The THP-1 human monocytic leukemia cell line (cat. no. FH0112) was procured from Shanghai Fuheng Biotechnology Co., Ltd., while the A549 human lung adenocarcinoma cell line (cat. no. CCL-185), LLC (cat. no. CRL-1642) and BEAS-2B human normal lung epithelial cells (cat. no. CRL-3588), human umbilical vein endothelial cells (HUVECs; cat. no. CRL-1730) were obtained from the American Type Culture Collection. Upon revival, the THP-1, BEAS-2B, A549 HUVEC and LLC cells were cultured in RPMI-1640 medium (Beijing Solarbio Science & Technology Co., Ltd.) supplemented with 10% fetal bovine serum (FBS; cat. no. 164210; Wuhan PuNuoSai Biotech Co., Ltd.) and maintained in a 37°C and 5% CO₂ incubator. Upon reaching 80-90% confluency, adherent cells were detached using 0.25% EDTA-trypsin or directly pipetted, while suspended cells were collected by centrifugation at 4°C and 1,200 x g for 5 min. Cells in the logarithmic growth phase were utilized for subsequent experiments.

The establishment of M1/M2 macrophage models was performed according to previous studies (35,36). THP-1 cells into M0 macrophages were induced using the widely employed phorbol 12-myristate 13-acetate (PMA; cat. no. P1585; Sigma-Aldrich; Merck KGaA), which was followed by polarization into the M1 or M2 macrophage phenotypes using interferon-γ (IFN-γ) or IL-4 (PeproTech China), respectively. For the generation of M0 macrophages, THP-1 cells in the logarithmic growth phase were centrifuged at 4°C and 1,200 x g for 5 min, the supernatant was aspirated and the cells were resuspended in RPMI-1640 medium supplemented with PMA (200 ng/ml) for 24 h. Subsequently, the cell concentration was adjusted to 1×10⁶ cells/ml before seeding into a 6-well plate. For the generation of M1 macrophages, the M0 macrophages were incubated in RPMI-1640 complete medium supplemented with a IFN-γ (20 ng/ml) for 24 h. Similarly, to induce the generation of M2 macrophages, M0 macrophages were cultured in RPMI-1640 complete medium supplemented with IL-4 (20 ng/ml) for 24 h.

Culture medium from the 6-well plate containing macrophages in various polarization states was aspirated, and the plate was washed twice with phosphate-buffered saline (PBS). Subsequently, the macrophages were incubated in serum-free RPMI-1640 medium at 37°C with 5% CO₂ for 24 h. Following this incubation period, the culture medium was harvested in a 5-ml sterile Eppendorf tube and centrifuged at 4°C and 13,800 x g for 15 min. The resulting supernatant, designated as the conditioned medium, was carefully transferred to another 5-ml sterile Eppendorf tube and stored at −80°C for future use.

For the CB treatment, macrophages were exposed to various concentrations of CB (Sigma-Aldrich; Merck KGaA; 10, 15, 20, 25, 30, 35, 40 and 50 ng/ml) for 24 h before being prepared for subsequent experiments. Similarly, for RSG treatment, macrophages were exposed to various concentrations of RSG (Sigma-Aldrich; Merck KGaA; 0.1, 0.5, 1, 5 and 10 μM) for 24 h before being prepared for further experiments. In certain investigations, macrophages were exposed to conditioned medium from M0, M1 or M2 macrophages for 24 h.

M0 macrophages and BEAS-2B, A549 and LLC cells were prepared as single-cell suspensions. After counting, the cells were seeded into a 96-well culture plate at a density of 1×10⁴ cells in 200 μl of medium per well, with 6 replicate wells per group. The next day, after cell adherence, various concentrations of CB or RSG were added to the wells for intervention, in which the control group was treated with an equivalent dose of DMSO (not exceeding 0.1% of the maximum dose). Following a 24-h incubation, 20 μl of 5 mg/ml MTT solution (Beijing Solarbio Science & Technology Co., Ltd.) was added to each well, and the plate was further incubated for ~4 h. Subsequently, 150 μl DMSO solution was added to each well and was used to dissolve the purple formazan, and the plate was shaken at room temperature for 15-20 min. The absorbance (A) values of each well were measured at 490 nm, with blank wells set to 0. The cell viability was then calculated using the following formula: Cell viability (%)=(experimental group A value/control group A value) ×100%.

