Ganoderma lucidum polysaccharide-decorated extracellular vesicle enables synergistical antitumor immunotherapy

Abstract

Ganoderma lucidum polysaccharide (GLP) holds considerable promise for tumor therapy, but its clinical application is limited by its poor tumor-targeting capability. Herein, we report the development of an immunoregulatory nano-bioconjugate formed by conjugating GLPs with low-pH culture medium reprogrammed CT26 tumor cell-derived extracellular vesicle (LTEV). Specifically, 4-carboxybenzeneboronic acid (CPBA), used as an intermediate coupling agent, is conjugated with the amino group of LTEV via its carboxyl group to obtain CPBA-LTEV. GLP is then covalently linked via boric ester linkages between its hydroxyl groups and the boronic acid groups of CPBA. After systemic administration, GLP@LTEV accumulates in homologous tumor tissues derived from the LTEV parent cells, remodeling the immunosuppressive tumor microenvironment (TME) by repolarizing M2-like macrophages towards the M1 phenotype, promoting dendritic cell (DC) maturation, activating cytotoxic T lymphocytes (CTLs), and inhibiting immunosuppressive regulatory T cells (Tregs). The synergism of tumor-targeting delivery and potent immunomodulatory effects in this combined manner therefore significantly inhibits subcutaneous tumor growth and lung metastasis with minimal side effects, providing a novel combination of polysaccharide and nanovesicle for robust tumor immunotherapy.

Graphical abstract

GLP@LTEV effectively accumulates in tumor tissues and significantly reverse the immunosuppressive TME via repolarizing M2 macrophages towards M1 phenotype, promoting DC maturation, activating CTLs, and inhibiting Tregs for robust tumor immunotherapy.

1. Introduction

Colorectal cancer (CRC) ranks third in global cancer incidence, characterized by a rising burden of incidence and mortality rates worldwide [1,2]. The tumor microenvironment (TME) of CRC is a complex ecosystem comprising tumor, immune, and stromal cells, alongside blood vessels and extracellular matrix (ECM) components [3,4]. This microenvironment exhibits a profoundly immunosuppressive phenotype, which manifests as poor immune cell infiltration and confers resistance to immunotherapeutic interventions [5]. Tumor-associated macrophages (TAMs) and immunosuppressive regulatory T cells (Tregs) are pivotal in establishing the immunosuppressive microenvironment of CRC [6,7]. Therefore, repolarizing TAMs from the M2 to the M1 phenotype and inhibiting Tregs represent effective strategies for reshaping the immunosuppressive microenvironment [[8], [9], [10], [11], [12]].

Over the past decades, Ganoderma lucidum-derived polysaccharide (GLP) has emerged as an efficient immunomodulatory agent for antitumor therapy because of its immunoactivity advantages [13,14]. GLP reprograms macrophages to the M1 phenotype, thereby promoting macrophage-mediated endocytosis of tumor cells [[15], [16], [17], [18]]. In addition, GLP enhances dendritic cell (DC) antigen presentation by promoting DC maturation, thereby potentiating cytotoxic T lymphocyte (CTL) activity and reducing the Treg ratio in antitumor immunotherapy [[19], [20], [21], [22], [23]]. However, GLP has several challenges, particularly its poor tumor-targeting ability, which limits its therapeutic potential in clinical application. Efforts in recent years have focused on enhancing GLP delivery, for example, liposome-based GLP delivery systems [24,25] and gold nanoparticles (AuNP) containing immunoactive GLP [26] significantly enhance immune responses, while rutin-carboxyphenyl boronic acid (CPBA)-GLP-dithiodipropionic acid (DPA)-dihydroartemisinin (DHA)/10-hydroxy camptothecin (HCPT) polymeric nanoparticles (RCGDDH NPs) improve cellular uptake efficiency and tumor growth inhibition in vivo [27]. Despite these advantages, GLP-based systems show limited efficacy in synergistic antitumor immunotherapy for CRC, which remains highly underexplored.

Recently, extracellular vesicles (EVs), including exosomes, microvesicles, and apoptotic bodies, have been extensively studied. They not only show remarkable performance in delivering various cargos such as RNAs, peptides, and small molecules [[28], [29], [30]], but also exhibit excellent stability and biocompatibility both in vitro and in vivo [31,32]. Almost all types of cells can release EVs. More importantly, tumor cell-derived EVs (LTEVs) are produced in substantially greater quantities than those secreted by normal cells [33]. Previous studies have revealed that low-pH culture medium reprogrammed LTEVs possess homologous tumor-targeting capability and high uptake efficiency, which can significantly enhance antitumor efficacy [34,35]. This finding motivates us to combine LTEV and GLP, thereby enabling a more efficient synergism enhancement of targeted reprogramming and antitumor immunotherapy.

Here, we construct a biomimetic nanosystem capable of remodeling the immunosuppressive TME, thus enhancing antitumor therapy of CRC. The LTEV is decorated with GLPs, allowing GLPs to accumulate at the tumor site through the homologous recognition capability of LTEV. This design of GLP@LTEV not only promotes TAM polarization towards the antitumor M1 phenotype, but also facilitates the DC maturation, activates CTLs, and suppresses Tregs in tumor tissues. Then, the activated CTLs secrete interferon-γ (IFN-γ), which further induces M2 macrophages to the M1 phenotype, generating robust tumor-specific immune responses, leading to reshaping the immune-inhibitory TME. As a result, both the CT26 subcutaneous tumor and lung metastasis are effectively suppressed with minor side effects (Scheme 1), showing a pioneering concept for immunotherapy in immune-desert tumors.

Scheme 1.

Schematic illustration of the preparation and synergistical antitumor immunotherapy of GLP@LTEV.

