Mouse prostate gross anatomy
10 male Balb/c athymic nude mice (5 weeks old) were purchased from Shanghai Lab Animal Research Center. Mice were acclimated for 3 weeks under pathogen-free conditions. Mice were anesthetized in an isoflurane chamber and placed in a supine position with their extremities fixed to the dissecting board. The abdominal region was disinfected, and the abdominal cavity was accessed via an incision made through the linea alba using Metzenbaum scissors. The bladder and seminal vesicles (SVs) were lifted with dissecting forceps to reveal the four lobes of the mouse prostate: AP, DP, lateral prostate (LP), and ventral prostate (VP). The prostate was viewed under a stereoscopic microscope (Zeiss Stemi 2000-C with image processing software). The AP, DP, LP, and VP were identified in the coronal, axial, cranio-lateral, and ventral views, respectively.
Tumor cell line
The LNCaP cell line was obtained from the China Center for Type Culture Collection. Cells were cultured in DMEM/F12 (Gibco, Grand Island, NY, USA) supplemented with 10% fetal bovine serum (FBS, Gibco) and maintained in an incubator (HERACELL 150i, Thermo Scientific, Rockford, IL, USA) at 37°C in a humidified atmosphere with 5% CO2. At approximately 80% confluence, cells were harvested with 0.25% trypsin/EDTA solution and washed twice in PBS. Cells (1×106) in 20μL PBS medium were mixed with 50% Matrigel (BD Biosciences) before inoculation.
Orthotopic injection of LNCaP cells
22 male Balb/c athymic nude mice (5 weeks old) were acclimated for 3 weeks before experimental manipulation. LNCaP cells were grafted into the mouse left or right DP under a stereoscopic microscope. Mice were anesthetized with 3% isoflurane in an induction chamber with flow rate of 0.8L/min and placed in a supine position. During surgery, heating pads were used to maintain body temperature. The lower abdomen was disinfected with 70% ethanol. A 1.5 cm transverse incision was made in the lower midline with microscissors above the presumed location of the bladder. Approximately 100μL intraperitoneal liquid spilled and was sponged with cotton balls. The intestine was pushed upward into the abdominal cavity using a sterile cotton swab. The SV was pulled outside the abdominal cavity with micro-tweezers. The SV was moved posteriorly and stabilized with a cotton ball to expose the DP. The trigonal bipyramidal DP was located, and the needle of a Hamilton syringe (sharp 10–12° 33G beveled needle) was inserted into one side of the DP at an angle of approximately 15° to the long axis of the DP. A 20μL suspension containing 1×106 LNCaP cells was slowly injected until the DP resembled a small blister (Fig. 1). The needle was retracted, and a cotton ball was placed over the injection site for about 1~2 sec to prevent bleeding, minimize leakage, and solidify the Matrigel within the lobe. The prostate and SV were returned to the peritoneum, and the abdominal wall and skin layer were sutured with a 5-0 braided absorbable suture (Covidien). The wound was disinfected with 70% ethanol. Mice were observed until recovery (approximately 1 h). The wound healed within 2 weeks. Starting 2 weeks post-injection, tumor growth was monitored by ultrasound (US) every 5 days.
Tumor surveillance by gray-scale US
US imaging was performed using a portable sonography device (GE LOGIQ BOOK-XP, MI 0.7, Tis 0.9, Frequency 9~11MHz, depth 2.0cm). Prewarmed US coupling gel was applied to the abdominal wall before imaging. 2D images of the mouse prostate and adjacent anatomy were acquired using a linear-array transducer (8L-RS) in B-mode with a center frequency of 11-MHz. The probe was positioned perpendicular to the abdominal wall in a sagittal orientation, and the bladder was identified as a round anechoic structure. The probe was moved until an echogenic tumor was identified on the dorsal side superior to the base of the bladder. Images of the bladder and tumor were acquired. Maximum sagittal length and height of the tumor was measured in the longitudinal plane. The probe was rotated to view the tumor in the transverse plane, and the width of the tumor was measured. Tumor volume was calculated as 0.52×maximum length×height×width.
