). We examined SIRP-α expression using western blot. While treatment with LPS alone resulted in a trend toward decreased expression levels, this change did not reach statistical significance. However, the combination of THGP and LPS led to significant down-regulation of SIRP-α expression, which was not observed with either treatment alone (Figure 5A). Next, we evaluated the antitumor effect of macrophages by coculturing RAW 264.7 cells with B16-F10 cells for two days and then performing a luciferase assay (Figure 5B). Coculture of macrophages with B16-F10 cells significantly reduced the number of viable B16-F10 cells (NC vs. Ctrl). When macrophages were stimulated with THGP or LPS alone for four days, no change in cytotoxicity against cancer cells was detected (Ctrl vs. THGP or LPS) (Figure 5B). Surprisingly, the combination of THGP and LPS markedly increased the cytotoxicity of macrophages to cancer cells compared with that of control macrophages (Ctrl vs. THGP and LPS). These results demonstrate that the combination of THGP and LPS induces the differentiation of macrophages into M1 macrophages, synergistically enhancing their phagocytic ability against foreign substances and their cytotoxicity against cancer cells.

Figure 5.
Figure 5.

Evaluation of the antitumor effects of THGP and LPS stimulation. (A) We assessed SIRP-α expression using western blotting after four days of stimulation with THGP and LPS alone or in combination. (B) We evaluated the antitumor effect of macrophages against B16-F10 cells using a luciferase assay after four days of stimulation with THGP and LPS alone or in combination. N=6, **p<0.01, *p<0.05.

Stimulation with THGP or LPS alone for 10 days has equivalent effects on M1 macrophage differentiation as stimulation with both agents. Previous results demonstrated that four days of combined stimulation with THGP and LPS promoted M1 macrophage polarization, enhancing the phagocytic activity of macrophages against foreign substances and their antitumor effects. To investigate the impact of prolonged stimulation, we extended the culture period from 4 to 10 days (Figure 6A). First, we examined macrophage morphology. During 1–4 days of culture, THGP stimulation alone increased the dendritic process length, whereas LPS stimulation alone increased the proportion of spindle-shaped cells. After 10 days of culture, THGP or LPS stimulation alone increased both the dendritic process length and the proportion of spindle-shaped cells (Figure 6B-D). These effects were similar to those observed with combined THGP and LPS stimulation. Next, we evaluated the phagocytic activity of the macrophages against foreign substances. Ten days of stimulation with either THGP or LPS alone had an enhancement effect equivalent to that of combined stimulation (Figure 6E). We also examined the antitumor effects of macrophages. While 4 days of stimulation with either THGP or LPS alone did not enhance the antitumor effects, 10 days of stimulation with either THGP or LPS alone enhanced the antitumor effects on B16-F10 cells (Figure 6F). This effect was equivalent to that observed with combined THGP and LPS stimulation. These results suggest that THGP and LPS stimulation alone can sufficiently induce M1 differentiation in long-term culture and that their combination can shorten the process.

Figure 6.
Figure 6.

Effects of 10 days of stimulation with THGP and LPS. (A) Experimental design for 10 days of stimulation with THGP and LPS. (B) Bright-field images after 10 days of stimulation with THGP and LPS alone or in combination. (C) Lengths of the dendritic processes in (B). (D) Percentage of spindle cells in (B). (e) Evaluation of the phagocytic activity of macrophages against foreign substances applying a phagocytosis assay after 10 days of stimulation with THGP and LPS alone or in combination. (F) Assessment of the antitumor effects of macrophages on B16-F10 cells applying a luciferase assay after 10 days of stimulation with THGP and LPS alone or in combination. Scale bar: 20 μm. N=6, **p<0.01, *p<0.05.

Discussion

In this study, macrophage differentiation induced by THGP and low-concentration LPS stimulation was investigated. Our results revealed that stimulation with THGP or LPS alone promoted the differentiation of M1 macrophages, increasing their phagocytic activity and antitumor effects. Furthermore, the combination of THGP and LPS had a more pronounced effect.

Undifferentiated macrophages (M0 macrophages) undergo dramatic morphological changes during their differentiation into M1 macrophages (30). M0 macrophages are small and round, whereas M1 macrophages are adherent and have an amoeboid shape and extended dendritic processes. It has been shown that long-term (over 10 days) treatment of RAW 264.7 cells with THGP progressively increases the length of dendritic processes (26). In our study, short-term (4 days) treatment with THGP increased the length of macrophage dendritic processes but did not increase the proportion of amoeboid-shaped adherent cells (Figure 1). Conversely, LPS stimulation increased the proportion of amoeboid-shaped adherent cells but did not affect dendritic process length. The combination of THGP and LPS resulted in cells exhibiting both characteristics. These findings suggest that the combination of THGP and LPS may significantly promote M1 macrophage polarization. Additionally, compared with stimulation with each agent alone, combination treatment significantly increased the gene expression of the M1 markers CD80 and CD86 (Figure 2A and B), further supporting the enhanced effect of combined THGP and LPS treatment on M1 macrophage differentiation.