2. Materials and methods

2.1. Regents and materials

Ganoderma lucidum polysaccharide (GLP) was purchased from Shanxi Ciyuan Biotechnology Co., Ltd (China). 4-carboxybenzeneboronic acid (CPBA), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC·HCl), and N-hydroxysuccinimide (NHS) were purchased from Aladdin (China). Lipopolysaccharide (LPS) was purchased from Sigma-Aldrich (USA). Recombinant murine granulocyte-macrophage colony-stimulating factor (GM-CSF) and interleukin 4 (IL-4) were purchased from PeproTech (USA). Dulbecco's modified eagle medium (DMEM), trypsin, penicillin-streptomycin, and fetal bovine serum (FBS) were purchased from VivaCell (China). 1,1-dioctadecyl-3,3,3,3-tetramethylindotricarbocyanine iodide (DiR) was purchased from Yeasen (China). Anti-CD9, anti-Alix, anti-CD63, anti-β-actin, HRP-conjugated anti-rabbit secondary, HRP-conjugated anti-mouse secondary antibodies, TNF-α, IL-6, and IFN-γ ELISA kits were purchased from Proteintech (USA). Red blood cell lysis buffer, PE- or APC-conjugated anti-mouse CD11b, FITC-conjugated anti-mouse CD86, APC-conjugated anti-mouse CD206, PE-conjugated anti-mouse CD11c, FITC-conjugated anti-mouse CD80, FITC-conjugated anti-mouse MHC-II, FITC-conjugated anti-mouse CD3, PE-conjugated anti-mouse CD8a, APC-conjugated anti-mouse CD4, and PE-conjugated anti-mouse Foxp3-antibodies were purchased from ThermoFisher (USA). Polyvinylidene fluoride (PVDF) membrane was purchased from Millipore (USA). Cell counting kit-8 (CCK-8) kit was purchased from TargetMol (China). Carboxyfluorescein succinimidyl ester (CFSE), 3,3′-dioctadecyloxacarbocyanine perchlorate (DiO), and hematoxylin and eosin (H&E) staining kit were provided by Beyotime (China). The mouse spleen lymphocyte separation solution, crystal violet staining, and TMR (red) tunel cell apoptosis detection kits were purchased from Solarbio (China). Calcein AM/PI double staining kit was purchased from Elabscience (China).

2.2. Cells and animals

The murine colon tumor (CT26), mammary carcinoma (4T1), and macrophage RAW 264.7 cells were purchased from Peking Union Medical College. These cells were cultured in DMEM supplemented with 10 % FBS and 1 % penicillin-streptomycin, and maintained in a humidified incubator at 37 °C with 5 % CO₂.

BALB/c and C57BL/6 mice were purchased from Hefei Qingyuan Biotechnology Co., Ltd (Hefei, China). All animal experiments were conducted in accordance with the guidelines of the Experimental Animal Ethics Committee of Anhui University of Chinese Medicine (Approval No. AHUCM-mouse-2023187).

2.3. LTEV isolation

EVs were isolated as described previously [34,36]. Briefly, CT26 cells were stimulated with complete DMEM at pH 4.0 for 15 min, then replaced this DMEM with normal complete DMEM. After 48 h, the supernatant from CT26 cells was sequentially centrifugated under the following conditions: 300×g for 15 min, 2000×g for 15 min, and 16000×g for 30 min. Subsequently, the pre-treated supernatant was ultracentrifuged at 120 000×g for 2 h (Beckman Coulter, USA. The precipitate, containing LTEVs, was resuspended in PBS and stored at −80 °C.

2.4. Preparation of GLP@LTEV

First, LTEV (0.018 g) was added to a dimethyl sulfoxide (DMSO) solution (pH 6.5) containing CPBA (0.27 g), EDC·HCl (0.145 g) and NHS (0.085 g) for the formation of amide bonds. After 3 h, GLP (0.18 g) were added into the mixture, followed by pH adjustment to 8.5 with 0.05 M NaOH solution. Following another 3 h of reaction, free GLP was removed via three rounds of ultrafiltration (5000×g, 10 min each) using a centrifugal ultrafiltration tube (100 kDa), yielding GLP@LTEV. The polysaccharide content of GLP@LTEV was quantified using the phenol-sulfuric acid method. The loading efficiency of GLP on LTEV was calculated through the equation below:

$$\text{Loading}\text{efficiency}(\%)=\frac{\text{Weight}\text{of}\text{loaded}\mathrm{G}\text{LP}}{\text{Weight}\text{of}\mathrm{G}\text{LP}@\text{LTEV}}\times 100\%$$

2.5. Characterization of boronic ester linkage and GLP@LTEV

First, GLP (0.18 g) and CPBA (0.27 g) were reacted in 10 mL of DMSO solution (pH 8.5). After 3 h, the reaction solution was dialyzed for 2 d in a dialysis bag (MWCO 3.5 kDa) to remove free CPBA. Then the dialyzed solution was collected and dried under a vacuum to obtain the product GLP-CPBA. And the boronic ester linkages between GLP and CPBA were subsequently characterized by FTIR, ¹H NMR, ¹B-NMR, and UV–vis absorption spectroscopy.

In addition, the morphology of GLP@LTEV was observed using transmission electron microscopy (TEM, HITACHI-HT7700, China). The size, zeta potential, and stability of GLP@LTEV were analyzed by dynamic light scattering (DLS, ZEN3690, USA).

2.6. In vitro GLP release from GLP@LTEV

A 1.5 mL aliquot of the GLP@LTEV solution was placed in a dialysis bag (MWCO 150 kDa) and dialyzed against buffers at pH 7.4 and 6.5. At predetermined time intervals, 1 mL of the external buffer was withdrawn for analysis and immediately replaced with an equal volume and pH of fresh buffer. The amount of released GLP was quantified using the phenol-sulfuric acid method.

