2.1 Major reagents and apparatus
UTI and placebo (phosphoric acid buffer solution containing sodium chloride and mannitol) were generous gifts from Techpool Bio-Pharma (Guangzhou, China). The RT-PCR kit (AQ201-01) was purchased from TransGen Biotech (China). P38 (14064-1-AP), ERK1/2 (16443-1-AP), JAK1 (66466-1-Ig), JAK2 (17670-1-AP), and Stat3 (10253-2-AP) were purchased from Proteintech. Phospho-p38 MAPK (CST,4511) and Phospho-p44/42 MAPK (Erk1/2) (CST, 4370) were purchased from CST. JAK1 (Phospho-Tyr1022) (AB11149-4), JAK2 (Phospho-Tyr1007) (AB11151-4), and STAT3 (Phospho-Tyr705) (AB11045-4) were purchased from Abcitech. UPA (AP30202b) and UPAR (AP8156c) were purchased from ABGENT. The Jak-Stat pathway inhibitor Ruxolitinib (INCB018424) (S1378) and S3I-201 (S1155) were purchased from Selleck Chemicals. ChemiDoc Gel Imaging System was purchased from Bio-Rad Company (CA, USA), and the inverted CX41 fluorescent microscope was purchased from Olympus (Tokyo, Japan).
2.2 Cell cultures
Human NPC cell lines S18, S26, 5-8F, and SUNE-1 were from our laboratory [22, 23]. S18-1C3, a subclone of S18 was generated by permanent transfection with a GFP-luciferase reporter, which was provided by Exploring Health LLC. S26 and S18 were isolated from their parental line CNE-2 [24], and 5-8F from its parental line SUNE-1 [25]. The human NPC cell lines S18, S26, 5-8F, SUNE-1, and the S18-1C3 with luciferase were cultured in DMEM (Gibco, USA) supplemented with 10% Fetal Bovine Serum (FBS, Gibco) with 0.1 mg/ml streptomycin and 100 U/ml penicillin in a humidified atmosphere of 5% CO2 at 37℃.
2.3 Grouping and drug administration
2.3.1 Cellular experiment
This experiment comprised of three groups: 1) a control group treated with physiological saline only; 2) a UTI medium dose group treated with UTI at a concentration of 800 U/mL (S18) or 3200 U/ml (5-8F); and 3) a UTI high dose group treated with UTI at a concentration of 1600 U/mL (S18) or 6400 U/ml (5-8F). All drugs were freshly prepared 4h before administration.
2.3.2 Animal experiment
A total of 50 female BALB/c nu/nu mice aged 4–5 weeks old were purchased from Guangdong Medical Animal Center (Production License No. SCXK [Yue] 2013-0002). FVB/N-Tg (MMTV-PyMT) 634Mul/J mice were purchased from the Jackson Laboratory (Stock No: 002374, https://www.jax.org/strain/002374). Nude mice and MMTV-PyMT mice were kept in a specific pathogen free environment at 22–25°C with 50–65% humidity. Drinking water, food, and experimental materials were sterilized, and the rule of aseptic operation strictly followed. This study was approved by the Institutional Animal Care and Use Committee of Sun Yat-sen University Cancer Center.
The spontaneous footpad to popliteal lymph node (LN) metastasis experiments were conducted as previously reported [24]. Briefly, 1 × 10 5 cells in 20 µl DMEM were subcutaneously injected into the footpad of the left hind limb of each mouse to generate a primary tumor. After 11 days, the mice were randomly divided into two groups for subsequent UTI treatment through intraperitoneal injections as follows: 1) The UTI group (n = 17) was injected with UTI at 1600 U/day/mouse for 38 consecutive days; 2) the control group (n = 16) was injected with an equal volume of placebo for 38 consecutive days. The animals were euthanized for sample collection 38 days after administration. At the termination of the experiment, the mice were injected with 150 mg/kg of D-Luciferin Potassium Salt (Mkbio, MX4603-100MG) before isoflurane anesthesia. The radiance was measured after substrate injection using the ChemiDoc Gel Imaging System (Bio-Rad, USA) and the popliteal LNs of the left hind feet were isolated. LNs were homogenized in TRIzol for total RNA extraction. Reverse transcription and real-time qPCR were performed to assess metastasis using specific primers for luciferase, which was only expressed in human nasopharyngeal carcinoma cells.
