Lipopolysaccharide suppresses lung metastasis by activating CD8⁺ T cells despite promoting effect via cancer-associated fibroblasts

Abstract

Background

Inflammation plays a dual role in tumor progression, and its overall effect depends on the complex interactions between the nature of the stimuli and the dynamic changes within the tumor microenvironment. Lipopolysaccharide (LPS) is a bacterial endotoxin whose immune effects on tumors may be either promoting or inhibiting, depending on the dose and timing. This study investigates the effects of LPS on tumor cells, fibroblasts, and immune cells in a mouse model of lung metastasis.

Methods

To investigate the effects of LPS on tumor progression, we employed C57BL/6 immune-competent mice, immune-compromised nude mice and immune-deficient NOD-SCID mice. We assessed metastasis through in vivo experiments, while in vitro studies were conducted to evaluate the proliferation of tumor cells in the presence of LPS and LPS-treated fibroblasts. Additionally, we analyzed immune cell infiltration and the role of specific immune cell populations, such as CD8⁺ T cells, macrophages, and dendritic cells, in mediating the effects of LPS.

Results

LPS treatment significantly suppressed metastasis in immune-competent C57BL/6 mice but had no effect in immune-compromised nude mice, and even promoted metastasis in severely immune-deficient NOD-SCID mice. In vitro, LPS inhibited tumor cell proliferation. However, when tumor cells were co-cultured with LPS-treated fibroblasts, their growth was promoted compared to those co-cultured with untreated fibroblasts. In vivo, LPS enhanced the infiltration of mature macrophages, neutrophils, and CD8⁺ T cells while reducing M2 macrophages in the tumor microenvironment. Depletion of CD8⁺ T cells, macrophages, or dendritic cells individually abolished the protective effect of LPS, demonstrating their essential roles. Furthermore, LPS boosted the cytotoxicity of CD8⁺ T cells against tumor targets.

Conclusion

LPS exerts a dual influence on tumors: overall LPS suppresses metastasis by activating cell-mediated immunity, specifically cytotoxic CD8⁺ T cells, despite promoting effect via inflamed fibroblasts.

Supplementary Information

The online version contains supplementary material available at 10.1186/s12885-026-15837-2.

Introduction

Inflammation plays a dual role in tumor progression, capable of both promoting and suppressing the disease. However, the net outcome of inflammation is complex and influenced by the timing, intensity, and duration of the inflammatory stimulus, as well as by dynamically altered stromal cells within the tumor microenvironment (TME). For instance, chronic inflammation is known to enhance cancer risk by fostering carcinogenesis, immunosuppression, and metastasis [1–5], whereas acute inflammatory stimuli can suppress tumor growth through immune system activation [6].

Lipopolysaccharide (LPS), a major component of the outer membrane of Gram-negative bacteria, consists of lipids and polysaccharides and retains most of the biological properties of bacterial endotoxins [7]. It induces acute airway inflammation by activating TLR4 in a MyD88-dependent manner [3]. TLR4 activation in macrophages by LPS stimulates the secretion of numerous pro-inflammatory factors, including IL-6 and IL-1β [8]. The dose and timing of LPS administration are critical; low-dose LPS (0.1 µg/mouse) induces a Th2-like immune response, while a high dose (10 µg/mouse) promotes a Th1 response in mouse models [2]. Intriguingly, plasmacytoid dendritic cell depletion during low-dose LPS treatment reduces tumor burden, whereas the same depletion during high-dose LPS increases it [5], underscoring how signal intensity dictates biological outcomes.

Substantial evidence indicates that persistent inflammation promotes tumor development. Within the TME, macrophages often polarize toward the M2 phenotype [9]. These tumor-associated macrophages (TAMs) secrete anti-inflammatory factors that impair CD8⁺ T cell cytotoxicity, reinforce immunosuppression, and facilitate tumor progression [10, 11]. TAM accumulation frequently coincides with increased numbers of cancer-associated fibroblasts (CAFs) and regulatory T cells [12]. CAFs, which are associated with chronic inflammation, have been shown to promote pre-metastatic niche formation in the lungs by remodeling the local immune microenvironment [13].