Macrophages play a crucial role in cancer. For example, M1 macrophages directly phagocytose cancer cells and inhibit cancer progression and metastasis (31, 32). Consequently, M1 macrophages are being targeted in cancer treatment research (33, 34). However, cancer cells express CD47 to evade macrophage phagocytosis. CD47, known as the “do not eat me” signal, binds to SIRP-α on macrophages, allowing cancer cells to escape phagocytosis (35). Inhibitory antibodies against CD47 or SIRP-α have been reported to increase macrophage phagocytosis and suppress cancer progression (36, 37). In our previous work, we demonstrated that prolonged exposure to THGP (10 days or more) resulted in the down-regulation of SIRP-α expression in macrophages. The results of the present study revealed that co-stimulation with THGP and LPS resulted in a significant decrease in SIRP-α expression (Figure 5A) within a shorter time frame than did treatment with THGP alone. Further studies using neutralizing antibodies are needed to determine whether the enhanced antitumor activity of macrophages following treatment with THGP and LPS is mediated through the SIRP-α/CD47 pathway. Previous studies have shown that macrophages treated with high concentrations of LPS (1 μg/ml) have strong antitumor effects on human lung cancer cells (38). This study revealed that macrophages stimulated with low-concentration LPS (1 ng/ml) for 10 days exhibited strong antitumor effects (Figure 6F). In addition, our study revealed that treatment with the combination of THGP and low-concentration LPS for four days enables macrophages to exert strong antitumor effects (Figure 5B). These findings indicate that the synergistic effect of THGP and LPS could be beneficial in cancer prevention strategies and as an adjuvant approach in cancer therapy.

Previous studies have suggested that THGP does not induce M1 macrophage differentiation in culture periods shorter than 10 days. Moreover, repeated administration of low-concentration LPS has been reported to increase macrophage phagocytosis both in vitro and in vivo (14, 39). In our study, 4 days of stimulation with THGP combined with LPS enhanced the antitumor effects of macrophages, whereas stimulation with THGP or LPS alone did not (Figure 5B). However, after 10 days of culture, stimulation with THGP or LPS alone resulted in changes that were comparable to those induced by the combined stimulation in terms of cell morphology changes, phagocytic activity against foreign substances, and antitumor effects (Figure 6). These results indicate that long-term stimulation with THGP and LPS induces M1 macrophage differentiation and that their combination shortens the time required for differentiation.

As hypothesized, the combination of THGP and LPS synergistically promoted M1 macrophage differentiation. This synergy likely stems from their distinct mechanisms of action. LPS is a well-known immunostimulatory substance that acts as a pathogen-associated molecular pattern (PAMP). It is recognized by TLR4, a pattern recognition receptor (PRR) on the cell surface (40). Upon recognition, LPS triggers immune cell activation through the MyD88-dependent pathway, primarily activating NF-κB. Furthermore, it has been reported that low-dose LPS activates macrophages through the activation of C/EBPδ, FoxO1 and CREB, which are transcription factors (41, 42). In contrast, THGP, a low-molecular-weight compound, is internalized by cells and functions intracellularly (43). In macrophages, THGP promotes M1 macrophage differentiation through the nuclear translocation of NF-κB (26). The difference in these stimulation mechanisms – LPS acting extracellularly and THGP acting intracellularly – may explain the observed synergistic effect when they are used in combination.

In this study, we demonstrated that the combination of THGP and LPS promoted M1 macrophage differentiation and antitumor activity in RAW264.7 cells. However, several limitations exist. For example, while the expression of M1 markers (CD80 and CD86) significantly increased at the mRNA level, no notable differences were observed at the protein level. This discrepancy might be attributed to time lags between mRNA and protein expression or posttranslational modifications. Additionally, we primarily evaluated the M1/M2 macrophage ratio via immunofluorescence image analysis, which has limitations in terms of quantitative accuracy. Therefore, implementing quantitative methods such as flow cytometry remains a future challenge. Furthermore, investigating changes in M2 markers (CD200, CD206, etc.) would help clarify M1 macrophage differentiation. In terms of phagocytic activity and antitumor effects, while the combination treatment resulted in statistically significant differences compared with the individual treatments, the increases were modest under our culture conditions. Evaluating synergistic or inhibitory effects under different concentration conditions would provide deeper insights into the effectiveness of combination therapy.