The culture medium from the 6-well plate containing macrophages in different polarization states was aspirated. The plate was then washed with PBS, which was repeated twice. The cells were scraped off using a cell scraper, repeating this step twice, and collected in a centrifuge tube. The collected cells were gently pipetted repeatedly and then centrifuged at 1,200 x g for 5 min at room temperature. After centrifugation, the cells were resuspended in PBS at a final concentration of 1×10⁵ cells/ml, then non-specific antigens were blocked using 5% BSA buffer (MilliporeSigma) for 20 min at room temperature. For intracellular CD86 staining, fixation in 4% paraformaldehyde for 20 min at room temperature and permeabilization with 0.1% Triton X-100 were performed prior to staining. The cells were then stained with monoclonal mouse anti-human CD86 (1:50; cat. no. PE-65155) and CD206 (1:100; cat. no. FITC-65165) antibodies (both from Proteintech Group, Inc.) for 30 min at 4^oC in the dark. Subsequently, the cells were analyzed using a BD FACSCanto II flow cytometer (BD Biosciences) and the FlowJo 10 software (BD Biosciences). A non-specific mouse Ig was used as an isotype control.

Total RNA in cells was extracted using the TRIzol method (Thermo Fisher Scientific, Inc.). The total RNA concentration of each sample was measured using a NanoDrop-2000 UV spectrophotometer. RT was performed using the RevertAid First Strand cDNA Synthesis Kit (cat. no. K1621; Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol and qPCR was conducted using the PowerUp™ SYBR™ Green Master Mix (cat. no. A25743; Thermo Fisher Scientific, Inc.). PCR thermocycling conditions were as follows: Denaturation at 94°C for 120 sec, followed by 40 cycles consisting of melting (95°C; 15 sec) and annealing/extension (60°C; 60 sec) phases. GAPDH was used as an internal reference to calculate the relative transcription level of the target gene by the 2^−ΔΔCq method (37). The primer sequences are shown in Table I.

Cells were seeded at a density of 5×10⁵ cells/ml in confocal culture dishes to prepare macrophages in different states. The culture medium was removed and the wells were washed twice with PBS. Next, 500 μl of 4% paraformaldehyde was added to each well and incubated at room temperature for 30 min, then 500 μl of 0.5% Triton X-100 was added to each well and further incubated at room temperature for 10 min. Blocking solution was next added to the wells and incubated at room temperature for 15 min. Then, primary antibodies against PPARγ (1:350; cat. no. 66936-1-Ig; Proteintech Group, Inc.) and macrophage receptor with collagenous structure (MARCO; 1:400; cat. no. bs-2659R; BIOSS) were added to each well and incubated overnight at 4°C. The next day, the corresponding fluorescent secondary antibody (1:200; cat. no. A23210, Abbkine Scientific Co., Ltd.) was added to the wells and incubated at room temperature for 1 h. DAPI (10 μg/ml) staining was performed for 5 min at room temperature in the dark, followed by imaging using a laser confocal microscope.

Protein levels of tumor necrosis factor (TNF)-α (cat. no. E-EL-H0109c; Elabscience Biotechnology Co., Ltd.), IL-6 (cat. no. E-EL-H0102c; Elabscience Biotechnology Co., Ltd.), IL-1β (cat. no. E-EL-H0149c; Elabscience Biotechnology Co., Ltd.), TGF-β (cat. no. MM-1774H1; Jiangsu Meimian Industrial Co., Ltd.) and IL-10 (cat. no. MM-0066H1; Jiangsu Meimian Industrial Co., Ltd.) were determined by respective ELISA assay kits.

Cells were seeded into a 6-well plate at a density of 8×10⁵ cells/ml. The macrophage-conditioned medium was thawed and mixed with RPMI-1640 medium with 1% FBS. After the cells had adhered to the plate and the cell confluency was >90%, a vertical scratch was made in the center of each well using a 200 μl pipette tip. The culture medium was then removed, the scratched cells were washed with sterile PBS to remove non-adherent cells and fresh mixed culture medium was added to the plate. The 6-well plate was placed horizontally in a 5% CO₂ and 37°C incubator. After 24 h, images were collected under a light microscope, and the results were measured and analyzed using ImageJ software version 1.48 (National Institutes of Health). Wound healing rate=(0 h scratch width-24 h scratch width)/0 h scratch width ×100%.

