Establishment of mouse subcutaneous RM-1 cell tumor model
All animal experiments were approved by the Ethics Committee of Hunan University of Chinese Medicine (LLBH-202303240004). Male C57BL/6 mice at the age of six weeks were procured from SJA Laboratory Animal Co. Ltd (Hunan, China). After one week of adaptive feeding, RM-1 cells (AW-CCM416, Abiowell) were injected subcutaneously. The number of cells injected into each nude mouse was 1 × 106; the injection volume was 100 µL, and the injection location was the left armpit. After tumor implantation, tumor measurements were performed 2–3 times per week.
Experiment 1, after obvious subcutaneous tumor formation (8 days), the mice were divided into 5 groups (n = 6 mice/group): PCa, ICA, CUR, ICA + CUR, and Docetaxel (DOC) groups. In the PCa group, mice were administered the same volume of vehicle control as the other experimental groups. In the ICA group, ICA (10 mL/kg; B21576, shyuanye, China) was intraperitoneally injected five times a week [12]. In the CUR group, mice were injected intraperitoneally with CUR (10 mL/kg; B20342, shyuanye, China) every two days [16]. In the ICA + CUR group: mice were administered with an equal dose of ICA + CUR as other experimental groups. After injecting ICA, CUR should be injected into the mice immediately. In the DOC group, mice were injected with DOC (10 mL/kg; S61817, shyuanye, China) intraperitoneally once a week as a positive control [30].
In experiment 2, mice were divided into 2 groups (n = 6 mice/group): ICA + CUR and ICA + CUR + SCFAs groups. In the ICA + CUR group, mice were injected with ICA + CUR. The mice were provided with water for normal feeding. In the ICA + CUR + SCFAs group, mice were injected with ICA + CUR. The mice’s drinking water was supplemented with a mixture of SCFAs: 67.5 mmol/L sodium acetate (S434883, Aladdin, China), 40 mmol/L sodium butyrate (303410, Sigma, USA), and 25.9 mmol/L sodium propionate (P1880, Sigma, USA) [9].
In experiment 3, mice were divided into 4 groups (n = 6 mice/group): ICA + CUR + IgG, ICA + CUR + SCFAs + IgG, ICA + CUR + anti-IGFBP2, and ICA + CUR + SCFA + anti-IGFBP2 groups. In the ICA + CUR + IgG group, mice were injected with ICA + CUR, on day 7, mice were intraperitoneally injected with IgG. In the ICA + CUR + SCFAs + IgG group, mice were injected with ICA + CUR, and on day 7, mice were intraperitoneally injected with IgG (10 mg/kg; 6-001-F, and systems, USA) once a day. The mice’s drinking water was supplemented with a mixture of SCFAs. In the ICA + CUR + anti-IGFBP2 group, mice were injected with ICA + CUR, and on day 7, mice were intraperitoneally injected with anti-IGFBP2 (10 mL/kg). In the ICA + CUR + SCFAs + anti-IGFBP2 group, mice were injected with ICA + CUR, and on day 7, mice were intraperitoneally injected with anti-IGFBP2 (10 mL/kg) [31]. The mice’s drinking water was supplemented with a mixture of SCFAs.
Twenty-Five days after tumor implantation, the experiment was completed, and the mice were sacrificed by intraperitoneal injection of pentobarbital sodium (150 mg/kg) [32]. The tumor was excised for further experiments. Stool and serum samples were collected for 16 S rRNA sequencing, metabolic analyses, and other analyses.
Fecal microbiota transplantation (FMT)
For FMT, after 3 days of antibiotic-containing water was administered to the mice, they were given normal water for 3 days. In the PCa or ICA + CUR groups, freshly excreted feces were collected and then mixed with sterile water. To remove solid impurities, a 70 μm nylon filter sieve was used. Oral injection of fecal bacterial suspension was performed via a tube-feeding needle at a rate of 200 µL/mouse/day (from the fourth day until the death of the mice) [33]. They were grouped into the FMT-PCa and FMT-PCa-ICA + CUR groups and five mice per group. In the PCa group, mice were administered the same volume of corn oil as the other experimental groups. In the FMT-PCa group, mice were administered the corn oil. In the FMT-PCa-ICA + CUR group, mice were injected with the same volume of ICA + CUR as the FMT-PCa group.