LPS and Ge-132, which are polymers of THGP, are widely known to stimulate the immune system. In oral administration experiments with mice, both have been reported to increase the phagocytic activity of intraperitoneal macrophages (26). However, a significant difference between LPS and Ge-132 is that LPS is hardly absorbed into the bloodstream (44), whereas approximately 20% of Ge-132 is absorbed (16). When orally ingested, LPS primarily acts directly on macrophages in the intestinal tract or indirectly activates macrophages by stimulating immune cells such as microfold cells and dendritic cells. It then influences immune cells throughout the body through a cell-to-cell contact-mediated signal transmission system (macrophage network system) originating in the intestinal tract, contributing to immune activation and health maintenance (11). However, Ge-132 induces the activation of macrophages and NK cells through oral ingestion. Additionally, it has been reported to stimulate the immune system similarly when it is administered intravenously (45). These results suggest that Ge-132 is absorbed from the intestinal tract and directly activates the immune system. Therefore, the effects of Ge-132 on the immune system may be widespread, acting both directly and indirectly from the site of absorption to the entire body. Since Ge-132 acts on immune cells throughout the body while LPS primarily acts on the intestinal tract, the combination of both may induce synergistic immune activation. In fact, the combination of Ge-132 with lactic acid bacteria with oligosaccharides, which have intestinal immune activity, have been reported to exert a synergistic effect, and a similar synergistic effect can be expected with LPS (46).

While we verified the combined effects of THGP and LPS via in vitro models, validating these results in vivo represents an important next step. Specifically, experiments using tumor models are necessary to confirm whether the observed antitumor effects are reproducible in living organisms. Furthermore, M1 macrophages have been reported to support the antitumor effects of T and NK cells through the secretion of cytokines such as IL-1β and IL-6 (47). Therefore, analyzing interactions between various immune cells represents an interesting task to strengthen our research findings. While we used single concentrations of THGP and LPS in this study, the optimal drug concentrations and administration schedules need to be determined. Additionally, particularly careful evaluation is needed regarding the possibility that immune stimulant combinations may not always have synergistic effects and could cause inhibitory effects (48).

LPS and THGP have similar effects on maintaining health. For example, the oral administration of LPS to diabetic mice has been reported to alleviate diabetes-related cognitive dysfunction (DRCD) (49). This effect is attributed to the ability of orally administered LPS to transform microglia into a neuroprotective phenotype. Beneficial effects on glucose and lipid metabolism have also been observed, along with a reduction in the levels of amyloid-β in the brain, which are closely associated with Alzheimer’s disease. Moreover, Ge-132 has been reported to decrease plasma glycated protein levels in type I diabetic rats when it is administered at 100 mg/kg (50). In type II diabetic model rats, Ge-132 administration at 100 mg/kg has been shown to inhibit Advanced Glycation End Products (AGE) accumulation in the kidneys and amyloid deposition in the cerebellum (51). Additionally, the oral administration of Ge-132 at 30 mg/kg or 300 mg/kg has been reported to dramatically mitigate age-related systemic spontaneous amyloidosis (52). These reports suggest that the oral administration of LPS and Ge-132 may effectively alleviate metabolic syndrome and cognitive impairment caused by aging and dietary habits. These findings warrant further investigation in this promising field of research.

Conclusion

This study demonstrated that THGP and LPS synergistically enhance macrophage phagocytic activity and antitumor effects by promoting their differentiation into M1 macrophages. Since THGP and LPS have different mechanisms of action (intracellular and cell surface), their synergistic effects suggest potential applications in immunotherapy. The findings from this study are promising as a novel therapeutic strategy, with the ability to promote M1 macrophage differentiation in a short period representing a significant clinical advantage. Furthermore, as THGP and LPS are expected to be effective against lifestyle-related diseases, such as diet-induced diabetes and aging-associated conditions, their immunomodulatory effects need to be elucidated in future research.

Acknowledgements

B16-F10/CMV-LUC#2 cells were developed and produced by Takashi Murakami. The Authors thank Satomi Sato at the Asai Germanium Research Institute for providing technical assistance with the experiments. The BZ-X810 fluorescence microscope was donated by Yoshiko Watanabe.

Footnotes

  • Authors’ Contributions

    J.A. designed the study, performed the experiments, analyzed the data and wrote the manuscript. T.T. performed the experiments and analyzed the data. Y.S., H.I., H.A. and T.N. interpreted all data and reviewed and edited the manuscript.