CEUS and Time-Intensity Curves (TIC) analysis
CEUS was performed using a LOGIQ E9 system (GE Healthcare, Milwaukee, Wis., USA) with a multiple frequency linear probe (ML 6~15 MHz) prior to sacrifice. Mice were anesthetized and were examined in the supine position. The focal depth was 1 cm, and the mechanical index was 0.08 to 0.10. The tumor was visualized in the sagittal plane, and the recommended dose (100μL) of SonoVue® (Bracco, Italy) was administered as a bolus with a 1 ml syringe through the retro-orbital venous sinus. Raw data generated for 120 s were saved, and imaging datasets were analyzed with quantification software (VueBox ™). A region of interest (ROI) was manually contoured to cover the whole tumor in the longitudinal plane. A time-intensity curve (TIC) was generated based on the signal intensities in the individual ROIs.
T2-weighted MRI
Magnetic resonance imaging (MRI) was performed using a 3.0 T MR system (Ingenia, Philips Medical Systems, Netherlands) with a dedicated 8-channel receive coil with a 5.0 cm inner diameter (Chenguang Medical Technologies Co., Shanghai, China) on Day 35 and Day 70 after orthotopic injection of LNCaP cells. T2-weighted spin-echo sequences were used to image the tumors. Transverse imaging (repetition time/echo time [TR/TE], 6.0 s/68 ms; flip angle, 90°; matrix, 64 × 64; field of view, 50×50 mm; slice thickness, 1.0 mm; slice gap, 0 mm; 2 signal averages) and coronal imaging (TR/TE, 3.0 s/80 ms; flip angle, 90°; matrix, 64 × 64; field of view, 100×100 mm; slice thickness, 1.0 mm; slice gap, 1 mm; 2 signal averages) were performed. Twenty coronal slices with a thickness of 500 mm each and an in-plane resolution of 100mm×100mm were acquired with a multi-slice, multi-echo sequence. The scan time was 13 min per data set. Orthotopic tumor volume was calculated as 0.5 × width × length × height.
Serum PSA, histological and immunohistochemistry
Blood samples were collected from the retroorbital sinus before sacrifice. PSA levels were analyzed using an Electro-ChemiLuminescence ImmunoAssay (ECLIA). Orthotopic tumors were removed, fixed in 10% neutral-buffered formalin, and embedded in paraffin. Serial sections (4 μm thick) were cut on a microtome and mounted on glass slides. Sections were deparaffinized in Histoclear (National Diagnostic, Atlanta, GA) and hydrated in a graded series of alcohols and running tap water. Histopathology was performed using standard hematoxylin and eosin staining. Immunohistochemical staining was performed with a Vectastain ABC Elite kit (Vector Laboratories, Inc., Burlingame, CA). Briefly, sections were deparaffinized and hydrated, and endogenous peroxidase activity was blocked with 0.3% hydrogen peroxide in methanol for 20 min. Antigen retrieval was performed using the Antigen Unmasking Solution (Vector Laboratories, Inc.). Sections were cooled and rinsed in PBS, incubated in blocking solution for at least 30 min at room temperature, and incubated with primary antibodies for CD31, α-smooth muscle (α-SMA; 1:50), Ki-67 (1:300), androgen receptor (AR), PSA, p63 (1:300), or P504S overnight at 4oC. Sections were incubated with the appropriate biotinylated secondary anti-goat, anti-mouse, or anti-rat immunoglobulin for 30 min at room temperature. The antigen-antibody reaction was visualized using 3,3’-diaminobenzidine tetrahydrochloride as a substrate. Sections were examined by light microscopy.
Statistical analysis
Statistical analyses were performed with MedCalc software (Version 12.3.0.0, Mariakerke, Belgium). Pearson’s correlation coefficient was used to compare tumor volumes measured on MRI and US on Day 35 and Day 70. Differences in the measurement techniques were plotted using the Bland-Altman method. p<0.05 was considered statistically significant.