The cell invasion assay was performed using a 6-well 8-μm Transwell chamber. For this, Matrigel was diluted with ice-cooled serum-free medium, and 150 μl of the matrix gel was added to each well. The 6-well plate was then placed in a cell culture incubator at 37°C with 5% CO₂ for 30 min. The 6-well plate was then placed in a cell culture incubator with 5% CO₂ at 37°C for 30 min. Next, 2 ml A549 or LLC cell suspension (5×10⁵ cells/ml) was added to the upper chamber, and 2 ml macrophage-conditioned medium was added to the lower chamber. After incubation for 48 h, the cells in the upper chamber were wiped off with a cotton swab, and the invasive cells were fixed with 4% paraformaldehyde at room temperature for 30 min, washed with PBS, stained with 0.1% crystal violet solution for 30 min at room temperature, and then observed and imaged under a light microscope. Cell counting was subsequently performed using ImageJ.

The culture medium was aspirated and the wells were washed twice with PBS. The 6-well plate was then transferred onto ice and 250 μl pre-prepared RIPA lysis buffer (cat. no. R0010; Beijing Solarbio Science & Technology Co., Ltd.) containing protease and phosphatase inhibitors (cat. no. P0100; Beijing Solarbio Science & Technology Co., Ltd.) was added to each well. The plate was further cooled on ice and the cells lysed for 30 min. The lysate was then transferred to a 1.5-ml sterile Eppendorf tube and centrifuged at 4°C and 13,800 g for 15 min. The protein concentration was determined using the BCA method and the proteins were then denatured. Subsequently, protein samples (50 μg for each lane) were separated on 10% SDS-polyacrylamide gels, followed by transferring onto PVDF membranes (MilliporeSigma). The PVDF membranes were blocked with 5% BSA (MilliporeSigma) for 2 h at room temperature. After that, primary antibodies against Arg-1 (1:1,000; cat. no. bs-23837R; BIOSS), inducible nitric oxide synthase (iNOS; 1:1,000; cat. no. bs-0162R; BIOSS); phosphorylated (p)-PPARγ (1:1,000; cat. no. bs-3737R; BIOSS), vimentin (1:1,000; cat. no. bsm-33170M; BIOSS), E-cadherin (1:1,000; cat. no. bs-1519R; BIOSS), N-cadherin (1:1,000; cat. no. bs-20623R; BIOSS) and GAPDH (1:3,000; cat. no. 60004-1-Ig; Proteintech Group, Inc.) were incubated with the membrane overnight at 4°C, followed by washing with Tris-buffered saline containing 0.05% Tween 20 and incubation with horseradish peroxidase-conjugated secondary antibodies goat anti-mouse IgG (1:10,000; cat. no. SA00001-1) and goat anti-rabbit IgG (1:10,000; SA00001-2; both from Proteintech Group, Inc.) at room temperature for 1 h. The western blot bands were visualized using an ECL kit (cat. no. PE0010; Beijing Solarbio Science & Technology Co., Ltd.). Densitometric analysis was performed using ImageJ software version 1.48 (National Institutes of Health).

For the preparation of conditioned medium, macrophages were treated with the aforementioned drugs, the drug-containing supernatant was removed and the cells were cultured for 24 h. The supernatant was then collected as conditioned medium for culturing cancer cells (A549 and LLC) for 24 h. After removing the supernatant, fresh culture medium was added to the cancer cells and the culturing was continued for another 24 h. The supernatant was then collected as conditioned medium for culturing HUVECs for 24 h. Matrigel Basement Membrane Matrix (BD Biosciences) was diluted with EBM-2 medium (Thermo Fisher Scientific, Inc.) and used to coat 24-well plates at 37°C for 1 h. Subsequently, 5×10⁴ HUVECs were treated with different conditioned media from the A549 or LLC cells for 24 h. The tube formation ability of HUVECs was then assessed by quantifying the number of meshes, tube length and number of branches.

All animal studies were conducted following protocols approved by the Animal Ethics Committee of Henan University of Chinese Medicine (Zhengzhou, China; approval no. IACUC-202302006). In this experiment, 18 6-8-week-old male BALB/c nude mice (18-21 g) were randomly assigned to 3 groups (n=6 per group) and housed in standard cages at 20-24°C with a 12/12-h light-dark cycle and ad libitum access to food and water. A 100-μl PBS suspension containing 5×10⁶ LLC cells was subcutaneously injected into the right flank of each mouse. To assess the effect of the LLC cells, data were collected every 5 days starting 4-5 days after tumor cell inoculation, which included animal weight and tumor dimension measurements. The mice were continuously fed for 25 days and received intraperitoneal injections of CB (10 mg/kg/day) (38,39) or DDP (2 mg/kg/day). For subcutaneous tumors, the maximum allowable diameter was 20 mm for each mouse. The tumor size was checked every other 1-2 days. The biggest tumor volume in the present study was 2,664.12 mm³. At the end of the experiments, the mice were euthanized using CO₂ (30-70% volume displacement of the chamber air per min) followed by cervical dislocation as a secondary method of euthanasia, in accordance with the approved protocol of the Experimental Animal Ethics Committee. The tumors from all animals were collected for subsequent analysis.