2.7. Western blot

The expression of CD9, Alix, and CD63 was evaluated by Western blot as described previously [34]. In brief, protein samples were extracted from NTEV and LTEV, and their concentrations were measured using a BCA assay kit. The proteins were separated by SDS-polyacrylamide gel electrophoresis and then transferred onto a PVDF membrane. After rapid blocking for 30 min, the membrane was incubated overnight at 4 °C with the following primary antibodies: anti-CD9 (1:1000, 20597-1-AP), anti-Alix (1:10000, 67715-1-Ig), and anti-CD63 antibody (1:500, 25682-1-AP). The membrane was washed with TBST three times and incubated with secondary antibody dilution (1:10 000) for 2 h. Next, the membrane was washed with TBST, covered with ECL reagent, and imaged using a chemiluminescence system (Tanon-5200, China).

For β-actin detection, the membrane was stripped of primary and secondary antibodies, washed, and then re-incubated with an anti-β-actin antibody (1:20 000, 66009-1-Ig). After incubation with an HRP-conjugated secondary antibody, the signal was again developed with ECL reagent for visualization.

2.8. Cytokine assay

First, RAW264.7 cells were polarized into M2 macrophages after being cultured in a medium containing 100 ng/mL IL-4 for 48 h. The M2 macrophages (1×10⁴ cells/well) were then seeded and cultured into a 96-well plate. After 12 h, the cells were treated with different concentrations of GLP@LTEV (GLP: 0, 0.15, 0.3, 0.6, 0.9 or 1.2 μg/μL, respectively) for 24 h. Cells treated with LPS (0.5 μg/mL) served as the positive control. Finally, the supernatants were collected, the concentrations of IL-6 and TNF-α were measured using ELISA kits.

2.9. Cell viability assay

CT26 cells (1×10⁴ cells/well) adhered to well plates after 12 h incubation in a 96-well plate and were individually treated with GLP and GLP@LTEV (GLP: 0, 0.15, 0.3, 0.6, 0.9 or 1.2 μg/μL, respectively) for 24 h. The cells treated with LPS (0.5 μg/mL) served as the positive control, while untreated cells used as the negative control. After treatment, the medium was replaced with 100 μL of fresh medium containing CCK-8 reagent, followed by incubation at 37 °C for 2 h. Absorbance was measured at 450 nm using a microplate reader, and cell viability was calculated according to the following formula:

$$\text{Cell}\text{viability}(\%)=\frac{\text{Sample}\text{absorbance}-\text{Blank}\text{absorbance}}{\text{Control}\text{absorbance}-\text{Blank}\text{absorbance}}\times 100\%$$

2.10. Macrophage polarization phenotype analysis

PBS, LTEV, GLP and GLP@LTEV (GLP: 0.6 μg/μL) were individually added to M2 macrophages seeded in 24-well plates. The cells treated with LPS (0.5 μg/mL) served as positive control. After 24 h, the cells were collected and labeled with FITC-conjugated anti-mouse CD86 and PE-conjugated anti-mouse CD206 antibody, respectively. The expression of CD86 and CD206 was assessed by flow cytometry and confocal microscopy.

2.11. Phagocytosis assay

M2-like macrophages and CFSE-labeled CT26 cells were seeded and co-cultured in 24-well plates. Then PBS, LTEV, GLP, and GLP@LTEV (GLP: 0.6 μg/μL) were individually added into each group for 24 h. After treatment, cells in each group were collected and labeled with APC-conjugated anti-mouse CD11b antibody. Phagocytosis rates were analyzed by flow cytometry and confocal microscopy.

2.12. Culture and detection of bone marrow-derived dendritic cells

First, bone marrow-derived cells were harvested from the femurs and tibiae of 6-8-week-old male C57BL/6J mice in a sterile environment. Erythrocytes were removed using erythrocyte lysis buffer. Bone marrow-derived dendritic cells (BMDCs) were obtained following a 7-day induction in complete DMEM medium containing 20 ng/mL GM-CSF and 20 ng/mL IL-4. After the differentiation period, the BMDCs were individually treated with LPS, PBS, LTEV, GLP and GLP@LTEV. After 24 h, DCs were stained with PE-conjugated anti-mouse CD11c, FITC-conjugated anti-mouse CD80, and FITC-conjugated anti-mouse MHC-II antibodies for flow cytometry analysis.

2.13. T-cell activation and proliferation study in vitro

CT26 cells were co-cultured with imDCs in the presence of PBS, LTEV, GLP or GLP@LTEV at 37 °C for 24 h. After removal of the treatment medium, T cells isolated from the spleens of C57BL/6J mice using a mouse spleen lymphocyte separation kit were added to the previously mixed cells at a ratio of 1:10 (DCs to T cells) for 48 h. After that, the T lymphocytes were stained with FITC-conjugated anti-mouse CD3 and APC-conjugated anti-mouse CD8a antibodies for flow cytometry analysis. To investigate T-cell proliferation, the isolated T cells were labeled with CFSE prior to co-culture, and analyzed by flow cytometry. The levels of IFN-γ in a mixed culture system were measured using an ELISA kit.

To analyze the antitumor effects of T lymphocytes, CT26 cells were co-cultured with activated T lymphocytes at a ratio of 1:10 for 24 h. The CT26 cells were stained with calcein AM/PI double staining kit and imaged by confocal microscopy (green indicated live, red indicated dead). The cell viability of CT26 was quantified according to the CCK-8 assay.

2.14. M2 macrophage repolarization of T lymphocyte-secreted IFN-γ in vitro

The activated T lymphocytes were added into the M2 macrophages for co-incubation. After 24 h, the macrophages were collected and stained with PE-conjugated anti-mouse CD11b, FITC-conjugated anti-mouse CD86, and APC-conjugated anti-mouse CD206 antibodies for flow cytometry analysis.

