Immunodynamic changes in a mouse model of malignant pleural effusion
Xiao-Lei Wei, Xu Guo, Chuang-Xin Zhang, Qi Wang, Xiao-Fan Liu, Ming-Ming Shao, Huan-Zhong Shi, Kan Zhai
TL;DR
This study tracks immune changes in mice with a cancer-related fluid buildup, showing how the immune system shifts from active to suppressed as the disease progresses.
Contribution
The study provides a detailed timeline of immune cell dynamics during MPE progression in a mouse model, identifying distinct early and advanced immunological stages.
Findings
Early MPE is marked by increased B cells, T cells, and natural killer cells but not macrophages or neutrophils.
Advanced MPE features immunosuppressive traits like reduced T cell activity and increased PD-1 and PD-L1 expression.
M2 macrophages and Th1 cells show functional decline in advanced MPE, suggesting a shift toward immune tolerance.
Abstract
Malignant pleural effusion (MPE), a common complication of advanced cancers, is associated with poor prognosis and reduced quality of life. Although host–tumor interactions are known to drive MPE development, the associated immune dynamics during disease progression remain unclear. Using a Lewis lung carcinoma-induced MPE model in C57BL/6JNidfc mice, we systematically evaluated general parameters and immune cell changes at two-day intervals throughout disease progression. The day of Lewis lung carcinoma cell injection into the pleural space was designated as day 0. By day 10 post-injection (p.i.), MPE-bearing mice exhibited ~ 10% body weight loss, marking the experimental endpoint. Pleural tumor mass and pleural effusion volume were minimal up to day 4 p.i. but increased sharply from day 6 onward. CD45⁺ immune cell counts rose over time, and days 6, 8, and 10 p.i. marked key stages of…
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Figure 4- —http://dx.doi.org/10.13039/501100001809National Natural Science Foundation of China
- —Beijing Scholars Program
- —Reform and Development Program of Beijing Institute of Respiratory Medicine
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Taxonomy
TopicsPleural and Pulmonary Diseases · Occupational and environmental lung diseases · Cancer Immunotherapy and Biomarkers
Background
Malignant pleural effusion (MPE) is a frequent and clinically significant complication of advanced malignancies, commonly caused by metastatic spread to the pleural cavity [1]. Although detailed epidemiological data are lacking, over 125,000 MPE-related hospitalizations occur annually in the United States, with lung cancer accounting for the highest proportion (37.8%) of cases [2]. The presence of MPE is associated with substantially reduced survival (5.5 vs. 17.7 months) and impaired quality of life, underscoring its prognostic importance [3, 4]. Current treatments for MPE involve a combination of antitumor therapies and palliative care. However, heterogeneity in immune cell composition and disease stage among patients often hampers therapeutic efficacy [5, 6]. Given the narrow therapeutic window in MPE and its rapid clinical progression, a deeper understanding of the immune microenvironment is urgently needed to inform optimal intervention strategies [7].
Human MPE constitutes a unique tumor microenvironment (TME) characterized by failed immune surveillance. Lymphocytic infiltration, particularly by CD4^+^ T cells, plays a central role in host-tumor interactions through complex inflammatory networks, which shape a dynamic and evolving TME [8, 9]. However, the intrinsic heterogeneity of human MPE samples, combined with their typical collection after substantial effusion accumulation, provides only a static snapshot of immune status, thus posing significant challenges for systematic investigation of immune dynamics during MPE progression. Therefore, well-controlled animal models are essential for analyzing these temporal immunological changes.
Several mouse models of MPE have been established, including genetically engineered mesotheliomas, xenografts in immunodeficient mice, and immunocompetent adenocarcinoma models [10]. Given the rarity of mesothelioma and the limitations of immunodeficient models in replicating host-tumor immune interactions, syngeneic immunocompetent systems offer greater translational relevance [11, 12]. In particular, intrapleural implantation of Lewis lung carcinoma (LLC) cells in C57B/6 mice induces pleural effusions with cellular and biochemical characteristics similar to those observed in human MPE [13]. Nonetheless, current studies have largely focused on immune responses at terminal disease stages, emphasizing tumor burden and effusion volume. This narrow temporal focus neglects the evolving immune landscape during disease development, thereby limiting mechanistic understanding of MPE pathogenesis.
To address this gap in the literature, we employed a mouse MPE model to longitudinally assess immune cell phenotypic and functional changes. Our study provides a detailed timeline of immunodynamic shifts, with particular emphasis on differences between early and advanced stages of MPE. These insights may inform the development of targeted, stage-specific therapies for MPE.
