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Cancer therapy has undergone notable evolution, progressing from conventional chemotherapy to contemporary immunotherapy strategies (Schirrmacher, 2019). Although immunotherapy has revolutionized cancer treatment, offering superior anti-cancer effects with minimal side effects compared to traditional approaches such as chemotherapy and radiotherapy (Tan
Tumor interacts with various immune cells in the tumor microenvironment (TME) to maintain an immunosuppressive milieu (Baghban
β-glucan, particularly mainly derived from
In this study, we obtained a soluble β-1,3/1,6-glucan (PPTEE) with high purity from
All animal experiments were conducted with the approval of the Institutional Animal Care and Use Committee of Kangwon National University (IACUC No. KW-221102-2). Female C57BL/6 mice, aged 5 weeks, were purchased from Koatech Co. Ltd (Pyeongtaek, Korea) and housed in the Specific Pathogen-Free facility at the Laboratory Animal Research Center of Kangwon National University. The mice received intramuscular immunizations every 2 weeks for a total of 6 weeks. Each mouse groups were administered 10 μg of Ovalbumin (OVA, Sigma Aldrich, St. Louis, MO, USA) alone, OVA+microemulsion (ME) and OVA+ME-containing 100 μg of PPTEE-glucan, as an adjuvant, for immunization.
A seed culture of
Treatment with pullulanase (Promozyme D2, Daejongzymes, Seoul, Korea) was carried out by incubating the supernatant at 30°C with agitation at 200 rpm for 24 h to enzymatically break down pullulan. Following this, the supernatant was subjected to protease treatment (Protamex, Daejongzymes) at 50°C for 24 h. To precipitate the proteins, 100% (v/v) trichloroacetic acid (TCA) was added to the treated supernatant to achieve a final concentration of 6% (v/v) TCA, which was then cooled at 4°C overnight. The TCA-treated mixture was centrifuged at 8,000×g for 20 min to pellet the precipitated polysaccharides. The supernatant was carefully decanted, leaving the precipitate behind.
The precipitate was resuspended in 2.5 volumes of ice-cold ethanol and stored at –20°C overnight to further facilitate polysaccharide precipitation. After incubation, the mixture underwent another round of centrifugation at 8,000×g for 20 min to collect the polysaccharide precipitate. The collected precipitate was reconstituted with distilled water and then subjected to a second round of ethanol precipitation by adding 2.5 volumes of ice-cold ethanol and incubating at –20°C overnight. Subsequently, the mixture was centrifuged at 8,000×g for 20 min to pellet the polysaccharides, which were washed with distilled water to remove any residual ethanol. Finally, the washed polysaccharide precipitate was lyophilized to obtain dry PPTEE-glucan samples for further analysis.
Following the manufacturer’s protocol, we quantified β-glucan levels using the Megazyme yeast β-glucan enzymatic kit (Megazyme, Bray, Ireland). The samples were dissolved in sodium hydroxide. After pH adjustment, the glucan was converted into glucose by β-glucanases, β-glucosidases, and chitinases, and then quantified using the GOPOD reagent. The entire process was conducted in accordance with the manufacturer’s guidelines, and each sample underwent the assay three times.
The total sugar content of β-glucan was determined using the phenol-sulfuric acid method (DuBois
The total protein content in β-glucan was determined using the Bradford method. In detail, 15 μL of the sample solution and 750 μL of the Bradford reagent were combined in a tube and incubated at room temperature for 5 min. Subsequently, the absorbance was measured at 595 nm. To quantify the total protein content, a standard calibration curve was established with Bovine Serum Albumin as the standard substance, and the total protein content was calculated based on this curve. The total protein content (%) of β-glucan was then calculated accordingly.
The molecular weight distribution of the β-glucan was analyzed using high-performance size-exclusion chromatography (HPSEC) with two series-connected Shodex columns, specifically KS-804 and KS-802 (Showa Denko, Tokyo, Japan). The samples were dissolved in a dimethyl sulfoxide/water mixture (90:10; v/v). The column was maintained at a constant temperature of 70°C, and the mobile phase (water) was delivered at a flow rate of 0.8 mL/min. The distribution was compared with pullulan standards (Shodex P-82, Showa Denko). These standards were prepared by allowing a 0.05% (w/v) aqueous solution to stand at 25°C for 24 h, resulting in complete particle swelling. The dispersion was stirred until all particles dissolved. Prior to use, the solution was filtered through a 0.45 μm filter.
