Cell culture and reagents
Human bladder cancer cell line Biu-87 was purchased from the American Type Culture Collection (ATCC, Manassas, USA). Mouse bladder cancer cell line MB49 was a gentle gift from Peking Union Medical Collage. All cancer cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco, MA, USA) containing 10% fetal bovine serum (FBS) (Gibco, MA, USA), and maintained at 37 °C in 5% CO2. HCPT was purchased from Sangon (Shanghai, China). Notch inhibitor SAHM1 (C94H162N36O23S) was purchased from MedChemExpress (MA, USA). Laminin-1 for cell culture was purchased from Sigma-Aldrich (L4544, MA, USA).
For 2D collagen (containing laminin or not) gels culture, type I collagen (Solarbio, Beijing, China) was diluted to 2.5 mg/ml with DMEM culture medium (containing 2 μg/ml laminin or not). Subsequently, 20 μl 1 M NaOH solution were subsequently added into 230 μl collagen solution. 250 μl of the collagen mixture was seeded into a 24-well plate and mixed thoroughly. After 37 °C incubation for 1 hour, cancer cells were seeded on top of the solid 2D collagen gels at a concentration of 1 × 104 cells/well and maintained in DMEM culture medium containing 10% FBS.
Human bladder tumor tissue sections were obtained from the First Affiliated Hospital, University of South China, and divided into NMIBC and MIBC according to the Guidelines for the Diagnosis and Treatment of Bladder Cancer (2019). All participants and/or thier legal guardians agreed to participate in the study and informed in prior. The clinical experiments were carried out according to the Declaration of Helsinki. This study was approved by the Ethics Committee of the First Affiliated Hospital of University of South China (#20170257). Survival information of 405 bladder cancer patients in The Cancer Genome Atlas Program (TCGA) was downloaded from https://www.cbioportal.org.
Cell proliferation assay
Cell proliferation was assessed by Cell Counting Kit-8 (CCK-8, Solarbio, Beijing, China). Briefly, MB49 or Biu-87 cells were seeded in 96-well plates (2500 per well) and cultured with DMEM culture medium supplemented with 10% FBS. Cell proliferation was examined at 0, 24, 48, and 72 hours according to manufacturer’s specifications. Absorbance of samples was quantified at 450 nm by microplate reader (Thermo Fisher, MA, USA). Cell proliferation was normalized to day 0 (2500 cells).
5 × 104 MB49 or Biu-87 cells were seeded in the 8 μm transwell insert (Corning, CA, USA) containing 100 μl culture medium (10% FBS). The bottom chamber was filled with 500 μl culture medium containing 20% FBS. After 24 hours, the migrating cells were fixed with paraformaldehyde and stained with crystal violet. The migrating cells numbers were counted under an optical microscope (Leica, Munich, Germany).
SiRNA to ITGB4 (#siRNA1: 5′-GGUCACCUCCAAGAUGUUC-3; #siRNA2: 5′-GGACUGGGUCCUUUCACAU-3′), ITGA6 (#siRNA1: 5′-GTGGGAAGTTTAATAGAGT-3; #siRNA2: 5′-CCTAGTGGGATATGCCTCCAGGTTA-3′), JAG1 (#siRNA1: 5′-GGAACAACCUGUAACAUAGCCCGAA-3; #siRNA2: 5′-CCACAGCAACGAUCACAAAUGACTT-3′), TRB3 (#siRNA1: 5′-CTTCGTCCAGCCCCAGTCC-3; #siRNA2: 5′-ATCTCTGGCTGCTTCTGCCGATGTT-3′) were purchased from Ruibo (Guangzhou, China). Transfections were performed in 24-well palates with 100 pmol siRNA and Invitrogen Lipofectamine 2000 (Thermo Fisher, MA, USA) according to manufacturer’s specifications. Quantitative Polymerase Chain Reaction (qPCR) was performed to examine the silence efficiency in Biu-87 cells.