For the xenograft tumor experiments, the S18 cell lines were harvested and washed with PBS, then resuspended in serum-free DMEM medium. The cell concentration was adjusted to 1 × 107 cells/mL. Cells were inoculated subcutaneously into the right armpits of 16 nude mice at 0.2 mL/mouse. Seven days after inoculation, the animals were randomly divided into two groups for subsequent intraperitoneal injections as follows: 1) The UTI group (n = 9) was injected with UTI at 1600 U/day/mouse for 17 consecutive days; and 2) the control group (n = 7) was injected with an equal volume of placebo for 17 consecutive days.
The development of breast cancer was spontaneous in the MMTV-PyMT mice. The nine week old MMTV-PyMT mice with breast cancer were randomly divided into two groups for subsequent intraperitoneal injections as follows: 1) The UTI group (n = 6) was injected with UTI at 1600 U/day/mouse for 5 weeks; 2) the control group (n = 16) was injected with an equal volume of placebo for 5 weeks. The animals were euthanized for sample collection 5 weeks after administration. Lung tissues were collected and fixed in Bouin’s solution (PHYGENE, PH0976) for 24 to 48 hours, and metastatic lung nodules were examined and counted.
2.4 Quantifying cell proliferation using MTS assay
Cells were seeded into 96-well plates at a density of 1 × 105 cells/well in 100 µl normal culture medium. UTI and PBS were added 24h later. Cell growth was determined using MTS (Cell Titer 96 Aqueous One Solution Cell Proliferation Assay solution; sigma), 10 µl MTS reagents were added to 100ul culture medium per well and incubated for 2–4 h at 37°C. The OD490 value was measured with a microplate reader.
2.5 Wound healing assays
Cells were seeded into a 6-well plate and cultured until 90% confluence. A sterile 200-µL tip was used to create artificial wounds in the cell monolayer, and the floating cells removed by a single wash with 1×PBS. UTI and PBS were added separately. Respective images were captured at 0 h and 24 h using an inverted microscope. Wound healings were monitored under a microscope and quantified at 0 h and 24 h.
2.6 Migration and invasion assays
Migration assays were conducted with Biocoat without Matrigel (Corning. Life sciences), and invasion assays were performed with Biocoat with Matrigel (Corning. Life sciences) following the manufacturer’s instructions. The harvested Biocoats were then stained with crystal violet, and invaded cells were counted under a microscope. Both experiments were repeated independently three times.
2.7 RNA isolation and real-time quantitative reverse-transcription PCR (qPCR)
Total RNA was isolated from cultured cells using TRIzol reagent (Invitrogen, USA). Complementary DNA (cDNA) synthesis was completed using the reverse transcription kit (Transgene, China) following the manufacturer’s instructions. qRT-PCR analysis was performed using the SYBR Green PCR Kit (Transgene, China). The relative mRNA levels are shown as the value 2−△Ct.
The following PCR primers were used:
Luciferase F: 5'-AGAGATACGCCCTGGTTCCT-3'′;
Luciferase R: 5'-ATCCCCCTCGGGTGTAATCA-3'′;
GAPDH-F: 5'- CTCATGACCACAGTCCATGC-3'′;
GAPDH-R: 5'- CAGTGAGCTTCCCGTTCAG-3'′;
uPA forward: 5′-GCCACACACT GCTTCATTGA-3′;
uPA reverse: 5′-TATACATCGA GGGCAGGCAG-3′;
uPAR forward: 5′-GCCTTACCGA GGTTGTGTGT-3′;
uPAR reverse: 5′-CATCCAGGCA CTGTTCTTCA-3′;
GAPDH was used as the internal control for measuring the relative level of luciferase.
2.8 Immunoblotting analyses
NPC cells treated with UTI or Placebo were lysed with a 25 µL lysis buffer and mixed with a 2 × sample buffer. Cell lysates/proteins were subjected to SDS-PAGE and then transferred onto a PVDF membrane. The membrane was incubated overnight at 4°C and subsequently with secondary antibodies for 1 h. After being washed with PBST, signals were visualized by incubation with ECL luminescence substrate and detected with the ChemiDoc Gel Imaging System (Bio-Rad, USA).
2.9 Statistical analysis
One-Way ANOVA was used to compare different independent groups of data. A p-value < 0.05 was considered statistically significant in all cases.