A female C57BL/6 mouse was subcutaneously injected with MC38 colon cancer cells (1×106). The mouse received intratumoral injections of QS-21 (1 μg) or a combination of PPTEE-glucan (100 μg) with ME, each in 50 μL, every 3 days starting from day 7 post-tumor inoculation until the conclusion of the experiment. Tumor size was monitored every other day until reaching a volume of 1,500 mm3 (calculated as length×width×2/2). Subsequently, the mice were euthanized to extract the tumors.
The tumor tissue was homogenized in a C-tube (Miltenyi Biotec, Bergisch Gladbach, Germany) containing 5 mL of digestion buffer [RPMI-1640 (Corning, NY, USA) with 2% Bovine serum (Gibco, Waltham, MA, USA), 10 mM HEPES (Welgene, Gyeongsan, Korea), 1% Penicillin/Streptomycin (Welgene), 400 U/mL Collagenase IV (Worthington Biochemical, Lakewood, NJ, USA), and 0.1 mg/mL DNase I (Roche Diagnostics, Basel, Switzerland)] using GentleMACS (Miltenyi Biotec). The digestion buffer was incubated in a shaking incubator at 200 rpm, 37°C for 45 min. The digested tumor tissues were filtered through a 100 μm pore size strainer, and 10 mL of PBS (Corning) was added before centrifugation at 1,400 rpm for 3 min at 4°C. After removing the supernatant, the pellet was suspended with 40% Percoll (Cytiva, Marlborough, MA, USA) and layered on 70% Percoll. Density gradient separation was carried out at 2,000 rpm for 20 min at room temperature without interruption. The white ring between the 40/70 Percoll layers was collected, washed with 10 mL PBS at 1,400 rpm for 3 min at 4°C, and the cell pellet was suspended in PBS for further analysis.
The extracted tumor cells were first incubated with PBS containing αCD16/CD32 (2.4G2) at room temperature for 10 min to block Fc receptors. Subsequently, the cells were incubated with a diverse combination of antibodies in PBS, including APC-Cy7-Ly6C (HK1.4), PE-PD-L1 (10F.9G2), PE-Cy7-Ly6G (1A8), PerCP-Cy5.5-CD45 (30-F11), BV421-CD11b (M1/70), and Fixable Viability Dye NIR (Invitrogen, Waltham, MA, USA), in the dark for 30 min at 4°C for cell surface staining. The tumor cells were then centrifuged at 1,400 rpm for 3 min at 4°C for washing. After removing the supernatant, the pellet was suspended in PBS. All fluorochrome-conjugated antibodies were obtained from BioLegend (San Diego, CA, USA). The stained tumor cells were analyzed using FACSVerse flow cytometry (BD Bioscience, Franklin Lakes, NJ, USA) and FlowJo Version 10.8.1 software (BD Bioscience).
The tibia and femur of a mouse were extracted to flush out bone marrow (BM) cells with PBS, followed by the removal of red blood cells (RBCs) using an RBC lysis buffer (Invitrogen) and filtration through a 100 μm pore size strainer. The isolated BM cells were cultured in a growth medium with GM-CSF (20 ng/mL) and IL-4 (20 ng/mL) for 6 days at 37°C with 5% CO2 to differentiate into dendritic cells (DC). Subsequently, the BM-derived DC (BMDC) were stimulated with PPTEE-glucan (10 μg/mL), QS-21 (1 μg/mL), and OVA (10 μg/mL) for 24 h at 37°C with 5% CO2 before further analysis. Similarly, the tibia and femur of a mouse were used to extract BM cells, which were then processed to remove RBCs and cultured in a growth medium with GM-CSF (20 ng/mL) and IL-6 (10 ng/mL) for 5 days at 37°C with 5% CO2 to differentiate into myeloid-derived suppressive cells (MDSC). The BM-derived MDSC were stimulated with PPTEE-glucan or QS-21 for 24 h at 37°C with 5% CO2 before subsequent analysis.
The stimulated BMDC were incubated with PBS containing αCD16/CD32 (2.4G2) at room temperature for 10 min to block Fc receptors. Subsequently, the cells were incubated with a diverse combination of antibodies, including PE-CD80 (16-10A1), APC-CD86 (GL-1), FITC-CD11c (N418), PE-Cy7-CD40 (3/23), PerCP-Cy5.5-H2-Kb (AF6-88.5), Pacific Blue-I-A/I-E (M5/114.15.2), and Fixable Viability Dye NIR (Invitrogen), in the dark for 30 min at 4°C for cell surface staining. The BMDC were then centrifuged at 1,400 rpm for 3 min at 4°C for washing. After removing the supernatant, the pellet was suspended in PBS. All fluorochrome-conjugated antibodies were purchased from BioLegend. The stained BMDC were analyzed using FACSVerse flow cytometry (BD Bioscience) and FlowJo Version 10.8.1 software (BD Bioscience).