2.15. In vivo fluorescence imaging of GLP@LTEV in the CT26 subcutaneous tumor model

CT26 cells were collected and adjusted to a density of 5.0 × 10⁶ cells/mL with PBS. Then CT26 cell suspension (100 μL each) was injected subcutaneously into the right flank region of female BALB/c mice. After two weeks, mice were randomly divided into three groups (3 animals per group): receiving low, medium, or high dosage of GLP@LTEV (GLP: 22.5, 45, or 90 mg/kg; LTEV: 2.5, 5, or 10 mg/kg, respectively). The different dosages of DiR-labeled GLP@LTEV were administrated via tail vein injection. The fluorescence intensity distribution of DiR in mice was monitored at different time points (3, 6, 12, 24 and 48 h) using an in vivo fluorescence imaging system (IVIS Spectrum, USA).

2.16. The distribution and therapeutic efficacy of GLP@LTEV in the CT26/4T1 bilateral tumor model

To establish a bilateral tumor model, female BALB/c mice were inoculated subcutaneously with 100 μL of CT26 cells (5.0 × 10⁶ cells/mL) in PBS on the right flank and 4T1 cells (5.0 × 10⁶ cells/mL) on the left flank, respectively. Two weeks later, the mice (n = 3) were administered an intravenous injection of 100 μL DiR-labeled GLP@LTEV (GLP: 90 mg/kg; LTEV: 10 mg/kg per mouse). In vivo fluorescence imaging (IVIS Spectrum, USA) was then performed to monitor the whole-body distribution and tumor accumulation at various time points.

100 μL of 4T1 and CT26 cells (5.0 × 10⁶ cells/mL) were individually injected into the left and right flanks of mice. One week later, mice were treated with PBS and GLP@LTEV (GLP: 90 mg/kg; LTEV: 10 mg/kg per mouse) on days 6, 9, 12, 15, and 18. During the treatment period, we monitored the tumor volume, body weight, and body temperature of the mice every three days. After the 21-day monitoring, all the tumor tissues and spleens from the mice were excised for analysis.

2.17. The growth inhibition and immune responses of GLP@LTEV in the CT26 subcutaneous tumor model

A CT26 subcutaneous tumor model was established according to the previous method [34]. When the tumor volume reached ∼60 mm³, mice were randomly divided into 4 groups (n = 7) and received intravenous injections of PBS, LTEV, GLP, or GLP@LTEV (GLP: 90 mg/kg; LTEV: 10 mg/kg per mouse). Treatments were administrated with 100 μL on days 6, 9, 12, 15, and 18. Tumor size, body weight, and body temperature of each mouse were monitored every 3 days. Tumor volume was determined following the formula: tumor volume (mm³) = (width² × length)/2.

On day 19, the mice were euthanized. Tumor tissues were weighted and subjected to H&E and TUNE staining for tumor apoptosis analysis. Next, the tumor tissues were ground into single-cell suspensions, which were then stained for flow cytometry and immunofluorescence analysis with the following antibodies: FITC-conjugated anti-mouse CD86, APC-conjugated anti-mouse CD206, PE/APC-conjugated anti-mouse CD11b, FITC-conjugated anti-mouse CD80, FITC-conjugated anti-mouse MHC-II, PE-conjugated anti-mouse CD11c, FITC-conjugated anti-mouse CD3, PE-conjugated anti-mouse CD8a, APC- conjugated anti-mouse CD4, and PE-conjugated anti-mouse Foxp3 antibodies. In addition, hematological analysis and H&E staining of major organs were performed to evaluate the biosafety of GLP@LTEV.

2.18. The therapeutic effects and immune responses of GLP@LTEV in the CT26 lung metastasis model

The CT26 lung metastasis model was established following a two-step approach. First, 100 μL of CT26 tumor cells (5.0 × 10⁶ cells/mL) were subcutaneously injected into the right flank of the female BALB/c mice as the primary tumor. Then mice were treated via tail vein injection with PBS, LTEV, GLP or GLP@LTEV (GLP: 90 mg/kg; LTEV: 10 mg/kg per mouse) on days 7, 10, 13,16, and 19. An additional 100 μL of CT26 cells were injected into BALB/c female mice via the tail vein on day 10. On day 23, mice were euthanized. The lung and primary tumor tissues were harvested, weighed, and analyzed by H&E staining. To analyze the immune responses induced by GLP@LTEV, primary tumor tissues were ground into single-cell suspensions, then stained with relevant antibodies for flow cytometry and immunofluorescence analysis.

2.19. Statistical analysis

All data were analyzed using GraphPad Prism 9 and Origin 2021, and presented as mean ± standard deviation (SD). The results of quantitative analysis of experimental data were performed using student's t-test and unpaired one-way ANOVA for statistical comparison. Significance levels were denoted as follows: ns (not significant), ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001, and ^∗∗∗∗p < 0.0001.