Experimental data were analyzed using GraphPad Prism 8.0 (Dotmatics) statistical software and are presented as the mean ± standard deviation. Normal distribution of the data was assessed by the Kolmogorov-Smirnov test. The unpaired Student's t-test was used for the comparison of differences between two groups, and one-way ANOVA followed by Bonferroni's multiple comparison tests was used for the comparison of differences among >2 groups. P<0.05 was considered to indicate a statistically significant difference.

The THP-1 cells appeared round and proliferated in suspension. Following induction with PMA for 24 h, the THP-1 cells underwent differentiation into M0 macrophages, which was accompanied by observable morphological changes. These changes included an increase in cell volume, resulting in an oval-shaped appearance, with a few fibroblast-like pseudopodia extending from the periphery. The cells also began to adhere to a surface. Upon the subsequent induction with IFN-γ, the M1 macrophages exhibited further changes, including an increased volume, enhanced surface adherence and an elongated morphology. Conversely, upon induction with IL-4, the M2 macrophages displayed an increase in volume, maintained an oval-shaped morphology, exhibited extending pseudopodia and tended to aggregate for growth (Fig. 1).

The M0 macrophages were exposed to varying concentrations of CB, resulting in a concentration-dependent decrease in cell viability (Fig. 2A). Similarly, treatment with CB for 24 h resulted in a decrease in normal lung epithelial cell (BEAS-2B) viability starting at a concentration of 30 ng/ml (Fig. 2B). Additionally, CB exhibited a concentration-dependent reduction in the viability of A549 and LLC cells, with a calculated IC₅₀ of 28.80 and 30.12 ng/ml, respectively (Fig. 2C and D). CB treatment inhibited the viability of A549 and LLC cells in a concentration-dependent manner, while concentrations below 30 ng/ml had no impact on the viability of M0 macrophages and BEAS-2B lung epithelial cells.

THP-1 monocytes differentiate into macrophages when induced by PMA, leading to changes in cell morphology and surface markers (40). Therefore, macrophage surface markers were assessed using flow cytometry in the present study. The findings revealed a higher percentage of CD86⁺ cells in M1 macrophages compared with M0 macrophages, and CB treatment did not influence the percentage of CD86⁺ cells in the M1 macrophages (Fig. 3A and B). Furthermore, the percentage of CD206⁺ cells was higher in M2 macrophages compared with M0 macrophages. Nevertheless, CB treatment significantly reduced the percentage of CD206⁺ cells in the M2 macrophages (Fig. 3C and D). The findings indicated that THP-1 monocytes, upon differentiation into macrophages via PMA induction, exhibit specific changes in surface markers.

The RT-qPCR results revealed significantly higher levels of CD206, Arg-1 and PPARγ mRNA expression in M2 macrophages compared with M0 macrophages (Fig. 4A-C). CB treatment failed to affect the CD206 and PPARγ levels in M0 macrophages, while Arg-1 mRNA expression was upregulated in the M0 + CB group compared with the M0 group (Fig. 4A-C). Notably, CB treatment led to a significant downregulation of MARCO mRNA expression in M2 macrophages (Fig. 4D). Consistent findings regarding the protein expression levels of Arg-1, PPARγ and MARCO were detected by western blotting (Fig. S1). Additionally, the TNF-α and IL-1β mRNA expression levels were upregulated in M1 macrophages compared with M0 macrophages. Furthermore, CB treatment significantly increased TNF-α and IL-1β mRNA expression in M0/M1 macrophages (Fig. 4E and F). These results indicated that M2 macrophages had elevated CD206, Arg-1 and PPARγ mRNA levels, which were unaffected by CB in M0 macrophages (except Arg-1 upregulation), while CB downregulated MARCO in M2 macrophages and increased TNF-α and IL-1β mRNA in both M0 and M1 macrophages.