Magnetic bead sorting of CD3+CD8+ T cells
A complete culture medium (2 mL) was taken to moisten a 40 μm cell strainer before placing the mouse spleen. The mouse spleen was cut with ophthalmic scissors and ground by pressing the bottom of the piston of a 5 mL syringe. The strainer was rinsed with 10 mL of RPMI-1640 complete medium (C3010-0500, Viva Cell). Cell precipitation was achieved by centrifugation at 1500 rpm. The supernatant was removed and 5 mL of red cell lysate was added, which was cleaved at room temperature and centrifuged to precipitate the cells. The supernatant was removed, and the cells were resuspended in 6 mL PBS and 1 mL buffer solution. Then 1.3 × 107 cells were counted. After the cells were suspended in 120 µL precooled buffer solution, 30 µL Biotin-Antibody Cocktail was added to the mixture, and the cells were incubated in the refrigerator. One milliliter of the buffer solution was added and centrifuged to collect the precipitates. A buffer solution (120 µL) was added for re-suspension, and then 60 µL Anti-Biotin MicroBeads were added to cells. The cells were placed in a refrigerator for incubation. The LS column was balanced on a magnetic rack with a buffer solution for equilibration. Subsequently, the aforementioned cells were loaded onto the LS column. After cleaning the LC column with buffer solution, the passed cells were collected for follow-up experiments (approximately 5.7 × 106 cells were counted after sorting). 1 × 105 cells /100 µL were taken into EP tubes, and washed with 1 mL PBS. After centrifuging at 1500 rpm, the cell pellet was retained. This step was once more. After resuspending the cell pellet in 100µL of basal culture medium, the corresponding antibody CD3-FITC (11-0032-82, eBioscience) was added, mixed, and incubated (to distinguish negative at this time). After PBS washing of the cells, they were centrifuged and the cell pellets were retained. Cells were precipitated in a basal medium and analyzed using a flow cytometer (A00-1-1102, Beckman).
Cell culture and treatment
Mouse PCa cells RM-1 and human PCa cells DU145 (AW-CCH043, Abiowell) were cultured in DMEM containing 10% FBS and 1% penicillin/streptomycin at 37℃ and 5% CO2 in a saturated humidity incubator.
In experiment 1, RM-1 and DU145 cells were grouped into the Control, ICA, CUR, and ICA + CUR groups. In the Control group, cells were cultured normally. In the ICA group, cells were treated with 30 µM ICA (N1705, APExBIO) for 48 h [34]. In the CUR group, cells were treated with 50 µg/mL CUR (HY- N0104, MCE) for 48 h [16]. In the ICA + CUR group, cells were treated with 30 µM ICA and 50 µg/mL CUR.
In experiment 2, RM-1 cells with CD8+ T cells were co-cultured. Then they were divided into RM-1 + T cells, ICA + T cells, CUR + T cells, and ICA + CUR + T cells groups. In the RM-1 + T cell group, cells were co-cultured with CD8+ T cells. In the ICA + T cell group, cells were exposed to 30 µM ICA for 48 h, followed by co-culture with CD8+ T lymphocytes. In the CUR + T cell group, cells were exposed to 50 µg/mL CUR for 48 h, followed by co-culture with CD8+ T lymphocytes. In the ICA + CUR + T cell group, cells were treated with 30 µM ICA and 50 µg/mL CUR for 48 h, followed by co-culture with CD8+ T lymphocytes.
