Nude mice and C57BL/6 mice used in this study were purchased from the Animal Centre of Ningxia Medical University (Yinchuan, China). GFP mice were purchased from the Model Animal Research Center of Nanjing University (Nanjing, China). The experiment was approved by the Ethics Committee of Ningxia Medical University. Animal management during the experiment strictly abided by the Guidance about Treating Experimental Animals issued by the Ministry of Science and Technology of the People’s Republic of China in 2006. Human BALL-1 cells were purchased from ATCC.
Construction of the nude mouse BALL model and observation of their survival state
Nude mice (male,4 weeks old) were randomly divided into eight groups (n = 10 mice per group): one normal control group and seven experimental groups that were injected with 102–108 human BALL cells via the caudal vein. The tumor model mice were executed by ip injection of pentobarbital (100 mg/kg) after the onset of cachexia. In addition, following BALL cell injection, one side of the testicular tissue was punctured every 7 days, with a total of three sites being punctured for smears and HE staining. BALL cells were detected until the testicular leukemia was diagnosed, and the onset time of testicular leukemia and the survival time of mice were recorded.
Independent marker verification
The markers of BALL cells and mouse SSCs were determined through a literature review, with immunofluorescence and flow cytometry methods used to verify the independent expression of the respective markers in BALL cells and SSCs. Finally, CD20 and CD38 were chosen as BALL cell markers and CD90 and CD49f as an SSC marker for subsequent experiments.
Sections (5 μm) were baked in a constant temperature oven at 60 °C for 4 h, dewaxed by dimethyl benzene, and rehydrated by gradient alcohol. The tissue sections were washed with phosphate-buffered saline (PBS) (3 × 5 min, similarly hereinafter). The tissue was repaired using 0.01 M citrate buffer antigen for 15 min, cooled to room temperature, repaired once again, and rinsed thoroughly with PBS. The tissue sections were blocked with 5% bovine serum albumin (BSA) blocking solution at room temperature for 1 h and added with primary antibody liquid overnight at 4 °C (anti-CD20, Invitrogen, 14020982, 1:200; anti-CD38, Invitrogen, 14038982, 1:200; anti-CD90, Bioss, bs-0778R, 1:200; anti-CD49f, Abcam, ab105669, 1:200). The next day, the tissue sections were held at room temperature for 1 h then washed with PBS. The secondary antibody was added at room temperature for 1 h, followed by PBS washes. 4′,6-Diamidino-2-phenylindole (DAPI) was added for 30 min, then the tissue was rinsed with PBS. DAPI was added to seal the section, which was photographed using fluorescence microscopy.
Sections (5 μm) were baked in a constant temperature oven at 60 °C for 4 h, dewaxed by dimethyl benzene, and rehydrated by gradient alcohol. The tissue sections were washed with PBS (3 × 5 min, similarly hereinafter). The tissue was repaired using 0.01 M citrate buffer antigen for 15 min, cooled to room temperature, repaired once again, and rinsed thoroughly with PBS. The tissue sections were blocked with 5% bovine serum albumin (BSA) blocking solution at room temperature for 1 h and added with primary antibody liquid overnight at 4 °C (anti-GFP, Proteintech, 66002-1-Ig, 1:200). The next day, the tissue sections were held at room temperature for 1 h then washed with PBS. The secondary antibody was added at room temperature for 1 h, followed by PBS washes. Hematoxylin was added for 3 min, then the tissue was dehydrated using graded ethanol, vitrification by dimethylbenzene, and seal sheet with neutral gum and photographed using microscopy.
Flow cytometry detection
Mouse blood (50 μL) in 2% EDTA as a coagulant was added to 1 mL PBS and centrifuged three times at 4 °C, 300g for 5 min. PBS (500 μL) was added to the pellet for re-centrifugation. Primary antibody was added (anti-CD20, Invitrogen, 14020982, 1:100; anti-CD38, Invitrogen, 14038982, 1:100; anti-CD90, Bioss, bs-0778R, 1:100; anti-CD49f, Abcam, ab105669, 1:100, anti-UTF1, Thermo Fisher, MFCDA84, 1:250; anti-PLZF, Bioss, Bs5971R, 1:250; CD117, Bioss, Bs20716R, 1:200), then cells were incubated at 4 °C for 1 h, washed with PBS, and centrifuged as described above. Fluorescent secondary antibody was added, and cells were incubated at 4 °C for 1 h, then centrifuged as above, except with the addition of 1 mL PBS for re-centrifugation. Cells were filtered through a 40-μm mesh and tested by an instrument (DM2000, Leica, USA).