3. Results and discussion

3.1. The preparation and characterization of GLP@LTEV

To engineer GLP with LTEV, the carboxylic group of CPBA was bonded with the amino group of LTEV, and the boronic acid group of CPBA established a boronic ester linkage with the hydroxyl groups of GLP (Fig. 1A). The formation of the boronic ester linkage in GLP-CPBA was verified by Fourier transform infrared (FT-IR), ultraviolet–visible (UV–vis) absorbance, proton nuclear magnetic resonance (¹H NMR), and boron nuclear magnetic resonance (¹¹B-NMR) spectra (Fig. 1B–E). According to the FT-IR spectra, the characteristic peak corresponding to the formation of the B-O bonds in boronic esters was observed at 1080 cm⁻¹. UV–vis absorbance spectra showed that characteristic absorbance peaks of the benzene ring in CPBA and GLP-CPBA appeared at 222 nm and 214 nm, respectively. The ¹H NMR spectra showed that the characteristic resonance signal of CPBA at 7.75–7.90 ppm was absent in GLP-CPBA, mainly ascribed to the protons of GLP. The boron signal of CPBA at ∼30 ppm appeared in GLP-CPBA via ¹¹B-NMR spectra, verifying the successful conjugation of GLP with CPBA. The obtained GLP@LTEV nanovesicles (Fig. 1F) were characterized as follows. The average diameter increased after GLP loading compared with LTEV, from approximately 118.7 to 210.1 nm, and the zeta potential decreased from −15 to −16.8 mV owing to the negative charge density of GLP (Fig. 1G and H). Transmission electron microscopy (TEM) images showed that the characteristic cup-shaped morphology of GLP@LTEV remained unchanged (Fig. 1I). The GLP loading capacity was determined to be 90.0 % by the phenol-sulfuric acid method (Fig. S1). The particle size and zeta potential of GLP@LTEV in phosphate-buffered saline (PBS) remained stable over a 7-day period, indicating good stability (Fig. 1J). However, we found that GLP was readily released from LTEV under the acidic condition (pH 6.5), compared with the physiological pH of 7.4. The release was particularly pronounced within the first 48 h, with a cumulative release rate reaching 82.2 % over 72 h, indicating the cleavable of the boric ester bonds within the TME (Fig. S2). Western blot analysis verified that the typical membrane protein (CD9, Alix, and CD63) of GLP@LTEV could be detected (Fig. 1K), indicating little protein loss during fabrication and the function of LTEV could be kept.

Fig. 1.

The preparation and characterization of GLP@LTEV. (A) Technological route for GLP@LTEV synthesis. (B) FT-IR spectra of CPBA, GLP, and GLP-CPBA. (C) UV–vis absorbance spectra of CPBA, GLP, and GLP-CPBA. (D) ¹H NMR spectra of CPBA, GLP, and GLP-CPBA. (E) ¹¹B-NMR spectra of CPBA, GLP, and GLP-CPBA. (F) Schematic model of GLP@LTEV. (G, H) Dynamic light scattering (DLS) detected the size and zeta potential of LTEV and GLP@LTEV. (I) TEM images of LTEV and GLP@LTEV. (J) Size and zeta potential stability of GLP@LTEV in PBS. (K) Western blot analysis of NTEV, LTEV, and GLP@LTEV.

3.2. GLP@LTEV triggering immune responses in vitro

GLP exerts its therapeutic effects primarily through immune modulation rather than direct cytotoxicity [37]. Fig. S3 shows that the combination of GLP and LTEV in GLP@LTEV does not inhibit CT26 cells. To explore the immune responses induced by GLP@LTEV, M2 macrophages were treated with different formulations, including LTEV, GLP, and GLP@LTEV for 24 h. The secretion of inflammatory cytokines tumor necrosis factor-α and interleukin-6 (TNF-α and IL-6) gradually increased with rising concentrations of GLP@LTEV, reaching their highest levels at 0.6μg/μL GLP (Fig. S4). To further confirm the M2 macrophage reprogramming capability of GLP@LTEV, we individually treated M2 macrophages with LTEV, GLP, and GLP@LTEV for 24 h. In addition, lipopolysaccharide (LPS), used as a positive control, is a commercial reagent that induces the repolarization of M2 macrophages. As shown in Fig. 2A, cellular morphology shifted from the elongated, spindle-like appearance characteristic of M2 macrophages to the rounded, flattened morphology typical of M1 macrophages in both the GLP and GLP@LTEV treatment groups. Immunofluorescence imaging and flow cytometry results demonstrated a significant increase in the expression of the M1-related costimulatory molecule CD86 and a substantial decrease in the M2-related costimulatory molecule CD206 in the GLP and GLP@LTEV treatment groups compared to the PBS group (Fig. S5A and Fig. 2B–E). Next, we investigated the phagocytic capability of re-educated macrophages using carboxyfluorescein succinimidyl ester (CFSE)-labeled CT26 cells. Compared with PBS- or LTEV-treated M2 macrophages, most of the tumor cells were effectively internalized and digested by GLP- or GLP@LTEV-treated M2 macrophages (Fig. 2F). Quantitative analysis demonstrated that the phagocytic rate of tumor cells reached 49.3 % in the GLP@LTEV group after 24 h of treatment (Fig. S5B and Fig. 2G). Collectively, these results indicate that GLP@LTEV promotes macrophage phagocytic capability by effectively repolarizing M2-like macrophages toward M1-phenotype macrophages.

Fig. 2.

In vitro effects of LTEV, GLP, and GLP@LTEV on M2 polarization. (A) Crystal violet staining images of M2 macrophages before and after treatment. LPS treatment used as a positive control group. Scale bar: 50 μm. (B) Representative confocal fluorescence images showing CD86 and (C) CD206 expression in M2 macrophages before and after treatment. Blue: cell nucleus, green: CD86 or CD206. Scale bar: 50 μm. (D) Flow cytometry quantitative analysis of CD86 and (E) CD206 expression on M2 macrophages before and after treatment. (F) Representative confocal fluorescence images of CT26 cells phagocytosed by M2 cells before and after treatment. CT26 cells were pre-labeled with CFSE (green), while macrophages were labeled with APC-conjugated anti-mouse CD11b (red). Scale bar: 20 μm. (G) Flow cytometry quantitative analysis of CT26 cells phagocytosed by M2 cells before and after treatment. ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. ns, not significant difference. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

The results of flow cytometry demonstrated that the expression levels of DC maturation markers (CD11c⁺CD80⁺ and CD11c⁺MHC-II⁺) following GLP@LTEV treatment were approximately 1.7- and 1.4-fold compared with the PBS group (Fig. S6), indicating that GLP@LTEV effectively promotes DC maturation. Studies have indicated that mature DCs activate T cells by presenting tumor-specific antigens, thereby initiating antitumor immunity. As shown in Fig. S7, CT26 cells were co-cultured with immature DCs (imDCs) in the presence of LTEV, GLP, or GLP@LTEV for 24 h. After removing LTEV, GLP, or GLP@LTEV, we added primary T cells to the matured DCs and then incubated them for another 48 h. Quantitative flow cytometry analysis revealed that the percentage of CD3⁺CD8⁺ T lymphocytes treated with GLP@LTEV was 22.4 %, representing a 1.6-fold increase compared to the LTEV group (13.9 %) and a 1.2-fold increase compared to the GLP group (18.4 %) (Fig. 3A and B). Subsequently, T lymphocytes were labeled with CFSE and analyzed using flow cytometry. The results revealed a slight increase in the percentage of proliferating CD8⁺ T cells from 10.5 % in the PBS group to 20.1 % in the GLP group. However, this rate increased further to 29.0 % in the GLP@LTEV group (Fig. 3C and D).