Methods
Cells and animals
LLC cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and were cultured in Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with 10% fetal bovine serum (FBS; Thermo Fisher Scientific) at 37 °C in a 5% CO_2_ atmosphere.
Male C57BL/6JNifdc mice (8–10 weeks old, 20–25 g) were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). The mice were acclimatized for 1 week in clean cages with a controlled temperature of 20–26 °C, a 12-h light/dark cycle, ad libitum access to water, and a standard chow diet at the Animal Care Facility of Beijing Chao-Yang Hospital, Capital Medical University. A total of 58 mice were used in this study. Due to low cell numbers at certain time points, cells from two mice were pooled for immunological analyses to ensure robust results. Corresponding parameters were averaged across the two samples. All animal procedures were approved by the Institutional Animal Care and Use Committee of Capital Medical University, Beijing, China (AEEI-2024-042).
Mouse MPE setup
The mice were anesthetized with 0.5% sodium pentobarbital. The anterior and lateral chest walls were shaved and disinfected. A 5-mm horizontal incision was made in the right anterolateral thorax at the xiphoid level. The fascia and muscle layers were retracted to access the pleural cavity. A suspension of 1.5 × 10^5^ LLC cells in 80 µL of phosphate buffered saline (PBS; Thermo Fisher Scientific) was injected into the pleural cavity through the 4th–5th intercostal space, approximately 3–5 mm lateral to the sternal stalk. The incision was closed with a continuous 5 − 0 Ethilon monofilament suture. The mice were monitored until full recovery. Sham-operated mice received 80 µL PBS without LLC cells. The day of LLC injection was designated as day 0.
Sample collection
Body weight, pleural tumors, and pleural lavage fluid or effusion were collected at two-day intervals. In cases of minimal effusion, pleural lavage was performed by injecting 1 mL of PBS into the pleural cavity, repeating this 3–4 times to ensure sufficient recovery. The mice were euthanized by CO_2_ asphyxiation. The abdominal wall was carefully opened, and the viscera were retracted to expose the diaphragm. Lavage fluid or pleural effusion was gently aspirated using a 1-mL syringe, and the volume was measured. Tumors within the pleural cavity were collected and weighed using an electronic scale (A&D, Tokyo, Japan).
Flow cytometry
Cells for flow cytometry analysis were isolated from pleural lavage fluid/effusion of mice. The antibodies used are listed in Table S1. CD45 was used to identify immune cells. Specific immune cell populations were defined as follows: B cells (CD19); T cells (CD3); natural killer (NK) cells (CD3⁻, NK-1.1); macrophages (CD11b, F4/80); neutrophils (CD11b, Ly-6G); CD4^+^ T cells (CD3, CD8⁻); CD8^+^ T cells (CD3, CD8); Th1 cells (CD3, CD8⁻, T-bet); regulatory T (Treg) cells (CD3, CD8⁻, Foxp3, CD25); and M2 macrophages (CD11b, F4/80, CD206). Absolute counting beads (Thermo Fisher Scientific) were used to determine absolute cell numbers in MPE.
For intracellular cytokine detection, the cells were stimulated for 5 h at 37 °C with a cell stimulation cocktail containing protein transport inhibitors (Thermo Fisher Scientific). After stimulation, the cells were stained with surface antibodies in PBS for 15 min at 4 °C. They were then fixed and permeabilized using fixation/permeabilization solution (Thermo Fisher Scientific) for 30 min at room temperature. After washing with permeabilization buffer (Thermo Fisher Scientific), the cells were stained with intracellular antibodies for 30 min at 4 °C, followed by additional washing and resuspension in PBS for flow cytometry. Flow cytometric analysis was performed on a FACS Canto II system (BD Biosciences, San Jose, CA, USA), and data were analyzed using BD FACSDiva Software (BD Biosciences) and FlowJo software (Version X, Ashland, OR, USA).
Phagocytosis assay
Cells were resuspended in Roswell Park Memorial Institute 1640 medium (Thermo Fisher Scientific) supplemented with 10% FBS at a concentration of 1 × 10^6^ cells/mL and incubated at 37 °C for 1 h. The pHrodo red or green zymosan conjugate bioparticles (Thermo Fisher Scientific) was added to the cultures as needed and incubated in the dark for 1.5 h. After incubation, the cells were collected and analyzed by flow cytometry to evaluate the phagocytic capacity of M2 macrophages and neutrophils.