Supernatant proteins were assessed using ELISA. There was a significant increase in the IL-6, IL-1β and TNF-α protein levels in the supernatant of M1 macrophages compared with M0 macrophages (Fig. 5A-C). Upon CB treatment, there was a further augmentation in IL-6, IL-1β and TNF-α protein levels in the supernatant of M1 macrophages (Fig. 5A-C). Additionally, the IL-10 and TGF-β levels were higher in the supernatant of M2 macrophages compared with M0 macrophages. However, CB treatment resulted in a reduction in IL-10 and TGF-β protein levels in the supernatant of M2 macrophages (Fig. 5D and E). The results indicated that IL-6, IL-1β and TNF-α levels were elevated in M1 macrophages and further increased with CB treatment, while IL-10 and TGF-β levels were higher in M2 macrophages but decreased with CB treatment.

In the complex TME, TAMs interact with the TME, undergo metabolic changes and alter the anticancer phenotype of early M1-TAMs (38). Typically, in the early stages of tumor development, M1-like TAMs predominate the TME and inhibit tumor growth by secreting certain inflammatory factors (39). As cancer progresses, late-stage M2-like TAMs begin to dominate the TME, promoting angiogenesis and aiding tumor cell dissemination and metastasis, thus exhibiting pro-tumor effects (40). In the present study, M0 macrophage-conditioned medium treatment significantly improved the wound healing of A549 and LLC cells compared with the control group (Fig. 6A-D). Additionally, M2 macrophage-conditioned medium further enhanced the migratory abilities of A549 and LLC cells compared with the M0 group (Fig. 6A-D). However, CB-treated M0 macrophage-conditioned medium showed no significant effect on the migration of A549, but attenuated the migration of LLC cells compared with the M0 group (Fig. 6A-D). Furthermore, CB-treated M2 macrophage-conditioned medium reduced A549 and LLC cell migration compared with the M2 group (Fig. 6A-D). The results indicated that M0 macrophage-conditioned medium significantly enhanced wound healing in A549 and LLC cells, with M2 macrophage-conditioned medium further boosting their migratory abilities, while CB-treated M0 medium had no significant effect on A549 migration but reduced LLC migration, and CB-treated M2 medium decreased migration in both cell types.

M2 macrophage-conditioned medium enhanced A549 and LLC cell invasion compared with the M0 group (Fig. 7A-D). However, CB-treated M0 macrophage-conditioned medium showed no significant effect on A549 and LLC cell invasion compared with the M0 group (Fig. 6A-D). Furthermore, CB-treated M2 macrophage-conditioned medium reduced A549 and LLC cell invasion compared with the M2 group (Fig. 7A-D). These results indicated that M2 macrophage-conditioned medium enhanced A549 and LLC cell invasion, whereas CB treatment had no significant effect on M0 macrophage-conditioned medium but reduced invasion in CB-treated M2 macrophage-conditioned medium.

First, lung cancer cells (A549 and LLC) were treated with conditioned medium from M0, M2 or CB-treated M2 macrophages. Then, the supernatants of the treated lung cancer cells were used to treat HUVECs. As shown in Figs. S2 and S3, the tube formation ability of HUVECs was significantly enhanced in the M2 group compared with the M0 group, and the tube formation ability was attenuated in the M2 + CB group compared with the M2 group.

Research has found that PPARγ can regulate the expression of the M2 macrophage-related factor, Arg-1 (25). Additionally, PPARγ can promote the progression of colon cancer cells and the growth of mouse tumors by affecting M2 polarization. RSG is an agonist of PPARγ. As demonstrated in Fig. 8A, RSG concentrations ranging from 0.1-10 μM had no effect on M0 macrophage viability. PPARγ mRNA expression in M2 macrophages was higher than that in M0 macrophages, which was significantly downregulated by CB treatment but not by RSG treatment (Fig. 8B). Additionally, CB treatment downregulated PPARγ mRNA expression in RSG-treated M2 macrophages (Fig. 8B). The nuclear translocation of PPARγ was enhanced in M2 macrophages compared with M0 macrophages. CB treatment decreased PPARγ expression in the nucleus, whereas RSG treatment increased PPARγ expression in the nucleus of M2 macrophages. Furthermore, CB treatment reversed the RSG-induced increase in nuclear expression of PPARγ in M2 macrophages (Fig. 8C). MARCO belongs to the class A scavenger receptor molecules. Research has revealed that macrophages express MARCO extensively on their surface, and MARCO⁺ TAMs in the TME tend to exhibit the M2 phenotype (40). In the present study, the expression of MARCO was elevated in M2 macrophages compared with M0 macrophages. CB treatment decreased MARCO expression in the nucleus, while RSG treatment increased MARCO expression in M2 macrophages. Furthermore, RSG treatment partially restored the CB-induced decrease in MARCO expression in M2 macrophages (Fig. 8D). These results indicated that CB treatment significantly downregulated PPARγ mRNA expression and nuclear translocation in M2 macrophages, whereas RSG treatment had opposite effects, with CB reversing the RSG-induced increase in PPARγ expression; additionally, CB reduced MARCO expression in M2 macrophages, while RSG partially restored CB-induced MARCO downregulation.