Fig. 3.

In vitro T cell activation and antitumor effects of LTEV, GLP, and GLP@LTEV. (A,B) Flow cytometry and quantification analysis of CD3⁺CD8⁺ T cells in vitro after incubation with different formulations for 48 h (n = 3). (C,D) Flow cytometry and quantification analysis of CFSE-labeled primary T-cell proliferation after incubation with different formulations (n = 3). (E) Representative fluorescence imaging of live (green)/dead (red) staining of CT26 cells after co-culture with T cells treated with different formulations. Scale bar: 50 μm. (F) Cell viability of CT26 cells after co-culture with T cells treated with different formulations (n = 3). (G,H) Flow cytometry analysis on the proportion and ratio of M1 macrophages and M2 macrophages after co-culture with activated T cells (n = 3). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. ns, not significant difference. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Furthermore, the pro-inflammatory cytokine interferon-γ (IFN-γ) secreted by CD8⁺ T cells was detected using an enzyme-linked immunosorbent assay (ELISA). The results indicated that IFN-γ level was 3.0 times higher in the GLP@LTEV and DCs group than with PBS and DCs treatment (Fig. S8). We assessed the inhibitory effect of activated CD3⁺CD8⁺ T cells on CT26 cells using live/dead staining and the cell counting kit-8 (CCK-8) assay. The results demonstrated higher cell viability in the LTEV-treated group (indicated in green), whereas the GLP@LTEV group exhibited increased cell death (indicated in red) (Fig. 3E). CCK-8 assay results indicated that CT26 viability was approximately 59.9 % when incubated with GLP@LTEV-treated CD3⁺CD8⁺ T cells, whereas the viability following treatment with PBS, LTEV, and GLP-treated CD3⁺CD8⁺ T cells were 100 %, 95.1 %, and 62.5 %, respectively (Fig. 3F). Given that M2 macrophages can be effectively reprogramed into M1 by INF-γ for antitumor therapy [36], we incubated M2 macrophages with the activated T lymphocytes for 24 h (Fig. S9). Flow cytometry analysis results indicated that the percentage of CD11b⁺CD86⁺-positive cells in the GLP@LTEV group was 66.8 %, which was substantially higher than in the PBS (33.0 %) and LTEV (47.9 %) groups. In contrast, CD11b⁺CD206⁺-positive cells in the GLP@LTEV group were 60.7 %, which was markedly lower than in the PBS (85.7 %) and LTEV (84.3 %) groups (Fig. 3G and H). Together, these results confirmed that GLP@LTEV effectively promotes DC maturation, amplifying antitumor effects of T lymphocytes and derives the re-polarization of M2 macrophages.

3.3. In vivo distribution and therapeutic efficacy of GLP@LTEV

To assess the tumor accumulation capability of GLP@LTEV, we established a subcutaneous CT26 tumor-bearing mouse model using CT26 cells. After two weeks, low-, medium-, and high-doses of GLP@LEV were labeled with 1,1-dioctadecyl-3,3,3,3-tetramethylindole tricarbonyl cyanide iodide (DiR) and then injected into mice via the tail vein. Time-dependent fluorescence signals of DiR in mice were detected using an in vivo imaging system. As shown in Fig. 4A, the signals of three groups gradually increased at the tumor sites over time. At 48 h post-injection, the low dosage-treated group displayed only faint signals, whereas the high dosage-treated group exhibited markedly stronger fluorescence within tumor tissues (Fig. S10). Meanwhile, tumor tissues and all organs were harvested to evaluate the biodistribution. As expected, ex vivo imaging and semi-quantitative analysis indicated that the cumulative level of tumor tissues in high dosage GLP@LTEV treatment was 3.9- and 1.8-fold higher than that in the low and medium dosage GLP@LTEV groups (Fig. 4B and C).

Fig. 4.

Tumor targeting and therapeutic efficacy of GLP@LTEV in the CT26 subcutaneous tumor model. (A) Fluorescence images of the mice receiving different concentrations of GLP@LTEV treatment (n = 3). (B) Imaging of isolated tumor and major organs at 48 h after intravenous injection of different concentrations of GLP@LTEV. (C) Quantitative analysis of fluorescence intensity in isolated tumors and major organs at 48 h after intravenous injection of different concentrations of GLP@LTEV (n = 3). (D) Treatment schedule of CT26 subcutaneous tumor in vivo. (E) Tumor volume recorded during the treatment course (n = 7). (F) Anatomical images of tumor masses on day 19. (G) Tumor weight on day 19. (H) Tumor inhibition rate on day 19. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

Given the favorable tumor distribution profile, the high-dose GLP@LTEV formulation was selected to evaluate its homotypic targeting capability in the bilateral tumor model. Mice bearing both CT26 (right) and 4T1 (left) tumors were intravenously injected with DiR-labeled GLP@LTEV. Fluorescence imaging revealed a markedly stronger DiR signal in CT26 tumors compared to the faint signal in 4T1 tumors (Fig. S11A and B). At 48 h, all major organs and tumor tissues were harvested for ex vivo imaging and quantitative analysis. The fluorescence signals indicated a CT26/4T1 signal ratio of up to 2.0 in the harvested tumors (Fig. S11C and D). To assess the antitumor efficacy of GLP@LTEV in this model, mice bearing tumors of approximately 90 mm³ were randomized into two groups and treated with either PBS or GLP@LTEV (Fig. S11E). Tumor volumes were recorded every three days. As shown in Fig. S11F and G, GLP@LTEV treatment significantly inhibited the growth of CT26 tumors but had a limited effect on 4T1 tumors. Correspondingly, the tumor inhibition rates (based on tumor weight) were 71.1 % for CT26 tumors, while only 16.3 % for 4T1 tumors (Fig. S11H and I). Furthermore, GLP@LTEV significantly decreased the spleen weight and index compared with the PBS-treated group (Fig. S11J-L). Moreover, the treatment did not alter the mice's body weight and body temperature (Fig. S11M and N).

