Establishment of rat model of bladder cancer and experimental groups

This study was in accordance with the ethical standards and was approved by the Henan Provincial People’s Hospital. A total of 28 wistar rats weighing 200 ± 20 g and 7 weeks of age were used in this study. The mice were bred according to the Guidelines for the Care and Use of National Institutes of Health (NIH) at the Animal Center of Zhengzhou University. A rat model of bladder tumor was induced by intravesical MNU (Sigma, USA) reperfusion, once every 2 weeks for 10 weeks (2 mg/kg). Tumor-bearing rats were randomly divided into four groups (n = 7): the normal saline group (control), the fisetin treatment group (positive control) [23, 24], the PA-MSHA treatment group and the PA-MSHA + fisetin treatment group. Each group was given tail vein injection of saline, fisetin, PA-MSHA or combination of PA-MSHA and fisetin, twice per week every 3 days.

Determination of bladder weight and expression of Caspase 3 and E-Ca

All rats were sacrificed in the 10th week after treatment, and then necropsy was performed. The total weight of the bladder was then determined as previously described [25]. Tumor tissues were fixed in 4% paraformaldehyde and embedded in paraffin. For morphological analysis, H&E staining and microscopic examination were performed on 3 µm-thick sections from paraffin-embedded tumor blocks. Immunohistochemical staining was probed with polyclonal anti-Caspase 3 antibody and polyclonal anti-E-cadherin antibody (Cell Signaling Technology, USA) according to the manufacturer’s instructions. Data was analyzed by Image-pro Plus Analysis system (Olympus, Japan). Macrophages were separated from tumor cells by their expression of CD11b and F4/80 for mouse macrophages and determined by flow cytometry [21].

The role of PA-MSHA in the expression of M1 cytokines and M2 cytokines

To detect the role of PA-MSHA in murine bone marrow (BM)-derived macrophages, macrophages were separated and stimulated with different concentrations of PA-MSHA in vitro. To further explore whether PA-MSHA stimulation efficiency is time-dependent, murine BM-derived macrophages were isolated and cultured with 106/mL PA-MSHA for 0, 6, 12, 24, 48, and 72 h. Then total RNA were extracted using TRIzol reagent (Invitrogen, USA), and cDNA were synthesized using one step PrimeScript miRNA cDNA Synthesis Kit (Takara Biotechnology, Dalian, China). The expression levels of cytokine (IL-12, TNF-α, IFN-γ, IL-4, IL-10 and TGF-β) were measured by qRT-PCR and the dates were determined by the 2−ΔΔCT method [26]. All qRT-PCR reactions were performed three times. Subsequently, the protein expression levels of the cytokines were determined by Western blotting. In brief, cells in culture were lysed in RIPA buffer (Sigma, USA), then, the proteins were loaded onto 10% SDS-PAGE prior to being transferred onto nitrocellulose (NC) membranes (Amersham Bioscience, UK). Following blocking with skimmed milk, the membranes were incubated with the corresponding horseradish peroxidase (HRP)-conjugated secondary antibody at room temperature for 1 h. GAPDH was used as a control. Each protein sample was examined in triplicate.

Apoptosis and invasion abilities assays in vitro

The mouse bladder cancer cell line BBN1617 cells or MB49 cells were incubated in the RPMI 1640 or culture supernatants of murine BM-derived macrophages treated with PA-MSHA. BBN1617 and MB49 were collected by low speed centrifugation (centrifuged at 2000 r/min for 3 min) and washed twice with ice-cold PBS. Then, 300 μL binding buffer were added to the cells. Afterward, the cells were stained with 5 μL propidium iodide (PI) and 5 μL annexin V at 4 °C for 5 min to 15 min in the dark. Apoptotic cells were counted by flow cytometry within 60 min using quadrant statistics of the annexin V-positive and propidium iodide-negative cells.

Cell invasion was determined by Transwell assays. The membrane of the chamber was coated with 30 mg/cm2 Matrigel (BD Biosciences, USA) for 1 h in 37 °C to form a matrix barrier. The lower chambers were filled with 600 μL of DMEM. The cells were suspended in DMEM to a concentration of 1 × 105 cells per well and were loaded into each upper well with a volume of 200 µL. After the chambers cultured in 5% CO 2 at 37 °C for 24 h, cells were fixed with methanol for 10 min and stained with crystal violet for 10 min. Subsequently, cells were washed with PBS. Counts were obtained from five random fields at 200× magnification.

Cell migration activity was analyzed using a scratch assay. BBN1617 and/or MB49 cells were plated onto 6-well plates at a density of 5 × 105 cells per well and cultured to 100% confluence. Afterward, cells were scraped with a pipette 200 μL tip in a cross in the center of each well, washed with PBS, and immediately replaced with fresh low-serum medium. 48 h after growth, the migration distances of the cells were observed and photographed under a Nikon microscope (Minato) at a magnification of 200× for each group.

Phagocytosis assay in vitro

To generate M1 or M2 macrophages, sorted BM cells were treated with recombinant mouse macrophage colony-stimulating factor (M-CSF). M1 polarization was achieved with further treatment through interferon gamma (IFN-γ) stimulation, followed by lipopolysaccharide (LPS). M2 polarization was obtained by further treatment with IL-4 and IL-13 [21].

Tumor cells were labeled with CFSE and co-incubated with murine BM-derived differentially polarized macrophages. Two different primary tumor cell lines (BBN1617 and MB49) were offered to either mouse M1 or M2 macrophages as targets. After the phagocytosis assays finished, staining of macrophages and the respective antibody master mix was done directly. The macrophage population was identified by macrophage markers CD11b and F4/80 targeted fluorescently labeled antibodies. Macrophages for each macrophage subtype was quantified by the percentage of CFSE+ events among CD206+ (mouse M2 macrophage), CD80+ (mouse M1 macrophages) events.

Statistical analysis

All date were analyzed with SPSS17.0 (SPSS Inc, USA) and expressed as the mean ± S.D. Student’s t test was used to analyze differences between two groups. One-way ANOVA analysis was used to determine the multi-sample analysis. All statistical tests were two-sided, and P value less than 0.05 was considered significant.