  • Conflicts of Interest

    J.A., T.T. and Y.S. were employees of Asai Germanium Research Institute Co., Ltd. H.A. received remuneration from Asai Germanium Research Institute Co., Ltd., as advisor. T.N. received remuneration from Asai Germanium Research Institute Co., Ltd., as an officer. The other author has no conflicts of interest associated with this manuscript to declare. The results described in the article were generated with funding from Asai Germanium Research Institute Co., Ltd.

  • Funding

    This research received funding from Asai Germanium Research Institute Co., Ltd.

  • Artificial Intelligence (AI) Disclosure

    During the preparation of this manuscript, a large language model (ChatGPT, OpenAI) was used solely for language editing and stylistic improvements in select paragraphs. No sections involving the generation, analysis, or interpretation of research data were produced by generative AI. All scientific content was created and verified by the authors. Furthermore, no figures or visual data were generated or modified using generative AI or machine learning–based image enhancement tools.

  • Received May 23, 2025.
  • Revision received June 5, 2025.
  • Accepted June 6, 2025.

This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

References

  1. ↵
    1. Mosser DM,
    2. Edwards JP
    : Exploring the full spectrum of macrophage activation. Nat Rev Immunol 8(12): 958-969, 2008. DOI: 10.1038/nri2448
    OpenUrlCrossRefPubMed
  2. ↵
    1. Chen Y,
    2. Hu M,
    3. Wang L,
    4. Chen W
    : Macrophage M1/M2 polarization. Eur J Pharmacol 877: 173090, 2020. DOI: 10.1016/j.ejphar.2020.173090
    OpenUrlCrossRefPubMed
  3. ↵
    1. Tugal D,
    2. Liao X,
    3. Jain MK
    : Transcriptional control of macrophage polarization. Arterioscler Thromb Vasc Biol 33(6): 1135-1144, 2013. DOI: 10.1161/ATVBAHA.113.301453
    OpenUrlAbstract/FREE Full Text
  4. ↵
    1. Liu YC,
    2. Zou XB,
    3. Chai YF,
    4. Yao YM
    : Macrophage polarization in inflammatory diseases. Int J Biol Sci 10(5): 520-529, 2014. DOI: 10.7150/ijbs.8879
    OpenUrlCrossRefPubMed
  5. ↵
    1. Mantovani A,
    2. Sica A
    : Macrophages, innate immunity and cancer: balance, tolerance, and diversity. Curr Opin Immunol 22(2): 231-237, 2010. DOI: 10.1016/j.coi.2010.01.009
    OpenUrlCrossRefPubMed
  6. ↵
    1. Wang N,
    2. Liang H,
    3. Zen K
    : Molecular mechanisms that influence the macrophage m1-m2 polarization balance. Front Immunol 5: 614, 2014. DOI: 10.3389/fimmu.2014.00614
    OpenUrlCrossRefPubMed
  7. ↵
    1. Cassetta L,
    2. Cassol E,
    3. Poli G
    : Macrophage polarization in health and disease. ScientificWorldJournal 11: 2391-2402, 2011. DOI: 10.1100/2011/213962
    OpenUrlCrossRefPubMed
  8. ↵
    1. Zipfel C
    : Early molecular events in PAMP-triggered immunity. Curr Opin Plant Biol 12(4): 414-420, 2009. DOI: 10.1016/j.pbi.2009.06.003
    OpenUrlCrossRefPubMed
  9. ↵
    1. Raetz CR,
    2. Whitfield C
    : Lipopolysaccharide endotoxins. Annu Rev Biochem 71: 635-700, 2002. DOI: 10.1146/annurev.biochem.71.110601.135414
    OpenUrlCrossRefPubMed
  10. ↵
    1. Resiliac J,
    2. Rohlfing M,
    3. Santoro J,
    4. Hussain SA,
    5. Grayson MH
    : Low-dose lipopolysaccharide protects from lethal paramyxovirus infection in a macrophage- and TLR4-dependent process. J Immunol 210(3): 348-355, 2023. DOI: 10.4049/jimmunol.2200604
    OpenUrlCrossRefPubMed
  11. ↵
    1. Inagawa H,
    2. Kohchi C,
    3. Soma GI