As shown in Fig. 9A, the effects of CB on macrophage polarization-related proteins were assessed. Arg-1 protein expression was upregulated in M2 macrophages compared with M0 macrophages. CB treatment repressed Arg-1 protein expression, whereas RSG elevated Arg-1 protein expression in M2 macrophages (Fig. 9A and B). Furthermore, CB treatment attenuated the RSG-induced increase in Arg-1 protein expression in M2 macrophages (Fig. 9A and B). No difference in iNOS protein expression between M0 and M2 macrophages was observed (Fig. 9A-C). CB treatment increased iNOS protein expression, while RSG had no effect on iNOS expression in M2 macrophages (Fig. 9A and C). Additionally, RSG treatment partially mitigated the CB-induced increase in iNOS expression in M2 macrophages (Fig. 9A and C). p-PPARγ expression was upregulated in M2 macrophages compared with M0 macrophages. CB treatment repressed p-PPARγ protein expression, while RSG elevated p-PPARγ protein expression in M2 macrophages (Fig. 9A and D). Furthermore, CB treatment attenuated the RSG-induced increase in p-PPARγ protein expression in M2 macrophages (Fig. 9A and D). These results revealed that in M2 macrophages, CB treatment decreased Arg-1 and p-PPARγ protein expression while increasing iNOS protein expression; conversely, RSG treatment increased Arg-1 and p-PPARγ expression without affecting iNOS, with RSG partially reversing the CB-induced effects on these proteins.

EMT is the process by which polarized epithelial cells transform into mesenchymal-like cells. During this transformation, cell surface epithelial markers such as E-cadherin and desmoplakin are downregulated, while mesenchymal markers such as N-cadherin and vimentin are upregulated, resulting in the loss of cell adhesion and apical-basal polarity, which promotes tumor progression and metastasis (41). As demonstrated in Fig. 10A, the effects of CB on EMT-related proteins in A549 lung cancer cells were assessed using western blotting. E-cadherin was downregulated in A549 cells treated with M2 macrophage-conditioned medium compared with M0 macrophage-condition medium (Fig. 10A and B). The E-cadherin protein level was increased in A549 cells cultured with CB-treated M2 macrophage-conditioned medium compared with M2 macrophage-conditioned medium, while RSG-treated M2 macrophage-conditioned medium had no effect on E-cadherin expression in A549 cells compared with M2 macrophage-conditioned medium (Fig. 10A and B).

Vimentin and N-cadherin were upregulated in A549 cells treated M2 macrophage-conditioned medium compared with M0 macrophage-condition medium (Fig. 10A, C and D). The vimentin and N-cadherin protein levels were decreased in A549 cells cultured with CB-treated with M2 macrophage-conditioned medium compared with M2 macrophage-conditioned medium, while RSG-treated M2 macrophage-conditioned medium increased vimentin and N-cadherin protein levels in A549 cells compared with M2 macrophage-conditioned medium (Fig. 10A, C and D). Furthermore, the RSG-mediated effects on vimentin and N-cadherin expression in A549 cells were attenuated by CB treatment (Fig. 10A, C and D). The results indicated that in A549 lung cancer cells, treatment with M2 macrophage-conditioned medium downregulated E-cadherin and upregulated vimentin and N-cadherin levels, effects attenuated by CB treatment but exacerbated by RSG treatment, indicating modulation of EMT-related protein expression.

The effects of CB on in vivo tumor growth were further examined using a nude mice xenograft model. As revealed in Fig. 11A-C, both DDP and CB treatments inhibited the in vivo tumor growth of LLC cells. However, DDP, but not CB, reduced the body weight of the nude mice. Additionally, both CB and DDP treatments significantly upregulated CD86 mRNA expression in the tumor tissues (Fig. 11E). However, CB, but not DDP, downregulated Arg-1 expression in the tumor tissues compared with the control group (Fig. 11F). These results indicated that both CB and DDP treatments effectively suppressed LLC tumor growth and increased CD86 mRNA expression in tumor tissues, with DDP additionally reducing body weight and CB uniquely downregulating Arg-1 expression compared with controls.