The stimulated BMDC (5×104) were seeded into a U-bottom 96-well plate. OT-I mice were sacrificed to extract the spleen and lymph nodes. The spleen and lymph nodes were minced on a 100 μm pore size strainer using a syringe piston in PBS. The PBS was collected into a 15 mL tube and centrifuged at 1,400 rpm for 3 min at 4°C. After removing the supernatant, the pellet was suspended with RBC lysis buffer (Invitrogen) to remove red blood cells. The sample was centrifuged at 1,400 rpm for 3 min at 4°C. The supernatant was removed, and the pellet was suspended in PBS. The PBS was filtered through a 100 μm pore size strainer. CD8+T cells in the sample were isolated using the CD8+T cell isolation kit (Miltenyi Biotec) and then stained with CellTrace Violet (CTV, Invitrogen) following the manufacturer’s instructions. The CD8+T cells were seeded into wells containing BMDC and co-cultured in growth medium for 2 days at 37°C with 5% CO2. The cells were collected, incubated with PBS containing αCD16/CD32 (2.4G2) at room temperature for 10 min to block Fc receptors. Subsequently, the cells were incubated with PBS containing a diverse combination of antibodies, including APC-CD8 (53-6.7), PE-CD45.1 (A20), and Fixable Viability Dye NIR (Invitrogen), in the dark for 30 min at 4°C for cell surface staining. The cells were then centrifuged at 1,400 rpm for 3 min at 4°C for washing. After removing the supernatant, the pellet was suspended in PBS. All fluorochrome-conjugated antibodies were purchased from BioLegend. The stained cells were analyzed using FACSVerse flow cytometry (BD Bioscience) and FlowJo Version 10.8.1 software (BD Bioscience).
The emulsion composition, consisting of oil, surfactant, and cosurfactant, was chosen based on our prior research (Lee
The spleen was minced on a 100 μm pore size strainer using a syringe piston in PBS. The PBS containing the minced spleen was collected in a 15 mL tube and centrifuged at 1,400 rpm for 3 min at 4°C. After removing the supernatant, the cell pellet was suspended in 1 mL of RBC lysis buffer (Invitrogen) and incubated for 1 min at room temperature. Following the addition of 4 mL of PBS, the tube was centrifuged at 1,400 rpm for 3 min at 4°C. After discarding the supernatant, the pellet was resuspended in 10 mL of PBS and filtered through a 100 μm pore size strainer, making the sample ready for further study. Splenocytes from OVA-vaccinated mice (2×105) were seeded into a round-bottom 96-well plate containing growth medium with 10 μg of OVA at 37°C with 5% CO2 for 3 days. The cells were then collected in a 1.5 mL tube, followed by intracellular staining as described above.
Harvested cells from the tumor or spleen were stimulated with 200 μL of growth medium, including Cell Stimulation Cocktail plus a protein transport inhibitor (eBioscience, Waltham, MA, USA), for 3 h in a 37°C, 5% CO2 incubator. The cells were then collected in a 1.5 mL tube and washed with 1 mL of PBS by centrifuging at 1,400 rpm for 3 min at 4°C. Subsequently, the cells were incubated with PBS containing αCD16/CD32 (2.4G2) at room temperature for 10 min to block Fc receptors. The cells were further incubated with a diverse combination of antibodies, including FITC-CD3 (145-2C11), PerCP-Cy5.5-CD4 (GK1.5), PE-Cy7-CD8 (53-6.7), PE-CD45 (30-F11), and Fixable Viability Dye NIR for T cells, and PE-Cy7-CD11b (M1/70), BV510-F4/80 (BM8), FITC-Ly6C (HK1.4), BV421-CD45 (30-F11), and Fixable Viability Dye NIR for MDSC. The cells were incubated in the dark for 30 min at 4°C for cell surface staining. Following this, the cells were treated with IC fixation buffer (eBioscience) for 30 min in the dark at 4°C for cell permeabilization. The fixed cells were washed with 1× permeabilization buffer (eBioscience) using a centrifuge as described in the previous step. For cytokine staining, the cells were incubated with 1× Permeabilization buffer containing APC-IFN-γ (XMG1.2) and APC-TNF-α (MP6-XT22) for T cells and MDSC, respectively, in the dark for 30 min at 4°C. Subsequently, the cells were washed with 1× Permeabilization buffer using a centrifuge as described earlier, and the pellet was suspended in 1× Permeabilization buffer. The stained cells were analyzed using FACSVerse flow cytometry (BD Bioscience) and FlowJo Version 10.8.1 software (BD Bioscience). All fluorochrome-conjugated antibodies were purchased from BioLegend.