Extraction and purification of SSCs
Following confirmation of the successful construction of the testicular leukemia model, mouse testicular tissue was collected under aseptic condition and the albuginea was stripped. The tissue was cut into 2–3-mm pieces, to which five volumes of 0.5% trypsin were added for digestion. When the tissues were digested to single cells, 2 mL serum was added to stop the digestion. The cells were centrifuged at 300g for 5 min, collected, washed with PBS three times, and filtered through a 40-μm mesh. The filtrate was collected, and SSCs were isolated and purified using the following methods according to each group.
Density gradient centrifugation for SSC screening
Percoll density gradient liquid was prepared in the order of 20%, 30%, 35%, 40%, 45%, 50%, and 60%. A 1-mL aliquot of each gradient was absorbed and added to a 15-mL centrifuge tube according to the gradient from large to small. A straw was used to carefully move the cell suspension to be separated, which was added on the separation medium at the top of the gradient fluid in the 15-mL centrifuge tube. The centrifuge tube was centrifuged at 4 °C, 500g for 10 min, and cells from the 35–45% gradient were collected, washed with PBS with three centrifugation steps at 300g for 5 min. The supernatant was abandoned, and the cells were re-suspended with 1.5 mL PBS. Cell count was determined, and the cell concentration was adjusted to 107/mL.
Immunomagnetic bead-based sorting for SSC screening
Cells of the obtained testicular cell suspension were re-suspended with magnetic bead separation buffer and precipitated into a centrifuge tube to obtain cell count. After centrifugation at 300g, the supernatant was abandoned. Antibody solution was added to the cells following instructions (CD20 microbeads, MACS, 130093452; CD38 microbeads, MACS, 130092263; CD90 microbeads, MACS, 12000295; anti-CD49f, Abcam, ab105669; anti-rat IgG microbeads, Miltenyi Biotec,130048502), incubated at 4 °C for 30 min after mixing, centrifuged at 300g, and washed three times. Then, 500 μL separation buffer was added to the final pellet to re-suspend the cells. A new centrifuge tube was placed into the grooves of the magnetic frame, and a pipette was used to carefully add the cell suspension. Magnetic bead separation buffer (500 μL) was added before the last drop of the filtrate, for a total of three times. Then, the cell separation tube was immediately removed from the magnetic field, and 1 mL of separation buffer was added, for a total of three times. Cells were centrifuged at 300g and counted, and the cell concentration was adjusted to 107/mL.
Flow cytometry for SSC screening
Cells after suspension and filtration were centrifuged, and the supernatant was abandoned. Primary antibody 200 μL (anti-CD20, Invitrogen, 14020982, 1:100; anti-CD38, Invitrogen, 14038982, 1:100; anti-CD90, Bioss, bs-0778R, 1:100; anti-CD49f, Abcam, ab105669, 1:100) was added to the pellet, mixed with cells, incubated at 4 °C for 30 min, centrifuged at 300g, and washed three times with PBS. Then, 200 μL FITC-labeled fluorescent antibody (1:100) was added, and cells were incubated at 4 °C for 30 min, centrifuged at 300g, washed three times in PBS, and screened by flow cytometry (FACS Vantage SE, BD Biosciences, Canada). Cells were screened and counted, and the cell concentration was adjusted to 107/mL.
Preparation of SSC transplant recipient mice and SSC transplantation
SSC transplant recipient mice (male, 4 weeks old) and SSC transplantation refer to the articles published by the research group , the incision was disinfected with alcohol and covered with iodine volts gauze and an elastic bandage. The skin incision was disinfected once daily. Penicillin was administered at 50,000 U/mouse via ip injection, for three successive days.
Tissues and cells were collected under aseptic condition and lysed with RIPA buffer. The protein concentration was determined using the BSA method after extraction according to the kit instruction (KGPBCA, Keygen Biotech, Nanjing China). Proteins were separated using 4–15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred onto polyvinylidene fluoride membranes, blocked with 5% skim milk powder for 1 h, then the primary antibody was added (anti-CD20, Invitrogen, 14020982, 1:2000). The next day, the membrane was kept at room temperature for 1 h and washed with PBS for 3 × 5 min. Corresponding secondary antibody (Proteintech, Rosemont, IL, USA) was added. The membrane was incubated at 37 °C for 1 h and washed with PBS. ECL was added and imaged by an instrument (ChemiDoc XRS+, Bio-Rad, Canada).