Reactive oxygen species (ROS) generation assessment
Cells were resuspended in Roswell Park Memorial Institute 1640 medium with 10% FBS at a concentration of 2 × 10^6^ cells/mL. CellROX™ reagent (Thermo Fisher Scientific) was added at a final concentration of 5 µM, and the cells were incubated for 30 min at 37 °C. Following incubation, the medium was discarded, and the cells were washed three times with PBS. ROS production by neutrophils was subsequently assessed using flow cytometry.
Statistical analysis
Data normality was assessed using the Kolmogorov-Smirnov test. For comparisons between two groups, we used Student’s t test; for multiple-group comparisons, one- or two-way ANOVA was applied. All analyses were performed in GraphPad Prism 8.0 (La Jolla, CA, USA). Data are presented as mean ± SEM, with P < 0.05 considered statistically significant.
Results
Mouse presentation over the course of MPE progression
The experimental design is illustrated in Fig. 1A. Up to day 8 post-injection (p.i.), the body weights of MPE-bearing mice did not differ significantly from those of sham controls. However, by day 10 p.i., the MPE-bearing mice exhibited an ~ 10% reduction in body weight compared to baseline (day 0; Fig. 1B), which was defined as the experimental endpoint. Pleural tumors and bloody MPE became visible by day 6 p.i., which grew progressively in both invasiveness and volume throughout disease progression (Fig. 1C–E).
Fig. 1. Mouse presentation following intrapleural injection of LLC cells. (A) Schematic overview of the experimental design. On day 0, mice were administered PBS (sham group, n = 3, with each value representing the mean of two mice) or 1.5 × 10^5^ LLC cells (LLC group, n = 3 per time point, with each value representing the mean of two mice) via intrapleural injection. Pleural tumors and lavage fluid/effusion were collected for quantification and analysis of immune cell composition and number. Immune profiling of CD8^+^ T cells, Th1 cells, M2 macrophages, and neutrophils was performed on days 6 and 10 post-injection (n = 4 per time point, with each value representing the mean of pooled effusions from two mice). (B) Body weight, (C) tumor weight, and (D) MPE volume are shown. (E) Representative macroscopic images of pleural tumors and MPE at the indicated time points. Data in (B)–(D) are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001
Progressive accumulation of CD45+ cells in MPE
The number of CD45^+^ immune cells gradually increased over the course of MPE development (Fig. 2A). At day 0, pleural lavage fluid primarily contained B and T lymphocytes, with smaller populations of macrophages, NK cells, and neutrophils (Fig. 2A). B cell counts exhibited a significant increase beginning at day 2 p.i., followed by a sustained rise from day 6 p.i. (Fig. 2B); while T cell counts remained stable initially but rose significantly from day 6 onward (Fig. 2C). NK cells increased after day 4, peaked at day 8, and then declined (Fig. 2D). Macrophage numbers gradually increased starting on day 4 (Fig. 2E), while neutrophils showed an initial rise at day 2, stabilized, and surged again on day 8, becoming the dominant immune cell population by day 10 (Fig. 2A, F). Together, these changes indicated days 6, 8, and 10 as critical timepoints in MPE immunodynamics, coinciding with the initial detection of MPE and a marked reduction in body weight.
Fig. 2. Immune cell dynamics during mouse MPE progression. (A) Quantification of CD45^+^ immune cells (left) and proportional distribution of immune cell subsets (right), with cell types distinguished by color. “Others” denotes remaining CD45^+^ cells not further measured in this study. (B–F) Temporal changes in B cells (B), T cells (C), NK cells (D), macrophages (E), and neutrophils (F). n = 3 per time point, with each value representing the mean of pooled effusions from two mice. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001
Immune cell subsets dynamics during MPE progression
The proportion of CD4^+^ T cells increased on day 2 but subsequently decreased, whereas the proportion of CD8^+^ T cells followed the opposite trend (Fig. 3A). Although their proportions shifted, the absolute numbers of both CD4^+^ and CD8^+^ T cells remained relatively stable until they began to increase progressively from day 6 onward (Fig. 3A). Among CD4^+^ T cell subsets, Th1 cells increased significantly in both proportion and number on day 6. Th1 cell proportions peaked on day 8, while their numbers continued to rise (Fig. 3B). In contrast, the proportion and absolute number of Treg cells increased significantly from day 6 onward, though we also observed a higher Treg frequency on day 4 compared to day 0 (but not day 2) (Fig. 3C). Meanwhile, both the frequency and absolute number of M2 macrophages increased progressively, beginning on days 2 and 4, respectively (Fig. 3D). We also observed days 6, 8, and 10 as critical timepoints in the temporal dynamics of immune cell subsets. Day 6 was characterized by the first appearance of MPE and the substantial increase in number of CD4^+^ T cells, CD8^+^ T cells, Th1 cells, Treg cells, and M2 macrophages, day 8 marked a turning point at which most populations continued to increase except for total CD4⁺ T cells, and day 10 represented the experimental endpoint. Therefore, we defined day 6 and day 10 as representative of early and advanced MPE stages, respectively.