LPS may also directly promote tumor cell growth, as TLR4 can be upregulated in some lung cancer cells. TLR4 stimulation by LPS has been reported to facilitate cell proliferation and promote carcinogenesis in models of skin [14], lung [15], breast [16], and colon cancers [1]. Thus, the overall effect of inflammatory signaling on tumor progression and metastasis is intricately linked to dynamic alterations in both tumor and stromal cells within the TME [17]. Factors that foster an immunosuppressive milieu promote tumor survival, while those that activate dendritic cells and CD8⁺ cytotoxic T lymphocytes (CTLs) enhance anti-tumor immunity [4]. Consequently, LPS likely exerts differential effects on stromal fibroblasts and immune cells within the TME. Understanding how inflammatory stimuli regulate these diverse cells could inform effective strategies for targeting the TME to inhibit tumor growth [5, 18, 19].

In this study, we investigate the effects of LPS treatment on tumor cells, fibroblasts, and immune cells in the TME using mouse models of lung metastasis, aiming to elucidate their respective roles in tumor promotion and suppression.

Materials and methods

Cell lines and cell culture

Primary mouse lung adenocarcinoma cells (S1601) were isolated from 4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK)-induced lung tumors of Gprc5a-knockout (KO) mice [20]. Eight-week-old Gprc5a-KO mice received two intraperitoneal (i.p.) injections per week of NNK (100 mg/kg body weight) dissolved in saline (0.9% NaCl). Twelve months later, tumors had developed in the lungs. The mice were anesthetized with 5% isoflurane and euthanized by cervical dislocation, followed by lung tissue collection. The tissues were digested into single cells, which were seeded onto culture plates. After three days, viable cells were passaged to establish the primary cell line. Gprc5a-KO mice were from our in-house breeding colony, which was established from founder stock originally provided by Dr. R. Lotan (University of Texas M.D. Anderson Cancer Center) and maintained under SPF conditions. Cells were cultured in DMEM (Invitrogen) supplemented with 10% fetal bovine serum (Gibco), 1% penicillin, EGF (5ng/mL) and bovine pituitary extract (50 mg/mL; Invitrogen), and streptomycin in a humidified incubator at 37 °C, 95% air, and 5% CO₂.

Flow cytometry analysis

Following euthanasia, lung tissues were collected via thoracotomy in experimental mice. The lung tissues were scissors cut into a meaty shape and digested with 150 m/mL collagenase IV and 20 m/mL DNase1 in PBS for 45 min at 37 °C. Single-cell suspensions were prepared through 70 μm and 40 μm nylon mesh. All the cells were incubated with red blood cell lysate for 5 min at room temperature and washed twice with PBS containing 10% FBS. Cells were stained with fluorescently labeled antibodies on ice for 30 min and then were analyzed by FACS (BD Biosciences). Finally, the data were analyzed in Flowjo 7.6.1 software. All antibody information is shown following: PE Rat Anti-CD11b (557397, BD), Alexa Fluor^® 647 Rat Anti-Mouse F4/80 (565854, BD), BV421 Rat Anti-Mouse CD8a (563898, BD), APC Rat Anti-Mouse Gr1 (553129, BD), PerCP/Cyanine5.5 anti-mouse CD3(100328, Biolegend), FITC anti-mouse CD8a (100706, Biolegend), PE anti-mouse CD11c (117308, Biolegend), FITC anti-mouse H-2 Antibody (125507 Biolegend), APC anti-mouse CD45 (157605, Biolegend).

Animal model

This article describes a total of eleven independent mouse experiments. The murine models used in each study are described below. (1) C57BL/6 mice received an intravenous (i.v.) injection of 1 × 10⁶ S1601-luc cells, followed by i.p. administration of three doses of LPS (5, 10 and 20 µg per mouse) at the indicated time points (n = 4 mice/group); (2) C57BL/6 mice received an i.v. injection of 1 × 10⁶ S1601 cells, followed by i.p. LPS (20 µg per mouse) at the indicated time points (n = 4 mice/group); 3,4) For detailed information, please refer to the survival experiment section in the Materials and Methods below; 5) C57BL/6 mice received an i.v. injection of 2 × 10⁶ S1601-luc cells, followed by i.p. LPS (20 µg per mouse) at indicated the times (n = 4 mice/group); 6) C57BL/6 mice received an i.v. injection of 2 × 10⁵ S1601-luc cells with single LPS dose (20 µg per mouse, i.p.) at day 40. Fluorescence imaging of lungs at day 47 revealed metastatic tumors (n = 4 mice/group); 7,8) Nude mice or NOD-SCID mice received an i.v. injection of 5 × 10⁵ S1601 cells, followed by i.p. LPS (20 µg per mouse) at the indicated times (n = 5 mice/group); 9) C57BL/6 mice received an i.v. injection of 1 × 10⁶ S1601 cells and subsequently received i.p. administration of LPS (20 µg per mouse) or anti-CD8 antibody (100 µg per mouse) at the indicated time points(n = 4 mice/group); 10,11) C57BL/6 mice were first intravenously injected with 1 × 10⁶ S1601 cells. At the indicated time points thereafter, they received the following treatments: i.p. LPS (20 µg/mouse), i.v. cytochrome C (5 mg/mouse) to deplete dendritic cells (n = 5 mice/group), or intranasal clodronate liposomes (40 µL/mouse) to deplete macrophages (n = 4 mice/group). Female mice were utilized in all animal experiments unless otherwise indicated. All mice were first anesthetized using 5% isoflurane inhalation. Euthanasia was then confirmed by cervical dislocation. All experimental mice (C57BL/6, NOD-SCID, and nude strains) used in this study were 8 weeks old, weighing approximately 20 g, obtained from Shandong Pengyue laboratory Animal Technology Co., Ltd., and housed under specific SPF conditions. All animal experiments were approved by the Ethics Committee of Shandong Second Medical University.