    : Oral administration of lipopolysaccharides for the prevention of various diseases: benefit and usefulness. Anticancer Res 31(7): 2431-2436, 2011.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Gibbons HM,
    2. Dragunow M
    : Microglia induce neural cell death via a proximity-dependent mechanism involving nitric oxide. Brain Res 1084(1): 1-15, 2006. DOI: 10.1016/j.brainres.2006.02.032
    OpenUrlCrossRefPubMed
  13. ↵
    1. Mizobuchi H,
    2. Yamamoto K,
    3. Tsutsui S,
    4. Yamashita M,
    5. Nakata Y,
    6. Inagawa H,
    7. Kohchi C,
    8. Soma GI
    : A unique hybrid characteristic having both pro- and anti-inflammatory phenotype transformed by repetitive low-dose lipopolysaccharide in C8-B4 microglia. Sci Rep 10(1): 8945, 2020. DOI: 10.1038/s41598-020-65998-8
    OpenUrlCrossRefPubMed
  14. ↵
    1. Yamamoto K,
    2. Mizobuchi H,
    3. Yamashita M,
    4. Inagawa H,
    5. Kohchi C,
    6. Soma GI
    : Attempt to construct an in vitro model of enhancement of macrophage phagocytosis via continuous administration of LPS. Anticancer Res 40(8): 4711-4717, 2020. DOI: 10.21873/anticanres.14472
    OpenUrlAbstract/FREE Full Text
  15. ↵
    1. Mizuno N,
    2. Nishibori E,
    3. Oka M,
    4. Jomori T,
    5. Takata M,
    6. Kumasaka T
    : Structural basis for polymer packing and solvation properties of the organogermanium crystalline polymer propagermanium and its derivatives. J Pharm Sci 104(8): 2482-2488, 2015. DOI: 10.1002/jps.24486
    OpenUrlCrossRefPubMed
  16. ↵
    1. Nakamura T,
    2. Shimada Y,
    3. Sato K
    : Bioorganic and medicinal organogermanium chemistry. Organogermanium Compounds: 839-865, 2023. DOI: 10.1002/9781119613466.ch19
    OpenUrlCrossRef
  17. ↵
    1. Filonova GE,
    2. Nikolaevskaya EN,
    3. Kansuzyan AV,
    4. Krylova IV,
    5. Egorov MP,
    6. Jouikov VV,
    7. Syroeshkin MA
    : Antioxidant properties of adrenaline in the presence of Ge-132. Eur J Org Chem 2019(26): 4128-4132, 2019. DOI: 10.1002/ejoc.201900331
    OpenUrlCrossRef
  18. ↵
    1. Vishtorskaya AA,
    2. Saverina EA,
    3. Pechennikov VM,
    4. Krylova IV,
    5. Lalov AV,
    6. Syroeshkin MA,
    7. Egorov MP,
    8. Jouikov VV
    : Assessing Ge-132 as an antioxidant in organic and water-containing media. J Organomet Chem 858: 8-13, 2018. DOI: 10.1016/j.jorganchem.2018.01.004
    OpenUrlCrossRef
  19. ↵
    1. Matsumoto H,
    2. Jiang GZ,
    3. Hashimoto T,
    4. Kuboyama N,
    5. Yamane J,
    6. Nonaka K,
    7. Fujii A
    : Effect of organic germanium compound (Ge-132) on experimental osteoporosis in rats: the relationship between transverse strength and bone mineral density (BMD) or bone mineral content (BMC). Int J Oral Med Sci 1(1): 10-16, 2002. DOI: 10.5466/ijoms.1.10
    OpenUrlCrossRef
  20. ↵
    1. Fujii A,
    2. Kuboyama N,
    3. Yamane J,
    4. Nakao S,
    5. Furukawa Y
    : Effect of organic germanium compound (Ge-132) on experimental osteoporosis in rats. Gen Pharmacol 24(6): 1527-1532, 1993. DOI: 10.1016/0306-3623(93)90447-6
    OpenUrlCrossRefPubMed
  21. ↵
    1. Hachisu M,
    2. Takahashi H,
    3. Koeda T,
    4. Sekizawa Y
    : Analgesic effect of novel organogermanium compound, Ge-132. J Pharmaco biodyn 6(11): 814-820, 1983. DOI: 10.1248/bpb1978.6.814
    OpenUrlCrossRef
  22. ↵
    1. Komuro T,
    2. Kakimoto N,
    3. Katayama T,
    4. Hazato T
    : Inhibitory effects of Ge-132 (carboxyethyl germanium sesquioxide) derivatives on enkephalin-degrading enzymes. Biotechnol Appl Biochem 8(5): 379-386, 1986.
