Patient tissue sample
This study was approved by the Sun Yat-sen Memorial Hospital Ethics Committee. From September 2011 to March 2021, our hospital (SYSMH) collected 190 ccRCC tissues and their matching adjacent tissues. The 190 ccRCC patients included 111 men and 79 women of age 21–89 years (average age: 53.2 years). Written informed consent was obtained from all participants. The patient inclusion criteria included clear cell renal cell carcinoma. Exclusion criteria included patients who did not receive any anti-tumor therapy before surgical resection. The pathologist of our hospital confirmed the ccRCC tissues and the adjacent non-tumor tissues.
Database
The database information used in this study is TCGA (https://portal.gdc.cancer.gov/). Since the data was sourced from an open public database, there was no need to obtain research approval from the ethics committee.
Reagents and antibodies
Recombinant human CXCL13 (300-47-50) was purchased from PeproTech (New Jersey, USA). PMA was obtained from Sigma (Darmstadt, Germany). The following antibodies are used for western blotting (WB), neutralization assay, flow cytometry, enzyme-linked immunosorbent assay (ELISA), and immunohistochemistry (IHC): CD11b and CD206 (BD Biosciences, San Diego, USA); CXCL13 ELISA (ab179881), rabbit anti-CXCL13 (Ab272874), rabbit anti-CXCR5 (ab203212), mouse anti-GAPDH (ab8245), rabbit anti-AKT (9272; CST); rabbit anti-p-AKT (Ser473; 4060, CST), rabbit anti-E-cadherin (3195; CST), rabbit anti-N-cadherin (13116; CST), and rabbit anti-vimentin (5741; CST). Akt inhibitor A3149 was purchased from APExBIO (Houston, USA).
Cell culture
The human ccRCC cell lines Caki-1, ACHN, and human THP-1 were obtained from the American Type Culture Collection (ATCC; Manassas, VA, USA). All cell lines were cultured in RPMI 1640 medium (Gibco), supplemented with 10% fetal bovine serum (FBS; Hyclone Technologies) and 1% penicillin and streptomycin. All cell lines are maintained in a humid environment at 37°C and under a 5% CO2 atmosphere.
THP-1 cells induced differentiation
The THP-1 cells were collected and inoculated into 6-well plates and then induced with PMA (100 ng/mL). M0 macrophages were obtained after culturing for 48 h. The Caki-1 and ACHN cells were seeded in the upper cavity of a Boyden chamber (0.4-μm pore size; Corning, USA) and co-cultured with M0 macrophages for 4 days to obtain M2 macrophages, after which flow cytometry was performed to determine whether the induction was successful.
Conditioned medium (CM)
After the macrophages are induced to differentiate, and then the macrophages were washed with PBS. Then, a fresh medium was added to the cells, followed by incubation for another 24 h. The medium supernatant was harvested and filtered, after which the complete medium was added at a ratio of 30% to produce the corresponding conditioned medium (M0-CM and M2-CM).
Co-cultivation program
The ccRCC cells and THP-1-derived macrophages were co-cultured in a Boyden chamber. M0 and M2 macrophages were seeded in the upper chamber, and the Caki-1 and ACHN cells were seeded in the lower chamber. Next, recombinant human CXCL13, CXCL13-neutralizing antibody, or AKT inhibitor A3149 were added to the lower chamber and incubated for12 h for co-cultivation. The RCC cells in the lower chamber were finally collected for PCR or WB.
RNA extraction and quantitative PCR (RT-qPCR).
TRIzol (Thermo Fisher Scientific) was used to extract the total RNA from cultured cells or tissues. With reference to the manufacturer's protocol, total RNA was reverse-transcribed into cDNA using the Prime Kit (Takara Biotechnology). The ABI7500 real-time PCR system (Thermo Fisher Scientific) was used to perform PCR with SYBR premixed real-time fluorescent quantitative PCR reagents (Taojiu Biotechnology). GAPDH was used as an internal control. The comparative 2-ΔΔCq method was used for relative quantification. The primer sequences of the analyzed genes are shown in Appendix 1.
Western blotting analysis
The total protein was separated from the cells with RIPA lysis buffer, separated by polyacrylamide gel, and then transferred onto the PVDF membrane. After blocking with 5% BSA solution, the membrane was incubated with primary antibodies (including E-cadherin, N-cadherin, Vimentin, AKT, and p-AKT) overnight. This membrane was then incubated with HRP-conjugated anti-rabbit antibody or anti-mouse antibody for 1 h at room temperature. The super signal WestFemto Maximum Sensitive Substrate (Thermo Fisher Science) was used to detect protein bands and the images were captured.