Animal treatment

LPS (HY-D1056, MCE, USA) treatment involved weekly intraperitoneal administration of 20 µg per mouse, while anti CD8 antibody (BE0061, BioXcell, USA) therapy was delivered via weekly intraperitoneal injection of 100 µg per mouse. In the dendritic cell depletion experiment, cytochrome C (C2506, Sigma, USA) was administered weekly to each mouse at a dose of 5 mg via tail vein injection. In the macrophage depletion experiment, Clodronate Liposomes (liposoma, Netherlands) was administered to each mouse at 40 µL per week via nasal drops.

Survival experiment

Survival experiments were conducted separately in female and male mice. The female mice included a control group (n = 14) and an LPS treatment group (n = 9), while the male mice contained 5 mice per group. All mice were eight weeks old and weighed approximately 20 g. After intravenous injection with 1 × 10⁶ tumor cells, the mice were monitored for survival. Following intravenous injection of 1 × 10⁶ tumor cells, mice were monitored and euthanized when body weight loss exceeded 20% of the initial weight, which was recorded as the event.

T cell-mediated tumor cell killing assay

Eight-week-old C57BL/6 mice were sacrificed and single-cell suspension from the spleen was prepared through 70 mm and 40 mm nylon mesh after sufficient grinding. In short, CD8⁺ T cells were obtained by CD8⁺ T Cell Isolation Kit (Miltenyi Biotec, Germany) and were activated with CD3/CD28 antibody-conjugated beads (#11453D, Thermo Fisher Scientific, USA) for 3 days. To analyze the killing effect of tumor cells by T cell inactivation, we co-cultured S1601 cells with T cells according to the ratio of tumor cells: T cells = 1:5 for 3 days in 24 well plates. Then the wells were washed twice with PBS to remove T cells. Cells were fixed with paraformaldehyde (Servicebio, G1101) for 30 min followed by crystal violet (Servicebio, G1014) staining.

In vivo bioluminescence imaging in mice

We performed lung metastasis in these mice by the intravenous injection of S1601-luc (luciferase) cells, followed by in vivo imaging. The dosage of D-Luciferin and imaging times were as previously described [21]. D-Luciferin (S7763) was from Selleck.cn.

IHC staining and H&E staining

A tissue microarray composed of tumor and adjacent normal tissue was stained to identify caspase3 and CD8 proteins. IHC and H&E staining were performed as previously described [22]. All antibodies were used in dilution according to manufacturers’ instructions.

Cell proliferation assay

STJ-1601 tumor cells were seeded in 96-well plates at equivalent numbers. One group served as a control, while the other groups were treated with 1 µg/mL, 4 µg/mL LPS. CCK-8 detection reagent was added to the culture medium on days 0, 1, 2, and 3, followed by incubation in a cell culture incubator for 2 h. Absorbance values for each well were subsequently measured.

Transfection

S1601 cells were seeded in 6-well plates at 5 × 10⁴ cells/well. After 24 h, 5µL of empty vector lentivirus lentiviruses (negative control, CON313 Genepharma, sequence: TTCTCCGAACGTGTCACGT) and 1 mL complete medium were added to each well. The viral supernatant was replaced with fresh complete medium at 48 h. Fluorescence microscopy was performed at 72 h to confirm universal fluorescence. Puromycin was supplemented in the medium at a 1:5000 ratio to select stably transfected cells.