In experiment 3, the RM-1 and DU145 cells were divided into Control, ICR + CUR, ICR + CUR + oe-NC, ICR + CUR + oe-DNMT1, ICR + CUR + oe-DNMT1 + siNC, ICR + CUR + oe-DNMT1 + si-IGFBP2 groups. In the Control group, cells were cultured under normal conditions. In the ICR + CUR group, cells were exposed to 30 µM ICA and 50 µg/mL CUR. In the ICR + CUR + oe-NC group, cells were treated with 30 µM ICA and 50 µg/mL CUR, then transfected with oe-NC. In the ICR + CUR + oe-DNMT1 group, cells were treated with 30 µM ICA and 50 µg/mL CUR, then transfected with oe-DNMT1. In the ICR + CUR + oe-DNMT1 + si-NC group, cells were treated with 30 µM ICA and 50 µg/mL CUR, then transfected with oe-DNMT1 + si-NC. In the ICR + CUR + oe-DNMT1 + si-IGFBP2 group, cells were treated with 30 µM ICA and 50 µg/mL CUR, then transfected with oe-DNMT1 + si-IGFBP2.
In experiment 4, the RM-1 and DU145 cells were divided into si-NC, si-DNMT1, si-DNMT1 + oe-NC, and si-DNMT1 + oe-IGFBP2 groups. In the si-NC group, cells transfected with si-NC. In the si-DNMT1 group, cells were transfected with si-DNMT1. In the si-DNMT1 + oe-NC group, cells were transfected with si-DNMT1 + oe-NC. In the si-DNMT1 + oe-IGFBP2 group, cells were transfected with si-DNMT1 + oe-IGFBP2.
In experiment 5, RM-1 and DU145 cells were grouped into the Control, Acetate, Propionate, Butyrate, and SCFAs groups. In the Control group, cells were cultured normally. In the Acetate group, the cells were treated with acetate for 72 h. In the Propionate group, cells were treated with propionate for 72 h. In the Butyrate group, cells were treated with butyrate for 72 h. In the SCFAs group, cells were treated with acetate, propionate, and butyrate together for 72 h.
16 S rRNA sequencing
Fecal samples from mice in the PCa, ICA, CUR, and ICA + CUR groups were collected to assess changes in microbial diversity. To obtain raw data, the Illumina NovaSeq PE250 was used for 16 S amplicon sequencing. DADA2 was invoked to denoise the raw data using the Qiime 2 analysis process. Denoised sequences were de-redundant to obtain the feature information directly. Species annotation was performed for each ASV sequence, and the species composition in the samples was measured by comparing species databases.
Detection of SCFAs
Fecal samples from mice in the PCa, ICA, CUR, and ICA + CUR groups were collected to detect changes in SCFAs (acetic, propionic, isobutyric, butyric, isovaleric, and valeric acid). An appropriate amount of feces (50 mg-100 mg) with magnetic beads and 300 µL saline (including 37.3 µg/mL d7 isobutyric acid) was homogenized at 60 Hz for 60 s. Supernatants were centrifuged at 4℃, and 200 µL was removed, acidified by adding 10 µL of 5 M HCl, and vortexed. Anhydrous ether (200 µL) was used for extraction, vortexed, and centrifuged at 4℃. The collected supernatant was subjected to analysis using an Agilent 7890B-5977B gas chromatograph.
Immunofluorescence (IF)
To assess Ki67 expression in the mouse tumors, The sections were immersed in a solution of sodium borohydride solution, soaked in 75% ethanol solution, and then placed in Sudan Black staining solution. The sections were incubated in a 5% BSA solution. Appropriate dilutions of primary antibody Ki67 (ab16667, 1:100, Abcam) were added dropwise overnight at 4℃. 50–100 µL CoraLite488-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) (SA00013-2, Proteintech) fluorescent antibody was incubated. DAPI was used to stain nuclei. The sections were sealed using buffered glycerol and subsequently examined using a fluorescence microscope.
