The cell culture reagents and products, such as the 35 mm temperature-responsive cell culture dishes, were purchased from Thermo Fisher Scientific (Rockford, IL, USA). Male nude mice (age 6 weeks) were provided by Shanghai SLAC Laboratory Animal Co., Ltd. and maintained in a barrier facility on high efficiency particulate air-filtered racks with 12 h dark–light cycles and allowed ad libitum access to food and water. All experimental protocols were approved by a named institutional review board and/or ethical licensing committee. Animal experiments were carried out according to the experimental procedures approved by the Committee for Animal Research of Shanghai Jiaotong University and followed the guidelines for the Care and Use of Laboratory Animals.
Cell culture and cell sheets harvesting
Human prostate carcinoma cell line DU145 (American Type Culture Collection, Manassas, VA, USA) were cultured in RPMI-1640 medium supplemented with 10% (v/v) fetal bovine serum and 100 U/ml penicillin and 100 μg/ml streptomycin at 37 °C with 5% CO2. The medium was changed every 2–3 days. When the cells reached 95% confluence, they were digested with 0.5% trypsin and then dissociated. To improve cell adhesion and cell sheets formation, 35 mm UpCell plates (Thermo Fisher Scientific, USA) were coated with 500 μg/ml rat tail collagen I for 24 h at 4 °C. DU145 cells (106/dish) were cultured at 37 °C in a humidified atmosphere with 5% CO2. After 4 days, when the cells reached over-confluency (> 100%), they were harvested as a contiguous cell sheet by holding the culture at 20 °C for 15 min.
Surface morphology of DU145 cell sheets
Morphological structures of DU145 cell sheets were observed by scanning electron microscopy (SEM, JSM-7800F Prime, JEOL, Japan). Briefly, samples were fixed in 2.5% glutaraldehyde, washed in deionized water, dehydrated in a graded series of ethanol, and dried by lyophilization. The specimens were then sputter coated with platinum and observed with a scanning electron microscope (SU8000 series; HITACHI, Tokyo, Japan).
Preparation of DU145 cell sheet fragments and cell suspension
After washing with Dulbecco’s phosphate-buffered saline (PBS; Sigma-Aldrich, St. Louis, MO, USA), DU145 cell sheets were chopped into small fragments for syringe injection. The fragment size was smaller than 1 mm × 1 mm so that it could be easily injected through 30 gauge needle. The fragments from a single cell sheet were treated with 0.25% trypsin–EDTA (Sigma-Aldrich, USA) at 37 °C for 10 min and the cell number was counted. After reaching 80–90% confluence, DU145 cells were washed with PBS, followed by a treatment with 0.25% trypsin–EDTA to produce cell suspension. The harvested DU145 cells were washed twice with PBS and then resuspended with BD Matrigel (BD, Bedford, MA, USA).
Ectopic and orthotopic transplantation by injection
The animals were anesthetized with 2% isoflurane in whole experiment. Twelve male nude mice (age 6 weeks) were randomly assigned to four groups. For ensure the equivalent cell number in different groups. A single cell sheet was harvested and chopped into fragments and the cell number was counted after treated with trypsin–EDTA. And then the equal number of DU145 cell suspension were used as control. For ectopic transplantation, DU145 cell sheet fragments (Group A) and cell suspension (Group B) with equal cell number in 100 μl were subcutaneously injected into the left back of the mice. For orthotopic transplantation, the nude mice were anesthetized and placed in supine position. After the prostate was exposed, 20 μl DU145 cell sheet fragments (Group C) and cell suspension (Group D) were injected into the ventral prostate capsule using a syringe with a 30-gauge needle (50 μl, BD, USA). The abdominal incision was closed using a 5–0 silk suture. Four weeks after injection, the nude mice were sacrificed, and the tumor, left upper limb, liver, lung, bladder, and prostate were harvested for further histological evaluation.