Fibroblast and cancer cell co-culture assay

NIH3T3 fibroblasts were pretreated with different concentrations of LPS (1 µg/mL, 4 µg/mL, 10 µg/mL), NF-κB inhibitor BAY 11-7082 (BAY, MCE, HY-13453, USA) (1µM), and BAY (1µM) combined with LPS (4 µg/mL), respectively. After 24 h, NIH3T3 cells were co-cultured with GFP-labeled S1601 cells at a ratio of 1:20. Fluorescence microscopy imaging was subsequently performed at 0 h and 48 h.

Statistical analysis

Statistical significance was determined using an unpaired two-tailed Student’s t-test, one-way ANOVA, two-way ANOVA, Fisher’s exact test, log-rank test, and the Pearson correlation coefficient as indicated. P-value < 0.05 was considered significant (NS, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001). All data were analyzed using GraphPad Prism 8 software. All the above experiments were performed in at least three independent replicates. All experimental values were represented as mean ± SD.

Results

LPS treatment suppresses lung metastases in immunocompetent C57BL/6 mice

Lung cancer development is frequently associated with chronic inflammation [23], which leads to the accumulation of numerous immune cells and proinflammatory factors in the TME. To investigate the effects of acute inflammation, we evaluated LPS treatment in a murine lung metastasis model. Immunocompetent C57BL/6 mice were intravenously injected with 1 × 10⁶ S1601-Luc mouse lung tumor cells. Remarkably, lung metastasis was significantly suppressed or even eliminated in LPS-treated mice, whereas untreated control mice developed extensive lung metastases (Fig. 1A-B). Furthermore, we observed a dose-dependent suppressive effect, with mice receiving 20 µg LPS exhibiting more pronounced suppression of lung metastases compared to those receiving 10 µg–5 µg (Fig. 1B). H&E staining confirmed that the lung tumor burden of mice treated with 20 µg LPS was dramatically reduced compared to controls, an effect that was consistent in both male and female mice (Fig. 1C-D). These findings demonstrate that acute inflammation induced by LPS treatment effectively suppresses lung metastasis in C57BL/6 mice.

Fig. 1.

LPS treatment suppresses lung metastasis in immunocompetent C57BL/6 mice. A Experimental timeline: C57BL/6 mice received an i.v. injection of 1 × 10⁶ S1601-luc cells, followed by i.p. administration of three doses of LPS (5, 10 and 20 µg per mouse) at the indicated time points. B. In vivo bioluminescence imaging of lung metastatic burden (n = 4 mice/group). C. Representative H&E-stained female mice lung sections. (n = 4 mice/group, Scale bars=100 μm). D. Representative H&E-stained male mice lung sections (n = 4 mice/group, scale bars=1000 μm). Statistical significance: NS (not significant), *P < 0.05, **P < 0.01, ***P < 0.001

LPS treatment prolongs survival of mice in a lung metastasis model

We next evaluated the effect of LPS treatment on the overall survival of mice with lung metastasis. Immunocompetent C57BL/6 mice were intravenously injected with 1 × 10⁶ S1601-luc cells and divided into two groups. One group received 20 µg of LPS per mouse weekly for three weeks, while the other group served as the untreated control. The results demonstrated that LPS treatment significantly prolonged overall survival, with similar results obtained in both male and female mice (Fig. 2A-C). Thus, LPS treatment not only suppresses lung metastasis but also prolongs survival.

Fig. 2.

LPS treatment prolongs survival of mice in a lung metastasis model. A Experimental timeline for survival study: C57BL/6 mice received an i.v. injection of 1 × 10⁶ S1601-luc cells and subsequently received i.p. administration of LPS (20 µg per mouse) at the indicated time points. B Kaplan-Meier survival curve of LPS treatment (n = 9 mice/group) versus untreated (n = 14 mice/group) female mice with lung metastasis. C Kaplan-Meier survival curve of LPS treatment versus untreated male mice with lung metastasis (n = 5 mice/group). D Experimental timeline for high tumor burden model: C57BL/6 mice received an i.v. injection of 2 × 10⁶ S1601-luc cells, followed by i.p. LPS (20 µg per mouse) at indicated the times. E In vivo luminescence imaging showing metastatic nodules in LPS (n = 5 mice/group) vs. control groups (n = 4 mice/group). F Experimental timeline for low tumor burden model: C57BL/6 mice received an i.v. injection of 2 × 10⁵ S1601-luc cells with single LPS dose (20 µg per mouse, i.p.) at day 40. Fluorescence imaging of lungs at day 47 revealed metastatic tumors. Statistical significance: NS, *P < 0.05, **P < 0.01, ***P < 0.001