    OpenUrlPubMed
  23. ↵
    1. Suzuki F,
    2. Brutkiewicz RR,
    3. Pollard RB
    : Ability of sera from mice treated with Ge-132, an organic germanium compound, to inhibit experimental murine ascites tumours. Br J Cancer 52(5): 757-763, 1985. DOI: 10.1038/bjc.1985.254
    OpenUrlCrossRefPubMed
    1. Suzuki F,
    2. Brutkiewicz RR,
    3. Pollard RB
    : Importance of T-cells and macrophages in the antitumor activity of carboxyethylgerma nium sesquioxide (Ge-132). Anticancer Res 5(5): 479-483, 1985.
    OpenUrlPubMed
  25. ↵
    1. Suzuki F,
    2. Brutkiewicz RR,
    3. Pollard RB
    : Cooperation of lymphokine(s) and macrophages in expression of antitumor activity of carboxyethylgermanium sesquioxide (Ge-132). Anticancer Res 6(2): 177-182, 1986.
    OpenUrlPubMed
  26. ↵
    1. Azumi J,
    2. Takeda T,
    3. Shimada Y,
    4. Zhuang T,
    5. Tokuji Y,
    6. Sakamoto N,
    7. Aso H,
    8. Nakamura T
    : Organogermanium THGP induces differentiation into M1 macrophages and suppresses the proliferation of melanoma cells via phagocytosis. Int J Mol Sci 24(3): 1885, 2023. DOI: 10.3390/ijms24031885
    OpenUrlCrossRefPubMed
  27. ↵
    1. Yi AK,
    2. Yoon JG,
    3. Hong SC,
    4. Redford TW,
    5. Krieg AM
    : Lipopolysaccharide and CpG DNA synergize for tumor necrosis factor-α production through activation of NF-κB. Int Immunol 13(11): 1391-1404, 2001. DOI: 10.1093/intimm/13.11.1391
    OpenUrlCrossRefPubMed
  28. ↵
    1. Ainai A,
    2. Ichinohe T,
    3. Tamura S,
    4. Kurata T,
    5. Sata T,
    6. Tashiro M,
    7. Hasegawa H
    : Zymosan enhances the mucosal adjuvant activity of poly(I:C) in a nasal influenza vaccine. J Med Virol 82(3): 476-484, 2010. DOI: 10.1002/jmv.21694
    OpenUrlCrossRefPubMed
  29. ↵
    1. Zhang W,
    2. Huang Q,
    3. Xiao W,
    4. Zhao Y,
    5. Pi J,
    6. Xu H,
    7. Zhao H,
    8. Xu J,
    9. Evans CE,
    10. Jin H
    : Advances in anti-tumor treatments targeting the CD47/SIRPα axis. Front Immunol 11: 18, 2020. DOI: 10.3389/fimmu.2020.00018
    OpenUrlCrossRef
  30. ↵
    1. Heinrich F,
    2. Lehmbecker A,
    3. Raddatz BB,
    4. Kegler K,
    5. Tipold A,
    6. Stein VM,
    7. Kalkuhl A,
    8. Deschl U,
    9. Baumgärtner W,
    10. Ulrich R,
    11. Spitzbarth I
    : Morphologic, phenotypic, and transcriptomic characterization of classically and alternatively activated canine blood-derived macrophages in vitro. PLoS One 12(8): e0183572, 2017. DOI: 10.1371/journal.pone.0183572
    OpenUrlCrossRefPubMed
  31. ↵
    1. Najafi M,
    2. Hashemi Goradel N,
    3. Farhood B,
    4. Salehi E,
    5. Nashtaei MS,
    6. Khanlarkhani N,
    7. Khezri Z,
    8. Majidpoor J,
    9. Abouzaripour M,
    10. Habibi M,
    11. Kashani IR,
    12. Mortezaee K
    : Macrophage polarity in cancer: A review. J Cell Biochem 120(3): 2756-2765, 2019. DOI: 10.1002/jcb.27646
    OpenUrlCrossRefPubMed
  32. ↵
    1. Pinto PC
    : The potential impact of new drug and therapeutic modalities on drug resistance to renal cell carcinoma. Anticancer Res 43(3): 983-991, 2023. DOI: 10.21873/anticanres.16242
    OpenUrlAbstract/FREE Full Text
  33. ↵
    1. Genard G,
    2. Wera AC,
    3. Huart C,
    4. Le Calve B,
    5. Penninckx S,
    6. Fattaccioli A,
    7. Tabarrant T,
    8. Demazy C,
    9. Ninane N,
    10. Heuskin AC,
    11. Lucas S,
    12. Michiels C
    : Proton irradiation orchestrates macrophage reprogramming through NFκB signaling. Cell Death Dis 9(7): 728, 2018. DOI: 10.1038/s41419-018-0757-9