The supernatant from stimulated BMDC and MDSC was collected in a 1.5 mL tube, then centrifuged at 13,000 rpm for 10 minutes at 4°C. The supernatant was transferred to a new 1.5 mL tube. TNF-α, IL-1β, IL-6, and IL-12p40 were measured using respective ELISA kits (Invitrogen) following the manufacturer’s instructions.
Immuno 96-well plates (Thermo Fisher Scientific, Waltham, MA, USA) were coated with 10 μg/mL of OVA in 0.05 M carbonate-bicarbonate buffer (pH 9.6, Sigma Aldrich) and incubated overnight at 4°C. The wells were washed three times using PBS containing 0.05% Tween-20 (PBS-T) and then blocked with 1% bovine serum albumin (BSA, MP Biomedicals, Solon, OH, USA) for 1 h at room temperature. After washing the wells with PBS-T, diluted serum samples were added to the wells and incubated overnight at 4°C. Subsequently, the wells were washed three times with PBS-T, and horseradish peroxidase-conjugated goat anti-mouse IgG1, anti-mouse IgG2b, anti-mouse IgG2c, or anti-mouse IgM (Southern Biotech, Birmingham, AL, USA) were added and incubated for 2 h at room temperature. Following five washes with PBS-T, TMB substrate (Surmodics, Eden Prairie, MN, USA) was added to the wells and incubated for 15 min at room temperature. The reaction was stopped by adding 0.5 M HCl. The optical density (O.D.) of the wells in the plates was measured at 450 nm using a microplate reader (Molecular Devices, San Jose, CA, USA).
Recipient mice were infused with CTV (Invitrogen)-stained OT-I CD8+T cells (1×106) via intravenous injection. The following day, the mice received subcutaneous administration of OVA (10 μg) along with either QS-21 (1 μg) or a combination of PPTEE-glucan (100 μg) with ME. Three days post-OVA injection, the mice were euthanized, and their spleens were harvested for the analysis of proliferated OT-I CD8+T cells using FACSVerse flow cytometry (BD Bioscience) and FlowJo Version 10.8.1 software (BD Bioscience).
The mouse tumor tissue was fixed in 4% formaldehyde for 24 h and dehydrated using ethanol in a Tissue Processor (Leica, Wetzlar, Germany). Subsequently, the tissues were embedded in paraffin and sectioned into 5 μm-thick slices. Slides containing these sections were incubated at 60°C for 1 h, deparaffinized using xylene, and rehydrated with graded ethanol. Apoptosis was assessed using a TUNEL assay kit (Thermo Fisher Scientific), following the manufacturer’s protocol. The slides were mounted with a mounting solution containing 4’,6-diamidino-2-phenylindole (DAPI, BioLegend) for confocal microscopy.
Statistical analyses were conducted using GraphPad Prism version 9 (GraphPad Software LLC, San Diego, CA, USA). The unpaired t-test was used to assess the differences between tow groups. The comparisons among multiple groups utilized Dunnett’s test and one-way analysis of variance (ANOVA). Furthermore, for comparisons involving groups with continuous values, two-way ANOVA was employed. The threshold for statistical significance was set at
To verify the immunogenicity of PPTEE, we stimulated BMDC with OVA for 24 h in the presence of PPTEE and QS-21 as positive controls. The mean fluorescence intensity (MFI) of CD80, CD86, CD40, H2-Kb, and I-A/I-E in PPTEE-treated BMDC was significantly increased compared to that in the other groups (Fig. 2A-2F). However, co-treating with ME and PPTEE to BMDC couldn’t enhance immune activation of PPTEE (Supplementary Fig. 1). Supernatants were collected from the stimulated BMDC to analyze pro-inflammatory cytokines levels. TNF-α, IL-1β, IL-6, and IL-12p40 were remarkably expressed in PPTEE-treated BMDC compared to OVA and OVA with QS-21 groups (Fig. 2G-2J). To assess the PPTEE-induced enhancement of antigen cross-presentation in BMDC, we co-cultured PPTEE-stimulated and OVA-treated BMDC with CellTrace Violet-stained OT-I CD8+T cells. PPTEE-treated BMDC showed significantly increased OT-I CD8+T cell proliferation at both ratios compared to that of BMDC treated with OVA alone, though not in QS-21-stimulated and OVA-treated BMDC (Fig. 2K). These data indicate that PPTEE can stimulate BMDCs into an immunogenic state, enhancing the expression of co-stimulatory markers, inflammatory cytokine production, and CD8+ T cell proliferation, ultimately increasing soluble antigen cross-presentation. These findings indicate the potential of PPTEE-glucan as an immune adjuvant that activates antigen-presenting cells.