To determine the effective range of this protection, we examined the effect of LPS treatment in a model with a higher tumor burden, injecting mice with 2 × 10⁶ S1601-luc cells. The results showed that LPS treatment significantly suppressed-though did not eliminate-lung metastasis in these mice (Fig. 2D-E). Additionally, we also tested a single LPS treatment in a model with a reduced tumor load, injecting 2 × 10⁵ S1601-luc cells. A single intraperitoneal injection of LPS administered on day 40 still substantially suppressed lung metastasis by day 47 (Fig. 2F). Together, these results demonstrate that LPS treatment can effectively suppress lung metastasis and significantly improve survival in mice.

LPS-induced suppression of lung metastases is dependent on host immunity

LPS is known to activate immune cells via TLR4. To investigate the role of host immunity, we repeated our experiments in immune-compromised nude mice, which lack T and B cells but retain macrophages and NK cells, and severely immune-deficient NOD-SCID mice, which lack most immune effector cells. First, we intravenously injected 5 × 10⁵ S1601-luc cells into nude mice. In vivo fluorescence imaging showed no significant difference in tumor metastasis between LPS-treated and untreated nude mice. Furthermore, lung weights and surface tumor counts were comparable between the two groups (Fig. 3A-B). H&E staining confirmed no significant differences in lung metastatic nodules between LPS-treated and control nude mice (Fig. 3C). These results indicate that LPS treatment neither protects against nor promotes lung metastasis in immune-compromised nude mice.

Fig. 3.

LPS-induced suppression of lung metastases is dependent on host immunity. A Experimental timeline: Nude mice or NOD-SCID mice received an i.v. injection of 5 × 10⁵ S1601 cells, followed by i.p. LPS (20 µg per mouse) at the indicated times. B In vivo bioluminescence imaging quantified lung metastatic burden in LPS vs. control groups (n = 5 mice/group). C H&E-stained lung sections (n = 5 mice/group, scale bar=1000 μm). D Luminescence imaging of lungs from NOD-SCID mice (fully immunodeficient) injected with 5 × 10⁵ S1601 cells ± LPS (n = 5 mice/group). E The effect of S1601 on proliferation after LPS (0, 1 µg/mL, 4 µg/mL) treatment. F NIH3T3 fibroblasts were pretreated ± LPS (1 µg/mL, 4 µg/mL, 10 µg/mL, 24 h), or with BAY (1µM, 24 h), or with BAY (1µM, 24 h) and LPS (4 µg/mL, 24 h) together, then co-cultured with GFP-labeled S1601-NC cells. Fluorescent tumor cells were quantified after 48 h (scale bar=100 μm). G Quantization of F diagrams Statistical significance: NS, *P < 0.05, **P < 0.01, ***P < 0.001. G Quantification of F-graphs

Next, we performed the same experiments in NOD-SCID mice. Fluorescence imaging demonstrated that LPS treatment significantly enhanced the metastatic area in the lungs of NOD-SCID mice, rather than suppressing it (Fig. 3D). These findings suggest that in the absence of a functional immune system, LPS-induced acute inflammation actually promotes tumor growth.

Given the complexity and diversity of cell types in the TME, we next investigated the specific effects of LPS on stromal and tumor cells. First, we assessed the direct effects of LPS on tumor cells. Notably, LPS treatment inhibited the proliferation of S1601 mouse lung tumor cells in vitro compared to untreated controls (Fig. 3E). However, this observation cannot explain the LPS-induced promotion of tumor growth observed in NOD-SCID mice. In addition, LPS level in vivo is dynamic and difficult to evaluate. A recent study indicates that LPS directly targets the TLR4 receptor on fibroblasts [24]. We therefore examined the role of fibroblasts in mediating the LPS effect. We treated NIH3T3 fibroblast cells with LPS for 24 h, replaced the medium, and then co-cultured them with GFP-labeled S1601 cells (S1601-NC). After two days, GFP⁺ S1601-NC tumor cells exhibited faster growth when co-cultured with LPS-treated fibroblasts compared to those co-cultured with untreated fibroblasts. This effect was blocked by the NF-κB inhibitor BAY 11-7082 (Fig. 3F), suggesting that LPS-activated fibroblasts promote tumor cell proliferation in an NF-κB-dependent manner.