Enzyme-linked immunosorbent assay (ELISA)
According to the kit’s instructions, ELISA was utilized to evaluate levels of IGFBP2 (CSB-E04589m, CUSABIO), interferon-γ (IFN-γ) (KE10001, Proteintech), interferon-α (IFN-α) (MFNAS0, R&D Systems), Perforin (Cbic-E13429m, CUSABIO), Granzyme A (CSB-E08717m, CUSABIO, China) and Granzyme B (CSB-E08720m, CUSABIO, China).
Western blot (WB)
To assess PD-L1, IGFBP2, DNMT1, EGFR, STAT3, p-EGFR, and p-STAT3 protein expression levels, the total proteins were first extracted using RIPA (#P0013B, Beyotime, China), and protein quantification was performed using the BCA protein assay kit (BL521A, Biosharp, China). Protein was mixed with SDS-PAGE loading buffer (AWB0055, Abiowell, China), and adsorbed on Nitrocellulose membrane. PD-L1 (28076-1-AP, 1:600, Proteintech), IGFBP2 (ab188200, 1:1000, Abcam), DNMT1 (AWA44628, 1:1000, Abiowell), EGFR (ab52894, 1:5000, Abcam), STAT3 (10253-2-AP, 1:2000, Proteintech), p-EGFR (ab40815, 1:1000, Abcam), p-STAT3 (ab76315, 1:5000, Abcam), and β-actin (28076-1-AP, 1:600, Proteintech) were incubated overnight at 4℃. HRP goat anti-mouse/rabbit IgG was added. An ECL chemiluminescence solution was used for color development. β-actin was used as an internal reference.
Flow cytometry (FCM)
A complete culture medium (2 mL) was taken to moisten a 40 μm cell strainer before placing the tumor. The tumor was cut with ophthalmic scissors and ground by pressing the bottom of the piston of a 5 mL syringe. The rest of the operation is as described above for magnetic bead sorting. RM-1 and DU145 cells were resuspended in 500 µL of 10% FBS 1640, added 1µL of Cell Stimulation Cocktail (plus protein transport inhibitors), and incubated at 37℃. The cells were resuspended in 500 µL 10% FBS 1640, and 1uL of Cell Stimulation Cocktail (plus protein transport inhibitors) was added and then stimulated at 37℃. After collecting the cells and centrifuging, the supernatant was discarded. Then, 1 mL of 0.5% BSA-PBS was added to wash the cells once, followed by centrifugation, and the supernatant was discarded. The corresponding antibodies CD3-FITC (11-0032-82, eBioscience, USA) and CD8-APC (17-0081-82, eBioscience, USA) were incubated, and light was avoided. Cells were washed with PBS. Next, the cells were resuspended in 500 µL of Intracellular Fixation buffer and fixed. After centrifuging and discarding the supernatant, the pellet was resuspended in 1× Permeabilization Buffer, followed by discarding the supernatant after centrifugation. Then, 100 µL of 1×Permeabilization Buffer was used to resuspend the cell pellet, and the corresponding antibody IFN-γ (12-7311-82, eBioscience, USA) or Ki67 (12-5698-82, eBioscience, USA) was added and mixed well. The mixture was incubated, protected from light, for 30 min. The cells were washed with 1 mL of 0.5% BSA-PBS, and the supernatant was discarded after centrifugation. Finally, the cells were resuspended in 150 µL of 0.5% BSA-PBS for FCM.
Cell counting Kit-8 (CCK-8) assay
RM-1 and DU145 cells were digested with pancreatic enzymes (AWC0232, Abiowell) and inoculated at a density of 1 × 104 cells/well. After adherent culture, 10 µL/well of CCK-8 (NU679, Dojindo) was added. The cells were incubated and analyzed using the Bio-Tek assay (MB-530, HEALES) at 0, 24, 48, and 72 h.