Fig. 3T cell subsets and M2 macrophages during mouse MPE progression. (A) Proportions and absolute numbers of CD4^+^ and CD8^+^ T cells among total T cells. (B and C) Frequencies and absolute counts of Th1 (B) and Treg (C) cells within the CD4^+^ T cells. (D) Frequency and quantification of M2 macrophages among total macrophages. n = 3 per time point, with each value representing the mean of pooled effusions from two mice. Data are presented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001
Early immune stimulation transitions to advanced immune suppression in MPE
To further characterize the immune state in MPE microenvironment, the phenotypic and functional profiles of CD8⁺ T cells, Th1 cells, M2 macrophages, and neutrophils were examined in early and advanced MPE stages. In advanced MPE, CD8⁺ T cells exhibited reduced production of effector cytokines (IFN-γ and TNF-α) and cytotoxic mediators (Granzyme B [GZMB] and Perforin), along with increased expression of exhaustion markers (PD-1 and CTLA-4) (Fig. 4A). These cells also showed reduced FasL expression, indicating suppressed FasL-mediated apoptosis, and decreased Ki-67, indicating impaired proliferative potential (Fig. 4A). Similarly, Th1 cells from advanced MPE showed diminished secretion of IFN-γ, TNF-α, and IL-2, along with reduced proliferation potential (Fig. 4B).
Fig. 4. Phenotypic and functional characterization of CD8^+^ T, Th1 cells, M2 macrophages, and neutrophils in early vs. advanced-stage mouse MPE. (A) Expression of IFN-γ, TNF-α, GZMB, Perforin, PD-1, FasL, CTLA-4, and Ki-67 in CD8^+^ T cells. (B) Median fluorescence intensity (MFI) of IFN-γ, TNF-α, IL-2, and Ki-67 in Th1 cells. (C) Expression of MHC-II, PD-L1, IL-10, and TGF-β in M2 macrophages, as well as phagocytic capacity. (D) Comparison of TNF-α secretion, phagocytosis, and ROS production in neutrophil at early versus advanced MPE stages. n = 4 per time point, with each value representing the mean of pooled effusions from two mice. Data represent mean ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001
In advanced MPE, M2 macrophages exhibited reduced MHC-II levels and increased PD-L1 expression, indicating immunosuppressive activity and impaired antigen presentation (Fig. 4C). IL-10 secretion was markedly elevated in the advanced stage, whereas TGF-β levels remained unchanged. Furthermore, M2 macrophages showed significantly reduced phagocytic activity by day 10 (Fig. 4C). Neutrophils in advanced MPE secreted significantly less TNF-α and exhibited reduced phagocytosis, though ROS production remained high (Fig. 4D). These findings suggested that the progression of MPE is driven by a coordinated shift in both lymphoid and myeloid compartments from an early immunostimulatory state to advanced-stage immunosuppressive and functionally exhausted phenotypes, thereby establishing a microenvironment that promotes disease progression.
Discussion
The significance of infiltrating immune cells and cytokines within the MPE has become increasingly recognized; however, comprehensive investigations into the temporal immune alterations accompanying disease progression remain limited [14]. In this study, we systematically examined the dynamic immune landscape during MPE progression using a mouse model induced by intrapleural injection of LLC cells. Our findings demonstrate that the MPE microenvironment undergoes continuous remodeling as the disease advances, characterized by both compositional and functional changes in immune cells. Specifically, we observed a transition from an immunostimulatory state in the early stage to a predominantly immunosuppressive environment in the advanced stage. These observations offer important insights into the immunopathogenesis of MPE and highlight potential windows for efficient therapeutic intervention.
Clinical studies have established a strong correlation between tumor burden, pleural effusion volume, and poor prognosis in MPE patients [3, 4]. Similarly, our mouse model recapitulated the key pathophysiological features of human MPE, including progressive systemic deterioration concomitant with tumor growth and effusion accumulation. Notably, a significant (~ 10%) reduction in body weight was observed by day 10 p.i., underscoring the model’s clinical relevance and validating its utility for analyzing the dynamic immune microenvironment associated with MPE progression.