LPS treatment enhances anti-tumor activities of innate immune cells in mouse lungs

The difference in LPS-induced effects on lung metastasis between immune-competent and immune-deficient mice suggests that host immunity plays a critical role in suppressing tumor growth, despite the pro-tumor influence of fibroblasts [17]. To characterize the effect of LPS on immune cell populations within the TME, we analyzed sorted cells from lung tissue using flow cytometry. Immune cells were isolated from the heterogeneous cell population and various immune cell types were labeled, sorted, and quantified using fluorescent markers. FACS analysis revealed three key findings: (1) A significant increase in mature F4/80⁺CD11b⁺ macrophages in LPS-treated lungs (Fig. 4A); (2) A significant reduction in CD206⁺ M2 macrophages in LPS-treated mice compared to controls (Fig. 4B), suggesting that LPS inhibits M2 polarization and promotes M1 macrophage polarization [25]; and (3) A marked increase in Gr1⁺ neutrophils in LPS-treated lungs (Fig. 4C), indicating that LPS enhances neutrophil recruitment. Collectively, these findings demonstrate that LPS treatment enhances the anti-tumor activities of innate immune cell populations in the lungs of C57BL/6 mice.

Fig. 4.

LPS suppresses lung metastasis through CD8 + T cells and enhanced innate immunity. A-C Flow cytometry of immune populations in LPS-treated lungs: A F4/80⁺CD11b⁺ macrophages, B CD206⁺ M2 macrophages, C Gr1⁺ neutrophils. D CD8⁺ T cell infiltration by IHC (scale bar=400 μm). E Experimental timeline for CD8⁺ T cell depletion: C57BL/6 mice received an i.v. injection of 1 × 10⁶ S1601 cells and subsequently received i.p. administration of LPS (20 µg per mouse) or anti-CD8 antibody (100 µg per mouse) at the indicated time points. F H&E-stained lung sections (n = 4 mice/group, scale bar=1000 μm). G Flow cytometry of CD8⁺ T cells in mouse lungs after CD8⁺ T cell depletion. Statistical significance: NS, *P < 0.05, **P < 0.01, ***P < 0.001

LPS-induced suppression of lung metastasis is CD8⁺ T cell-dependent

Within the immune system, CD8⁺ T cells play a critical role in suppressing tumor development and mediating the killing of cancer cells [26]. We next assessed CD8⁺ T cell infiltration in the lung tumor microenvironment using IHC staining. The results demonstrated a significant increase in CD8⁺ T cells within the tumor tissues of LPS-treated mice compared to untreated controls (Fig. 4D). Although chronic TCR stimulation is known to impair CD8⁺ T cell function in cancer [26], our findings indicate that LPS treatment enhances the infiltration of activated CD8⁺ T cells with cytotoxic potential against lung cancer cells.

To determine whether CD8⁺ T cells are essential for the LPS-mediated suppression, we performed depletion experiments. After administering tumor cells, mice were injected with LPS, or LPS plus anti-CD8 antibody, once a week for three weeks. Notably, while LPS treatment alone suppressed metastasis, depletion of CD8⁺ T cells substantially reversed this protective effect (Fig. 4E-G). These data confirm that the LPS-induced suppression of metastasis is dependent on CD8⁺ T cells.

Macrophages and dendritic cells are essential for LPS-induced suppression of lung metastasis

The priming of CD8⁺ T cells depends on antigen-presenting cells, such as macrophages and dendritic cells. We next analyzed the effect of macrophage depletion via injection of clodronate liposomes (C.L.) in the lung metastasis model. The results showed that depletion of macrophages reversed the LPS-induced suppression of lung metastasis (Fig. 5A-C). Similarly, we examined the effect of dendritic cell depletion by injection of cytochrome C (Cytc), and found that depletion of dendritic cells also reversed the LPS-induced suppression of lung metastasis (Fig. 5A, D, E). Thus, both macrophages and dendritic cells are essential for the LPS-induced suppression of lung metastasis.

Fig. 5.