Transwell assays
First, a transwell assay was performed to determine the migratory capacity of the cells. A complete medium (500 µL) was placed in the lower layer of a Transwell (3428, Corning). The treated cells were digested with trypsin, serum-free medium was added to resuspend the cells at 2 × 106 cells/mL, and 100 µL of cells were added to each well. After being placed in incubation, the upper chamber was removed, and placed in a new well with PBS. Then the upper chamber was washed with PBS. The cells in the upper chamber were wiped with cotton swabs. The cells were fixed with 4% paraformaldehyde. Cells were stained with 0.1% crystal violet (AWC0333, Abiowell) and washed 5 times with water. Cells on the surface of the upper chamber were observed under an inverted microscope, and three fields of view were obtained. In the invasion assay, Matrigel-coated Transwell chambers (354262, BD) were used, following the same experimental procedures as the migration assay.
Real-time quantitative polymerase chain reaction (RT-qPCR)
The expression levels of perforin, granzyme A, and granzyme B in sorted CD8+ T cells were detected. Total RNA was extracted from CD8+ T cells using Trizol (15596026, Thermo, America), and cDNA was obtained using a reverse transcription kit (CW2569, CowinBio, China). Finally, we performed specific amplification using the SYBR method on a fluorescence quantitative RT-PCR instrument (SPL0960, Thermo, USA). The primer information used in the experiment can be found in Table 1.
Immunohistochemistry (IHC)
The expression of DNMT1 (bs-0678r, 1:200, Bioss) and IGFBP2 (ab188200, 1:200, Abcam) in mouse tumors was detected. Tumor sections were sequentially placed in xylene and gradient ethanol, followed by washing with distilled water. The sections were immersed in 0.01 M citrate buffer (pH 6.0) and boiled using an electric stove, then cooled to room temperature and removed from the slides, followed by washing with PBS. Next, 1% periodic acid was added to the sections and left at room temperature for 10 min, followed by rinsing with PBS. Diluted primary antibodies against DNMT1 and IGFBP2 were added to the sections and incubated overnight at 4℃. After washing the sections with PBS, 50 ~ 100 µL of HRP goat anti-rabbit-IgG was added and incubated at 37℃ for 30 min, followed by washing with PBS. Then, 50 ~ 100 µL of pre-made DAB working solution was added to the sections and incubated at room temperature, followed by washing with distilled water. The sections were counterstained with hematoxylin, rinsed with distilled water, and then blued in PBS. The sections were subsequently subjected to a graded series of alcohol solutions for dehydration. Finally, the sections were removed and placed in xylene, covered with neutral gum, and observed under a microscope.
Co-immunoprecipitation (CO-IP)
To detect the interaction between DNMT1 and IGFBP2, we first extracted total protein from RM-1 and DU145 cells. The protein supernatant was then divided into 2 tubes and a certain amount of antibody DNMT1(AWA44628, 1:1000, Abiowell) and IGFBP2 (ab188200, 1:1000, Abcam) was added, followed by thorough mixing and incubation overnight at 4℃ with rotation. Next, Protein A/G agarose beads were taken and mixed with IP lysis buffer (AWB0144, Abiowell, China), then centrifuged to collect the precipitate. The sample was supplemented with the pre-treated Protein A/G agarose beads and gently shaken at 4℃. After immunoprecipitation, the mixture was centrifuged, and the bottom of the agarose bead tube was retained to remove the supernatant, followed by washing the agarose beads with 400 µL IP lysis buffer for 4 times, and retaining the final precipitate. In the agarose bead precipitate, 30 µL of IP lysis buffer was added, followed by mixing with 10 µL of 5× loading buffer. The mixture was boiled, and then quickly cooled in an ice box for subsequent WB analysis.
Statistical analysis
GraphPad Prism 8.0 software was applied for statistical analysis. Data were expressed as mean ± standard deviation. Student’s t-test was used between the two groups, and one-way analysis of variance (ANOVA) was performed for inter-group comparison. A correlation analysis was performed using the Spearman correlation coefficient to examine the relationship among gut microbiota with the IGFBP2/EGFR/STAT3/PD-L1 pathway and immune-related indicators. P < 0.05 indicated that the difference was statistically significant.
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