    OpenUrlCrossRefPubMed
  34. ↵
    1. Goshima T,
    2. Ieguchi K,
    3. Onishi N,
    4. Shimizu T,
    5. Takayanagi D,
    6. Watanabe M,
    7. Fujimoto Y,
    8. Ohkuma R,
    9. Suzuki R,
    10. Tsurui T,
    11. Mura E,
    12. Iriguchi N,
    13. Ishiguro T,
    14. Shimokawa M,
    15. Hirasawa Y,
    16. Kubota Y,
    17. Ariizumi H,
    18. Horiike A,
    19. Yoshimura K,
    20. Tsuji M,
    21. Kiuchi Y,
    22. Kobayashi S,
    23. Fujishiro J,
    24. Hoffman RM,
    25. Tsunoda T,
    26. Wada S
    : Non-classical monocytes enhance the efficacy of immune checkpoint inhibitors on colon cancer in a syngeneic mouse model. Anticancer Res 44(1): 23-29, 2024. DOI: 10.21873/anticanres.16784
    OpenUrlAbstract/FREE Full Text
  35. ↵
    1. Chao MP,
    2. Weissman IL,
    3. Majeti R
    : The CD47-SIRPα pathway in cancer immune evasion and potential therapeutic implications. Curr Opin Immunol 24(2): 225-232, 2012. DOI: 10.1016/j.coi.2012.01.010
    OpenUrlCrossRefPubMed
  36. ↵
    1. Yanagita T,
    2. Murata Y,
    3. Tanaka D,
    4. Motegi SI,
    5. Arai E,
    6. Daniwijaya EW,
    7. Hazama D,
    8. Washio K,
    9. Saito Y,
    10. Kotani T,
    11. Ohnishi H,
    12. Oldenborg PA,
    13. Garcia NV,
    14. Miyasaka M,
    15. Ishikawa O,
    16. Kanai Y,
    17. Komori T,
    18. Matozaki T
    : Anti-SIRPα antibodies as a potential new tool for cancer immunotherapy. JCI Insight 2(1): e89140, 2017. DOI: 10.1172/jci.insight.89140
    OpenUrlCrossRef
  37. ↵
    1. Tseng D,
    2. Volkmer JP,
    3. Willingham SB,
    4. Contreras-Trujillo H,
    5. Fathman JW,
    6. Fernhoff NB,
    7. Seita J,
    8. Inlay MA,
    9. Weiskopf K,
    10. Miyanishi M,
    11. Weissman IL
    : Anti-CD47 antibody-mediated phagocytosis of cancer by macrophages primes an effective antitumor T-cell response. Proc Natl Acad Sci USA 110(27): 11103-11108, 2013. DOI: 10.1073/pnas.1305569110
    OpenUrlAbstract/FREE Full Text
  38. ↵
    1. Taniguchi H,
    2. Shimada Y,
    3. Sawachi K,
    4. Hirota K,
    5. Inagawa H,
    6. Kohchi C,
    7. Soma G,
    8. Makino K,
    9. Terada H
    : Lipopolysaccharide-activated alveolar macrophages having cytotoxicity toward lung tumor cells through a cell-to-cell binding-dependent mechanism. Anticancer Res 30(8): 3159-3165, 2010.
    OpenUrlAbstract/FREE Full Text
  39. ↵
    1. Inagawa H,
    2. Kobayashi Y,
    3. Kohchi C,
    4. Zhang R,
    5. Shibasaki Y,
    6. Soma GI
    : Primed activation of macrophages by oral administration of lipopolysaccharide derived from Pantoea agglomerans. In Vivo 30(3): 205-211, 2016.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    1. Takeuchi O,
    2. Akira S
    : Pattern recognition receptors and inflammation. Cell 140(6): 805-820, 2010. DOI: 10.1016/j.cell.2010.01.022
    OpenUrlCrossRefPubMed
  41. ↵
    1. Maitra U,
    2. Gan L,
    3. Chang S,
    4. Li L
    : Low-dose endotoxin induces inflammation by selectively removing nuclear receptors and activating CCAAT/enhancer-binding protein δ. J Immunol 186(7): 4467-4473, 2011. DOI: 10.4049/jimmunol.1003300
    OpenUrlAbstract/FREE Full Text
  42. ↵
    1. Morris MC,
    2. Gilliam EA,
    3. Button J,
    4. Li L
    : Dynamic modulation of innate immune response by varying dosages of lipopolysaccharide (LPS) in human monocytic cells. J Biol Chem 289(31): 21584-21590, 2014. DOI: 10.1074/jbc.M114.583518
    OpenUrlAbstract/FREE Full Text
  43. ↵
    1. Takeda T,
    2. Doiyama S,
    3. Azumi J,
    4. Shimada Y,