Macrophages and dendritic cells are essential for LPS-induced suppression of lung metastasis. A Experimental timeline for macrophage depletion or dendritic cell depletion. C57BL/6 mice were first intravenously injected with 1 × 10⁶ S1601 cells. At the indicated time points thereafter, they received the following treatments: intraperitoneal LPS (20 µg/mouse), intravenous cytochrome C (5 mg/mouse) to deplete dendritic cells, or intranasal clodronate liposomes (40 µL/mouse) to deplete macrophages. B H&E-stained lung sections (n = 5 mice/group, scale bar=1000 μm). C Flow cytometry of macrophages in mouse lungs after macrophages depletion. D H&E-stained lung sections (n = 4 mice/group, scale bar=1000 μm). E Flow cytometry of dendritic cells in mouse lungs after dendritic cells depletion. Statistical significance: NS, *P < 0.05, **P < 0.01, ***P < 0.001

LPS treatment enhances the cytotoxicity of CD8⁺ T cells

To determine the effect of LPS treatment on immune cell cytotoxicity, we first examined the apoptotic features of tumor cells in vivo. Caspase-3, a pro-apoptotic protein, serves as a reliable apoptosis marker in cancer cells [27]. IHC staining revealed significantly elevated caspase-3 expression in lung tumor tissues from LPS-treated mice compared to untreated controls (Fig. 6A). Based on previous studies indicating that the major cellular components of tumor tissue include tumor cells and immune cells [28], we collected tumor tissue from the lungs of mice 21 days after tumor cell injection, a single-cell suspension was prepared, and following the removal of immune cells via CD45 labeling, the remaining cells were predominantly tumor cells (S1, A). Furthermore, flow cytometry analysis demonstrated a substantial increase in PI⁺/Annexin V⁺ apoptotic cancer cell populations in LPS-treated lungs (Fig. 6B). These findings indicate that LPS treatment not only boosts immune cell infiltration but also enhances their tumor-killing capacity.

Fig. 6.

LPS treatment enhances the cytotoxicity of CD8⁺ T cells. A Caspase-3⁺ apoptotic cells by IHC (scale bar=400 μm). B Flow cytometry of tumor apoptosis: Annexin V⁺ (early apoptotic), PI⁺ Annexin V⁺ (late apoptotic/necrotic), PI⁺ (necrotic). C CTL assay: S1601 cells co-cultured with CD8⁺ T cells from control (Con-T) or LPS-treated mice (LPS-T). Residual tumor cells were stained with crystal violet (scale bar=100 μm). Statistical significance: NS, *P < 0.05, **P < 0.01, ***P < 0.001

To specifically evaluate the effect of LPS on CD8⁺ T cell cytotoxicity, we conducted a T cell killing assay. CD8⁺ T cells were isolated from both control and LPS-treated C57BL/6 mice and then co-cultured with target S1601 mouse lung tumor cells. Notably, CD8⁺ T cells from LPS-treated mice exhibited a markedly enhanced tumor-killing capacity compared to those from untreated controls (Fig. 6C). This result demonstrates that LPS treatment directly potentiates the cytotoxic function of CD8⁺ T cells.

Discussion

In this study, we demonstrate that LPS treatment suppresses lung metastasis in immune-competent C57BL/6 mice but not in immune-compromised nude mice and immune-deficient NOD-SCID mice. This suppression is mediated by enhanced host immunity, characterized by increased infiltration of macrophages and neutrophils and the activation of CTLs. These findings suggest that the induction of acute inflammation may represent a strategy to enhance therapeutic outcomes in cancer patients.

As a potent inflammatory stimulus, LPS triggers a well-characterized response involving the release of pro-inflammatory factors like TNF-α and IL-1β, the generation of reactive oxygen species, and the activation of immune cells. In the context of lung metastasis, we found that LPS treatment increases F4/80⁺CD11b⁺ macrophages and Gr1⁺ neutrophils, promotes M1 macrophage polarization, and enhances CD8⁺ T cell cytotoxicity. Thus, despite LPS-activated fibroblasts promotes tumor growth, the dominant effect in an immune-competent host is the LPS-induced activation of CD8⁺ T cells and innate immune cells, which leads to the suppression of metastasis.

CD8⁺ T cell activation is known to involve pro-inflammatory signaling. Our study confirms that TLR4 activation via acute LPS treatment increases CD8⁺ T cell infiltration into tumors and enhances their cytotoxic activity against lung adenocarcinoma cells. We propose that LPS treatment activates CD8⁺ T cells and promotes their tumor infiltration, where they work synergistically with other activated innate immune cells to eliminate metastatic cells. This stands in stark contrast to the effect in NOD-SCID mice, which lack these immune cells and where LPS instead promotes metastasis. However, the therapeutic application of LPS requires caution, as excessive amounts can trigger damaging immune over-activation, as seen in conditions like sepsis [29].