After transplantation, tumor loading was observed and recorded every 3 days. The maximum diameter (a) and minimum diameter (b) of the tumor were measured with micrometer caliper. The tumor volume was assumed to be a semi-ellipsoid and was calculated by the following formula : Tumor volume (V) = a × b2 × π/6.
Magnetic Resonance Imaging (MRI) of the tumor
MRI was used to observe the growth of tumors in vivo and whether there was local invasion or distant metastasis. The nude mice were examined by MRI at the 2nd and 4th week after inoculation and all animals were anesthetized with 2% isoflurane. The MRI was done in 7.0 T small animal MR scanners (Biospec 70/20 USR; Bruker Biospin MRI, Inc., Billerica, MA, USA). T2-weighted MRI was performed by a fast spin-echo sequence, based on specific parameters as follows: TE = 32 ms, TR = 2050 ms, slice thickness = 1 mm, FoV = 30 × 32 mm, and matrix = 256 × 256, scan time≈20 min.
For cross-sectional observations, the harvested cell sheets, resected tumors and organs were fixed with 4% paraformaldehyde and embedded in paraffin. The specimens were sliced into 5 μm sections, followed by haematoxylin and eosin (HE) staining and Masson staining. For immunohistochemical staining, the sections were blocked with 1% bovine serum albumin (BSA) and 0.5% Triton-X100, then treated with desmin, vimentin, CK-8, CD31 and type I collagen IgG antibody (rabbit anti-human, 1:500, Abcam, Cambridge, MA, USA) at 4 °C overnight. After washing with PBS, the sections were incubated with a horseradish-peroxidase-conjugated goat anti-rabbit IgG antibody (1:1000; Invitrogen, Carlsbad, CA, USA) for 1 h at room temperature. Finally, the sections were stained with 3,3 N-diaminobenzidine tertrahydrochloride (Sigma-Aldrich, USA) and counterstained with hematoxylin. Images were captured using an upright metallurgical microscope (Olympus, Japan).
Tumor cell apoptosis analyzed by TUNEL and cleaved caspase-3 immunofluorescence
According to the instructions supplied by the manufacturer, TdT-mediated dUTP nick end labelling (TUNEL) assay was performed with cell apoptosis in situ detection kit (YEASON, China). Briefly, the slides were firstly incubated with proteinase K for 10 min at room temperature, then incubated with Alexa Fluor 488–12-dUTP in TdT buffer for 1 h at 37 °C with protecting from light, and followed by staining with DAPI solution (1 μg/ml, Invitrogen) at room temperature for 5 min. The slides were observed immediately under a fluorescence microscope. Green fluorescence was observed at 520 ± 20 nm with a standard fluorescent filter and DAPI was observed at 460 nm. The cell apoptosis was observed under a fluorescence microscope (Olympus, Japan). In order to detect active caspase-3, cleaved caspase-3 immunofluorescence was used to detect the active (cleaved) form of caspase-3. The resected tumors in each group were fixed with 4% paraformaldehyde and embedded in paraffin. Sections were then permeabilized with 0.5% Triton X-100 (Invitrogen) and blocked with 5% bovine serum albumin for 30 min. Then, sections were incubated with an optimal concentration of rabbit monoclonal anti–cleaved caspase-3 antibody (1:300, Abcam, Cambridge, MA, USA) overnight at 4 °C, then incubated with anti–rabbit secondary antibody (1:500; Alexa Fluor 470; Invitrogen, Carlsbad, CA, USA) for 45 min at 37 °C, followed by washing 3 times with PBS. The cell nucleus was stained with DAPI (1 μg/ml, Invitrogen) for 30 s. The cell apoptosis was observed under a fluorescence microscope (Olympus, Japan).
SPSS version 19 (IBM, USA) was used for statistical analysis. All data are presented as mean ± SD. An unpaired Student’s t test was performed to compare differences between two groups. One-way analysis of variance was performed for the comparison of multiple groups. P < 0.05 was considered a significant difference.