    5. Tokuji Y,
    6. Yamaguchi H,
    7. Nagata K,
    8. Sakamoto N,
    9. Aso H,
    10. Nakamura T
    : Organogermanium suppresses cell death due to oxidative stress in normal human dermal fibroblasts. Sci Rep 9(1): 13637, 2019. DOI: 10.1038/s41598-019-49883-7
    OpenUrlCrossRefPubMed
  44. ↵
    1. Ghoshal S,
    2. Witta J,
    3. Zhong J,
    4. De Villiers W,
    5. Eckhardt E
    : Chylomicrons promote intestinal absorption of lipopoly saccharides. J Lipid Res 50(1): 90-97, 2009. DOI: 10.1194/jlr.M800156-JLR200
    OpenUrlAbstract/FREE Full Text
  45. ↵
    1. Aso H,
    2. Shibuya E,
    3. Suzuki F,
    4. Nakamura T,
    5. Inoue H,
    6. Ebina T,
    7. Ishida N
    : [Antitumor effect in mice of an organic germanium compound (Ge-132) when different administration methods are used]. Gan To Kagaku Ryoho 12(12): 2345-2351, 1985.
    OpenUrlPubMed
  46. ↵
    1. Nakamura T,
    2. Saito M,
    3. Aso H
    : Effects of a lactobacilli, oligosaccharide and organic germanium intake on the immune responses of mice. Biosci Biotechnol Biochem 76(2): 375-377, 2012. DOI: 10.1271/bbb.110655
    OpenUrlCrossRefPubMed
  47. ↵
    1. Tong N,
    2. He Z,
    3. Ma Y,
    4. Wang Z,
    5. Huang Z,
    6. Cao H,
    7. Xu L,
    8. Zou Y,
    9. Wang W,
    10. Yi C,
    11. Yin Z,
    12. Wang Q
    : Tumor associated macrophages, as the dominant immune cells, are an indispensable target for immunologically cold tumor-glioma therapy? Front Cell Dev Biol 9: 706286, 2021. DOI: 10.3389/fcell.2021.706286
    OpenUrlCrossRefPubMed
  48. ↵
    1. Morrissey KM,
    2. Yuraszeck TM,
    3. Li CC,
    4. Zhang Y,
    5. Kasichayanula S
    : Immunotherapy and novel combinations in oncology: current landscape, challenges, and opportunities. Clin Transl Sci 9(2): 89-104, 2016. DOI: 10.1111/cts.12391
    OpenUrlCrossRef
  49. ↵
    1. Mizobuchi H,
    2. Yamamoto K,
    3. Yamashita M,
    4. Nakata Y,
    5. Inagawa H,
    6. Kohchi C,
    7. Soma GI
    : Prevention of diabetes-associated cognitive dysfunction through oral administration of lipopolysaccharide derived from Pantoea agglomerans. Front Immunol 12: 650176, 2021. DOI: 10.3389/fimmu.2021.650176
    OpenUrlCrossRefPubMed
  50. ↵
    1. Shimojo N,
    2. Pan JW,
    3. Yoshioka K,
    4. Ishii T,
    5. Nakajima C,
    6. Naka K
    : Effects of organic germanium compounds on glycated plasma proteins in diabetic rats. Clin Chem Enzym Commun 5: 179-184, 1993.
    OpenUrl
  51. ↵
    1. Nakamura K,
    2. Fujita Y,
    3. Akiba M,
    4. Hosokawa T,
    5. Kakimoto N,
    6. Osawa T
    : Suppression of ages formation in spontaneous diabetic OLETF rat by organic germanium compound [Poly-trans-(2-carboxyethyl) Germasesquioxane] (Ge-132). Phosphorus Sulfur Silicon Relat Elem 150(1): 375-391, 1999. DOI: 10.1080/10426509908546406
    OpenUrlCrossRef
  52. ↵
    1. Kuga N,
    2. Oboshi S,
    3. Sato H,
    4. Sato R
    : Inhibition of senile amyloidosis of mice by bis-carboxyethyl germanium sesquioxide. Acta Pathol Jpn 26(1): 63-71, 1976. DOI: 10.1111/j.1440-1827.1976.tb03293.x
    OpenUrlCrossRefPubMed
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Combination of 3-(Trihydroxygermyl)propanoic Acid (THGP) and Lipopolysaccharide Promotes Macrophage Differentiation to M1, and Antitumor Activity
JUNYA AZUMI, TOMOYA TAKEDA, YASUHIRO SHIMADA, HISASHI ASO, HIROYUKI INAGAWA, TAKASHI NAKAMURA
Anticancer Research Aug 2025, 45 (8) 3425-3440; DOI: 10.21873/anticanres.17704
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