We developed a ME system containing PPTEE-glucan (ME+PPTEE) and assessed its potential as a vaccine adjuvant. We confirmed OVA+ME contains comparable amount of OVA compared to the OVA control (Supplementary Fig. 2). The particle properties of the ME+PPTEE and OVA+ME+PPTEE formulations were investigated (Supplementary Fig. 3). The hydrodynamic size of the ME+PPTEE group was 318.1 ± 56.33 nm, whereas that of the OVA+ME+PPTEE group was 410.37 ± 44.28 nm. Additionally, the zeta potential value of the ME+PPTEE group was 2.66 ± 0.11 mV, while that of the OVA+ME+PPTEE group was 11.90 ± 0.63 mV. The spherical shapes and comparable diameters (to hydrodynamic diameters in Supplementary Fig. 3) of ME+PPTEE and OVA+ME+PPTEE were observed in TEM image (Supplementary Fig. 4). The average content of PPTEE in ME, analyzed by HPLC system, was 63.22%. Mice were intramuscularly injected with OVA-containing ME or ME+PPTEE. PPTEE alone treatment failed to induce vaccination (Supplementary Fig. 5). Both ME-and ME+PPTEE-treated groups exhibited higher titers of IgG1, IgG2b, and IgG2c compared to the OVA control group. Notably, the IgG2b titer was significantly higher in the serum of ME+PPTEE-treated mice (Fig. 3A-3D). The higher IgG2b(Area Under Curve, AUC)/IgG1(AUC) ratio observed in ME+PPTEE-treated mice compared to that in ME-treated mice suggested the induction of a Th1 immune response in C57BL/6 mice (Fig. 3E). To evaluate antigen-specific T cell responses in vaccinated mice, splenocytes were stimulated with OVA. While IFN-γ expression in CD4+T cells was comparable among all groups, CD8+T cells from ME+PPTEE-treated mice showed significantly increased IFN-γ expression compared to that of the ME-treated group (Fig. 3F). To confirm the immunogenicity of PPTEE
Building on the observed activation of BMDCs and enhanced antigen-specific immune responses following PPTEE treatment, we evaluated the potential of PPTEE in mitigating cancer growth using a murine tumor model. Intratumoral injections of ME formulations, including ME alone, QS-21-containing ME (ME+QS-21), and PPTEE-containing ME (ME+PPTEE), were administered to MC38-bearing mice. Although ME alone failed to induce anti-cancer effects, the mice treated with ME+QS-21 and ME+PPTEE exhibited reduced cancer growth and tumor mass (Fig. 4A, 4B). Studies have reported that β-glucan treatment in cancer-bearing mice can reduce cancer growth by modulating MDSCs to become immunogenic (Albeituni
Our findings demonstrated enhanced expression of co-stimulatory markers, pro-inflammatory cytokines, and cross-presentation abilities in PPTEE-treated BMDCs
MDSCs of the myeloid lineage share specific characteristics with monocytes and neutrophils and their immunosuppressive function contributes to the pathogenesis of cancer by inhibiting anti-cancer T cells, promoting regulatory T cell differentiation, supporting angiogenesis, and creating a metastatic environment (Umansky
Our data demonstrate an increased CD11b+Ly6C+ cell population in tumors of PPTEE-glucan-treated mice, as well as
The immunologic activity and binding to its receptors, CR3 and dectin-1, of β-glucan are determined by variable molecular characteristics such as the degree of polymerization, branches, length, and solubility, depending on its sources (Han
β-glucan has demonstrated its potential as a vaccine adjuvant. In the context of hepatitis B vaccination, curdlan, a β-glucan derived from the cell wall of
Cancer is characterized by the establishment of a TME that suppresses anti-cancer immune responses (Shimizu
In the late 1990s, the emulsion adjuvant MF59 was licensed and showed the potential to prevent pandemic influenza by enhancing cytotoxic immune activity (Khurana
In summary, we confirmed the potential of highly purified soluble PPTEE-glucan generated by