Encouraged by this homotypic targeting, we further investigated therapeutic efficacy and immune responses in the subcutaneous CT26 tumor model (established as shown in Fig. 4D). When tumors reached ∼60 mm³, tumor-bearing mice were randomized into four groups receiving PBS, LTEV, GLP, or GLP@LTEV. As shown in Fig. 4E and F, compared with other groups, the GLP@LEV group had significantly smaller tumor volumes, thus demonstrating the highest antitumor efficacy in inhibiting the growth of CT26 subcutaneous tumors. GLP@LTEV also exhibited the highest tumor inhibition rate based on the evaluation of average tumor weight. Specifically, the results of the inhibition rate individually were 24.0 %, 62.6 %, and 80.4 % for LTEV, GLP, and GLP@LTEV (Fig. 4G and H).

3.4. The antitumor immune responses of stimulated by GLP@LTEV in the CT26 subcutaneous tumor model

To assess whether GLP@LTEV could remodel the TME upon reaching CT26 subcutaneous tumors, we first examined immune cell distribution by immunofluorescence staining (Fig. S12). Flow cytometry analysis of TAMs demonstrated a progressive increase in the CD11b⁺CD86⁺ population in the following order: PBS (20.2 %), LTEV (24.8 %), GLP (29.4 %), and GLP@LTEV (45.4 %). In contrast, the population of CD11b⁺CD206⁺ decreased in the order of PBS (76.6 %), LTEV (75.4 %), GLP (69.2 %), and GLP@LTEV (51.0 %) (Fig. 5A and B). Correspondingly, the GLP@LEV group showed a significant increase in the ratio of M1 macrophages to M2 macrophages, showing a 3.4-fold increase compared to the PBS group (Fig. 5C). Furthermore, following GLP@LTEV treatment, the double-positive flow cytometric events for CD11c⁺CD80⁺ and CD11c⁺MHC-II⁺ was individually 26.8 % and 15.0 % (Fig. 5D and E), which in turn significantly increased the proportion of CD3⁺CD8⁺ T cells. Typically, the CD3⁺CD8⁺ T cells reached 14.0 % after GLP@LTEV treatment, which was 7.3 times that of the PBS group (1.9 %) and 1.5 times that of the GLP group (9.3 %) (Fig. 5F). Meanwhile, the GLP@LTEV treatment substantially decreased the CD3⁺CD4⁺Foxp3⁺ cell population in tumor tissues (Fig. 5G). As a result, in GLP@LTEV-treated tumors, the ratio of CD3⁺CD8⁺ T cells to Tregs was significantly increased, being 4.1-fold higher than in the LTEV group and 1.9-fold higher than in the GLP group (Fig. 5H). H&E and TUNEL staining further demonstrated that tumor tissue in the GLP@LTEV-treated group exhibited more extensive cellular necrosis compared to all other groups (Fig. 5I). Taken together, these results indicated that GLP@LTEV effectively repolarizes M2 macrophages, promotes DC maturation, enhances T cell activation, and suppresses Tregs within tumor tissues. Furthermore, GLP@LTEV treatment had no abnormal effects on body weight, body temperature, spleen weight, organ histology, or hematological analysis in mice (Figs. S13–16), further confirming the excellent safety of the GLP@LTEV formulation.

Fig. 5.

Immune responses of GLP@LTEV in the CT26 subcutaneous tumor model. (A) Flow cytometry analysis of the proportion of M1 macrophages in tumor tissues (n = 3). (B) Proportion of M2 macrophages (n = 3). (C) Ratio of M1 to M2. (D,E) Percentage of mature DC in tumor tissues (n = 3). (F) Frequency of CD3⁺CD8⁺ T cells in tumor tissues (n = 3). (G) Frequency of CD3⁺CD4⁺Foxp3⁺ in tumor tissues (n = 3). (H) Ratio of CD3⁺CD8⁺T cells to Tregs. (I) Representative images of tumor tissue sections stained with H&E and TUNEL. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

3.5. The therapeutic efficacy and immune responses of GLP@LTEV in the CT26 lung metastasis model

Considering the therapeutic effects and robust immune activation of GLP@LTEV, we further investigated whether GLP@LTEV can elicit potent performance in the CT26 lung metastasis model. CT26 tumor-bearing mice were randomly grouped and received different treatments, including PBS, LTEV, GLP, and GLP@LTEV. On day 10, these mice were rechallenged by CT26 cells via intravenous injection (Fig. 6A). Images of the isolated lungs showed that the lung tissues in the GLP@LTEV-treated group exhibited the fewest metastatic foci compared to other groups (Fig. 6B and C). Statistics analysis further demonstrated that lung tissue weights was lowest in GLP@LTEV group (Fig. 6D). In addition, the lung index, the ratio of lung tissue weight to mouse body weight, was significantly reduced in the GLP@LTEV group compared with the LTEV group (Fig. 6E). As shown in the H&E-stained images of lung tissues, the potent therapeutic efficacy was achieved with few metastatic foci in the group of GLP@LTEV, while different extent of abnormalities were observed in the PBS, LTEV, and GLP groups (Fig. 6F).

Fig. 6.