Previous reports have shown that the predominant immune infiltrates in human MPE typically include lymphocytes (mainly CD4^+^ T cells, along with CD8^+^ T cells, B cells, and NK cells), followed by macrophages and neutrophils [15]. Interestingly, approximately 20% of human MPE cases exhibit neutrophil predominance, which may result from infection-driven inflammation [16, 17] or patient-specific variability [5]. In contrast, our mouse MPE model revealed neutrophils as the dominant population at the advanced stage, followed by lymphocytes (including B cells, CD4^+^ and CD8^+^ T cells, and NK cells) and macrophages, in the absence of infection. This discrepancy likely stems from inherent differences between human and murine immune systems. Despite genomic conservation, mice exhibit divergent immune ontogeny, activation, and responsiveness to immunological stimuli [18]. Additionally, mice generally have a higher proportion of peripheral B cells compared to T cells [19, 20], which may explain the observed B cell dominance among lymphocyte subsets in our MPE model. While interspecies differences must be acknowledged, the mouse model remains indispensable for delineating the dynamics of specific immune subsets during MPE progression.
One previous study on lung adenocarcinoma progression reported a gradual shift from an immunostimulatory TME to an immunosuppressive TME [21]. Based on changes in body weight, pleural tumor burden, MPE volume, and immune cell dynamics, we identified day 6 and day 10 p.i. as representative of early and advanced MPE stages, respectively. Comparative analysis between these stages revealed a similar immunological shift in our murine MPE model, marked by CD8^+^ T cell exhaustion, reduced NK cell prevalence, impaired Th1 and neutrophil functions, and increased proportions of immunosuppressive M2 macrophages and Treg cells. These findings are consistent with observations in human lung adenocarcinoma and merit further mechanistic investigation. Although the roles of CD4^+^ T cell subsets in MPE have been well documented [22], our results of advanced mouse MPE align with single-cell RNA sequencing data from human MPE, which show diminished IFN-γ and GZMB expression in cytotoxic CD8^+^ T cells, elevated PD-1 and CTLA-4 expression in exhausted CD8^+^ T cells, and impaired antigen presentation and phagocytosis by M2 macrophages [9, 23, 24]. These similarities affirm the translational relevance of our murine MPE model. Based on the immune characteristics of the TME, our findings suggest that tumor-targeted therapy may be more effective during the early stage of MPE, whereas combining it with immunotherapy could be more beneficial at the advanced stage.
Additionally, the shift from immunostimulation to immunosuppression during MPE progression appears to be largely driven by cytokine-mediated regulatory cascades [25]. Immunosuppressive cytokines such as TGF-β and IL-10 promote the recruitment and activation of tumor-associated macrophages and Treg cells while simultaneously inhibiting Th1 cell activity and suppressing CD8^+^ T cell cytotoxicity [25]. Moreover, M2 macrophages and neutrophils may cooperate to enhance TME immunosuppression [26], thereby facilitating tumor invasion into pleural tissues, blood vessels, and lymphatics [27]. However, clinical samples offer only a static snapshot of immune status, limiting their utility in revealing the dynamics of tumor-immune interactions. In contrast, our mouse model allows for longitudinal immune monitoring, making it a valuable platform for decoding immunoregulatory mechanisms throughout MPE progression and for guiding precision-targeted therapies.
This study has some limitations. Our analyses focused on individual immune subsets without assessing their interactions with tumor cells or among themselves. We also did not evaluate soluble mediators within the MPE, which are critical for understanding microenvironmental remodeling during disease progression. Furthermore, reliable immunological biomarkers or pannels that precisely distinguish early MPE stage from advanced MPE stage remain to be identified and validated for clinical application. To address these limitations, future studies using the mouse MPE model should incorporate transcriptomic and proteomic approaches to comprehensively characterize MPE progression and optimize therapeutic interventions.
Conclusions
This study characterized the temporal evolution of body weight, pleural tumor burden, effusion volume, and immune cell profiles during MPE development in a mouse model. Based on systemic and immunological changes, we defined day 6 and day 10 p.i. as early and advanced MPE stages, respectively, which exhibited distinct immunostimulatory and immunosuppressive features. These findings provide insights into the immunomodulatory mechanisms underlying MPE progression and offer valuable guidance for the development of stage-specific, TME-based therapeutic strategies.
Supplementary Information
Below is the link to the electronic supplementary material.
Supplementary Material 1: Table S1. List of antibodies used for flow cytometry
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