The promotion of lung metastasis in NOD-SCID mice is consistent with reports that LPS can enhance cancer cell survival, growth, and invasiveness in various models through pathways like NF-κB [30], JNK [31], and MAPK [32]. However, we found that this pro-tumor effect is not due to direct impact on tumor cells, as LPS attenuated their proliferation in vitro. Instead, we found that co-culture with LPS-activated fibroblasts promotes tumor cell proliferation. LPS levels in vivo are dynamic and difficult to evaluate, while LPS treatment promotes tumor growth in NOD-SCID mice. Thus, we conclude that LPS exerts divergent effects on different cell types within the TME. Collectively, LPS plays a dual role in vivo: it suppresses lung metastasis via activated innate immune cells and CD8⁺ T cells, while it can promote tumor growth via inflamed fibroblasts.

In clinical, bacterial infections are implicated to stimulate antitumor immunity, leading to tumor regression in some contexts [33]. High-grade gliomas (HGG) are primary brain tumors with relatively high incidence and poor prognosis [34]. The median overall survival (OS) time is approximately 14–16 months in patients with glioblastoma (GBM) [35]. Interestingly, a recent report shows that two cases of long-term survival (5 and 14 years, respectively) in glioblastoma patients with concomitant prolonged bacterial wound infection. This implies that bacterial-induced immune activation may contribute to suppression of tumor growth [36].

This study has several limitations. First, as it did not include human cell lines or tissues, it remains uncertain whether similar effects occur in humans given the known differences between animal models and patients. Furthermore, there are currently no systematic clinical data demonstrating an association between LPS exposure or gram-negative bacterial infections and improved tumor prognosis in humans. Second, the relationship between cancer cell TLR4 expression and Gram-negative bacterial infections in modulating cancer progression requires further investigation. Third, since fibroblasts cannot be easily depleted in vivo, their specific contribution needs to be further elucidated through pathway analysis. Four, while the direct application of LPS is not advised, alternative strategies such as the targeted delivery of immune stimulators to specific immune cells could more effectively potentiate anti-tumor immunity while minimizing off-target effects. For example, attenuated or inactivated bacteria can serve as delivery vehicles or adjuvants that stimulate innate immune receptors, thereby boosting the immunogenicity of tumor antigens [37]; Bacterial adjuvants can reprogram TAMs from an immunosuppressive phenotype to a pro-inflammatory, tumoricidal state through trained immunity mechanisms [38, 39]. Overall, bacteria-based immune adjuvants represent a promising strategy to potentiate tumor-specific immune responses and overcome tumor immune evasion. Last, we acknowledge that the sample size (mostly n = 4–5 per group) limits the generalizability of our findings. However, the high consistency of the data within groups and the large effect sizes provide preliminary but compelling evidence for our conclusions.

In conclusion, our results demonstrate that acute inflammation induced by LPS can inhibit lung metastasis in immunocompetent mice by stimulating CD8⁺ T cell-mediated immunity and prolonging survival. This finding suggests that the calibrated induction of acute inflammation could be explored as an approach to improve therapy for lung cancer.

Abbreviations

LPS

Lipopolysaccharide

TME

Tumor microenvironment

TAM

Tumor-associated macrophage

CAF

Cancer-associated fibroblast

CTL

Cytotoxic T lymphocytes

NNK

4-(Methylnitrosamino)-1-(3-pyridyl)-1-butanone

KO

Knockout

C.L.

Clodronate liposomes

Cytc

Cytochrome C

i.p.

Intraperitoneal

i.v.

Intravenous

Authors’ contributions

CWW and LY were responsible for experimental design, experimental operations, data analysis, and manuscript drafting; HM verified experimental data; ZTT conducted literature search and data analysis; WZP provided technical support; DJ performed manuscript revision and funding support; WT contributed to experimental conceptualization and funding acquisition.

Funding

This work was supported by grants from National Nature Science Foundation of China (No. 82172565 (JD), 82103571 (TW); Key Projects of Shandong Provincial Natural Science Foundation/Joint Fund (ZR202306150012 to JD). Youth Natural Science Foundation of Shandong Province (No. ZR2021QH295 to TW). Development Plan for Youth Innovation Teams in Colleges and Universities of Shandong Province of China (2023KJ249 to TW).

Data availability

The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.

Declarations

Ethics approval and consent to participate

All animal experiments were approved by the Ethics Committee of Shandong Second Medical University (Approval No. 2021SDL265).

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.