Therapeutic efficacy of GLP@LTEV in the CT26 lung metastasis model. (A) Treatment schedule of CT26 tumor lung metastasis in vivo. (B) Representative images of lung tissues at the end of the experiment. The yellow arrows indicate metastatic nodules. (C) Metastatic nodules of lung tissues (n = 3). (D) Lung weight (n = 3). (E) Lung index (n = 3). (F) Representative H&E staining images of the lung tissues following different treatments. ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

As shown in Fig. 7A, LTEV or GLP treatment produced only mild inhibition of primary tumor growth compared to the PBS group, while GLP@LTEV-treated achieved the strongest antitumor effect. The average tumor weight occurred in the following order: PBS > LTEV > GLP > GLP@LTEV (Fig. 7B). We calculated the tumor growth inhibition rate of GLP@LTEV from final tumor weight, reached 86.6 %, which was substantially higher than that of LTEV (29.0 %) or GLP (67.8 %) treatment (Fig. 7C). Moreover, H&E and TUNEL staining results demonstrated that GLP@LTEV induced the highest level of apoptosis (Fig. 7D).

Fig. 7.

Immune responses of GLP@LTEV in the CT26 lung metastasis model. (A) Photographs of tumor tissues after different treatments. (B) Tumor weight. (C) Tumor inhibition rate. (D) The representative H&E and TUNEL images of tumor tissues collected from mice at the end of various treatments. (E,F) Flow cytometry analysis of macrophages in tumor tissues (n = 3). (G) The ratio of M1 macrophages to M2 (n = 3). (H,I) Flow cytometry analysis of DC maturation in tumor tissues (n = 3). (J) Flow cytometry analysis of CD3⁺CD8⁺ T cells in tumor tissues (n = 3). (K) Flow cytometry analysis of CD3⁺CD4⁺Foxp3⁺ in tumor tissues (n = 3). (L) The ratio of CD3⁺CD8⁺ T cells to Tregs (n = 3). ∗p < 0.05; ∗∗p < 0.01; ∗∗∗p < 0.001; ∗∗∗∗p < 0.0001.

To investigate the antitumor immune responses motivated by GLP@LTEV, we performed immunofluorescence analysis of immune cell distribution within primary tumors (Fig. S17). Flow cytometry results indicated that the frequencies of CD11b⁺CD86⁺ and CD11b⁺CD206⁺ in the GLP@LTEV group individually reached 42.2 % and 34.6 %, compared with the PBS group (35.7 % and 68.3 %) (Fig. 7E and F). Quantitative analysis revealed that GLP@LTEV significantly increased the ratio of M1 macrophages to M2 macrophages, reaching 2.3-, 2.1-, and 1.5-fold levels relative to the PBS, LTEV, and GLP groups respectively (Fig. 7G). In addition, the proportions of CD11c⁺CD80⁺ and CD11c⁺MHC-II⁺ DCs in the GLP@LTEV group significantly increased to 28.2 % and 26.4 % respectively, representing 2.4- and 2.2-fold higher than the PBS group, respectively (Fig. 7H and I). As expected, GLP@LTEV treatment also significantly increased the proportion of CD3⁺CD8⁺ T cells, which was 2.4-, 1.4- and 1.4-fold higher than those in the PBS, LTEV, and GLP groups, respectively (Fig. 7J). The GLP@LTEV-treated group showed the lowest proportion of CD3⁺CD4⁺Foxp3⁺ (6.6 %), while those of PBS, LTEV, and GLP reached 17.1 %, 11.3 %, and 9.9 %, respectively (Fig. 7K). Consistently, GLP@LTEV also significantly increased the ratio of CD3⁺CD8⁺ T cells to Tregs, representing a 2.1-fold increase compared to the GLP group (Fig. 7L). Overall, GLP@LTEV demonstrated markedly stronger immunostimulatory ability than single LTEV or GLP treatment in the CT26 tumor lung metastasis model, thus shedding light on its potential antitumor immunotherapeutic efficacy.

4. Conclusion

Many types of carriers have been studied in depth for polysaccharide delivery, including hollow nanoparticles [38,39], gold nanoparticle [26], liposome [24,25,40], hydrogel [41], as well as some polysaccharide self-assembly methods [42,43], which are vital to drug loading, delivery, and disease therapy. Nevertheless, homologous tumor-targeting capability of drug delivery vehicles may be clinically meaningful. In this study, an engineered LTEV-targeting delivery system was successfully constructed, capable of delivering GLP for synergistic antitumor therapy. Owing to the homologous tumor-targeting properties of LTEV, GLP@LTEV effectively accumulates in tumor tissues upon intravenous administration. GLP@LTEV significantly reversed the immunosuppressive TME via repolarizing M2 macrophages towards M1 phenotype, promoting DC maturation, activating CTLs, and inhibiting Tregs. Benefiting from the synergistic effects of tumor-targeting and immunomodulation, GLP@LTEV demonstrated the remarkable efficacy in eliminating subcutaneous tumor and preventing lung metastasis. Overall, this nanosystem exhibits favorable safety and biocompatibility, and can be readily adapted to functionalize various other fungus-derived polysaccharides. We believe that this personalized strategy holds considerable promise for future clinical translation.

CRediT authorship contribution statement

Zhaorong Ouyang: Writing – original draft, Methodology, Formal analysis, Data curation. Siyu Li: Writing – original draft, Methodology, Formal analysis, Data curation. Ao Zhang: Validation, Formal analysis. Shu Ye: Validation, Formal analysis. Wanqiu Ye: Methodology, Investigation. Guolei Wen: Methodology, Investigation. Tao Wei: Methodology, Investigation. Biao Cai: Writing – review & editing, Supervision. Houli Liu: Writing – review & editing, Supervision, Project administration, Funding acquisition.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Appendix A. Supplementary data

The following is the supplementary data to this article:

Data availability

Data will be made available on request.