MTS determination
Cell proliferation was measured by the MTS assay. Cells (1 × 103; 100 µL per well) were seeded into 96-well plates. Different groups of cells were added with different (fresh) conditioned media (PBS, M0-CM, and M2-CM) and the corresponding additives (recombinant human CXCL13, neutralizing antibody of CXCL13, or AKT inhibitor A3149) daily. Next, 20% MTS solution was added to each well, followed by incubation at 37°C in the dark for 2 h. The optical density of the cells was then measured at 492 nm. The measurements were performed at 0, 24, 48, 72, 96, and 120 h to generate growth curves.
Wound healing assay
The cells were seeded in 12-well plates and cultured to produce a confluent monolayer. By using a 200-μL pipetting tip, the wound area was scratched and then collected tissues were washed thrice in PBS to remove debris. The washed cells were placed in a 12-well Boyden chamber and then inoculated with M0 and M2 macrophages in the upper chamber and FBS-free medium and additives (recombinant human CXCL13, neutralizing antibody to CXCL13, or AKT inhibitor) in the lower chamber. The wound closure was observed under an inverted microscope and the images were captured during 0–12 h.
Migration and intrusion detection
In the migration test, 1 × 105 cells were suspended in 200-μL of serum-free medium per well and seeded in the upper chamber of a 24-well Boyden chamber. In the invasion test, 1 × 105 cells suspended in 200 μL of serum-free medium were inoculated into the upper chamber of a Matrigel-coated cell. At the beginning of the cell migration assay, 1 × 104 M0 and M2 macrophages were seeded in the lower compartment. Next, recombinant CXCL13, anti-CXCL13 neutralizing antibody, and Akt inhibitor were added to the lower compartment. After 48 h or 72h of incubation, the membrane was fixed with 4% paraformaldehyde and then stained with crystal violet solution. At least 5 randomly selected areas were counted at a magnification of 100X.
ELISA
According to the manufacturer's instructions, the secretion of human CXCL13 in the supernatant of M0 and M2 macrophages was detected. After color development, the absorbance at 450 nm was measured on a microplate reader.
Flow cytometer
According to the manufacturer's instructions, the digested cells were washed with PBS and incubated with flow cytometry antibodies CD11b and CD206 for 20 min in the dark, followed by washing with PBS and resuspension on the machine. Finally, the CellQuest software version 7.5.3 (FACS Vantage-SE, BD Immunocytometry Systems, San Diego, CA) was employed to analyze the cells by multicolor flow cytometry.
Immunochemistry
The ccRCC and adjacent tissues were fixed with formalin, and the paraffin-embedded tumor tissues and the adjacent sections were stained with anti-CD206 antibody and anti-CXCR5 antibody, respectively. CD206 was used as a marker for M2 macrophages. The stained cells were counted in each field of view, and each slice had a field of view of at least 400 times. The average number of positive cells from the 5 highest field measurements was used for subsequent data analysis. The expression of the target protein was evaluated by the ratio and intensity of positive cells.
Xenotransplantation experiment
All animal experiments were performed following the protocol approved by the Animal Care and Use Institutional Committee of Sun Yat-sen Memorial Hospital, Sun Yat-sen University (Guangzhou, China). In order to study the effect of CXCL13 on tumor growth, 5-week-old female BALB/c nude mice were randomly divided into negative and CXCL13 groups (n = 5 in both groups). Approximately 5.0 × 106 Caki-1 cells were injected subcutaneously into the upper back of nude mice. When the xenograft was palpable (approximately 0.5-cm in diameter), 0.1 mg/kg of PBS or CXCL13 was injected weekly for 4 consecutive weeks [9]. The following formula was used to calculate the tumor volume (V) every 3 days: V = (W2 × L)/2. Finally, mice were sacrificed (place the mice in the euthanasia chamber, and then introduce 100% CO2 gas at a flow rate 25% of chamber volume per minutefor 4 min.).
Statistical analysis
All statistical calculations were performed using the SPSS 20.0 (IBM Corp.) and R software. Quantitative data and categorical data were analyzed by Student's t-test and Fisher's exact test, respectively. One-way analysis of variance and Tukey's post-test were performed for statistical analysis on more than 2 groups. The cumulative survival rate was calculated by Kaplan–Meier analysis and log-rank test. P < 0.05 was considered to indicate a statically significant difference.