Quantitative polymerase chain reaction
MB49 or Biu-87 were cultured on dish or 2D laminin/collagen gels for 5 days. Cells were then harvested and total RNA was extracted using RNA Extraction Kit (Thermo Fisher, MA, USA) according to the manufacturer’s instructions. Reverse transcription of total RNA was performed using cDNA synthesis kits (Takara Bio, Tokyo, Japan) following the manufacturer’s instructions. PCR was performed with SYBR Green Supermixes (Biorad, MA, USA). Primer sequences were downloaded from https://pga.mgh.harvard.edu/primerbank/.
Cancer cells were lysed by RIPA buffer (25 mM Tris, pH 7.6, 150 mM NaCl, 1% NP40, 1% DOC, 0.1% SDS). Protein samples were resolved by SDS–polyacrylamide gel electrophoresis and blotted on polyvinylidene difluoride membranes. Some of the blots were cut prior to hybridisation with antibodies. Primary antibodies, including anti-integrin α6 (ab181551), anti-integrin β4 (ab133682), anti-Notch1 (ab52627, cleaved intracellular domain of Notch1), anti-JAG1 (ab109532), anti-TRB3 (ab137526) and anti-β-actin (ab8226) were purchased from Abcam (Cambridge, UK). Full blots containing markers were included in supplementary materials.
Immunohistochemistry and immunofluorescence
Bladder tumor tissues were fixed in 10% formalin solution. The samples were processed, embedded in paraffin, and sectioned at 5 μm for immunohistochemical and immunofluorescence staining. Sections of tumor tissues were then dewaxed, rehydrated, quenched of endogenous peroxidase, blocked, and incubated with the primary antibody: anti-Laminin (ab11575, Abcam, Cambridge, UK), anti-integrin α6 (ab181551, Abcam, Cambridge, UK), anti-integrin β4 (ab133682, Abcam, Cambridge, UK) and anti-Notch1 (ab52627, Abcam, Cambridge, UK) at 4 °C overnight. Samples were then incubated with secondary antibodies and stained with hematoxylin/ 4′, 6-diamidino-2-phenylindole (DAPI). The intensity of protein expression was quantified by Image J 2.0 (N.J, USA) and Image-pro Plus 6.0 software (MA, USA). 10 fields were included in each sample. The mean of brown intensity in 10 fields were identified as the expression intensity in this sample. 15 samples from 15 patients were included in each group.
Dual luciferase activity assay
Activation of Notch1 signaling in tumor cells were determined by luciferase reporter assay. MB49 or Biu-87 cells were seeded on dish or 2D laminin/collagen gels for 3 days, and then co-transfected with control/pGL3 vector containing firefly luciferase reporter gene and the 30 UTR of Notch1 gene (Yunzhou, Beijing, China) using lipofectamine 2000 (Invitrogen, MA, USA). 48 hours later, a luciferase assay kit (Promega, MA, USA) was used for luciferase activity assay.
Orthotopic animal models
Female C57BL/6 mice (6–8 weeks old) were purchased from Huafukang (Beijing, China). To establish orthotopic bladder cancer model, 1 × 106 MB49 cells in 100 μl PBS were intravesical instilled into the bladders of C57BL/6 mice by venous indwelling needles. On day 6 and 8, mice were treated with PBS, HCPT (0.5 mg/ml), SAHM1 (0.5 mg/ml) or combining treatment by intravesical instillation. On day 10, the occurrence of hematuresis was recorded (n = 10). On day 12, mice were sacrificed for tumor weight analysis (n = 6). The tumor weight was calculated according to the formula: tumor weight = total bladder weight – normal bladder weight (21 mg). Survival of tumor bearing mice was recorded on a daily basis (n = 6). All animal experiments of this study were approved by the Institutional Animal Care and Use Committee of University of South China (20150223-154). The animal studies were conducted in accordance with the Public Health Service Policy and complied with the WHO guidelines for the humane use and care of animals.
Each experiment was performed for three independent times. Data were presented as the mean ± SEM and statistical significance was analyzed using GraphPad 7.0 software (L.J, USA). Statistical significance between groups was calculated by Student’s t test for two groups or by one-way ANOVA for more than two groups. Bonferroni analysis were further used for the post hoc test. The survival rates were analyzed by Kaplan–Meier survival analysis. The survival information of clinical bladder patients was downloaded from https://www.cbioportal.org/. *p < 0.05; **p < 0.01; ns, no significant difference.