Animals, cell culture and reagents
Female athymic nude mice were purchased from Shanghai SLAC Laboratory Animal Co., Ltd. (Shanghai, China) and maintained under specific pathogen-free (SPF) conditions. The human pancreatic cancer cell line used in this study, SW 1900, was obtained from the Cell Bank of the Chinese Academy of Science (Shanghai, China). Both RPMI medium and fetal bovine serum (FBS) were supplied by Gibco Invitrogen (Carlsbad, CA, USA), and cells were maintained in RPMI medium supplemented with 10% FBS. An ATO solution containing 0.9% sodium chloride was provided by Harbin Yida Pharmaceutical Co., Ltd. (Lot.20190102); 2,3-dimercapto-1-propanol was supplied by Tokyo Chemical Industry Co., Ltd. (Lot.WG5MB-AT) and stored in anhydrous alcohol. Other reagents were obtained from Sigma-Aldrich unless otherwise specified. Protocols for animal experiments were approved by the Animal Experimental Ethic Committee of the Ninth People’s Hospital, School of Medicine, Shanghai Jiao Tong University.
Preparation of the formula
The combination of dimercaprol and ATO was prepared in water, and the chemical structures formed were detected in an abiotic condition by mass spectrometry. Briefly, the water solution containing ATO and alcohol solution containing BAL were mixed in a tube with a molar ratio of ATO:BAL = 1:6 (an ATO molecule contains double arsenic ions) and stored at 4 °C. Then, a mass spectrometer (UPLC/SFC-MS, Waters, MA, USA) was used to examine the possible chemical structures.
Survival and tumor volume analysis
To establish animal tumor models, sub-confluent hormone-independent SW 1990 cells (5 × 106 per mouse) were injected into 5 mice in one of their flanks. After 3 weeks, mice bearing tumors were sacrificed, and the tumors were harvested. Two of the larger tumors were cut into approximately 2 mm × 2 mm × 2 mm pieces and then transplanted into mice.
The mice bearing tumors were randomized into the following 4 groups (n = 6): control group: injection of saline containing 1% alcohol (v:v), radiation-treated (RT) group: 1 Gy of total body X-ray radiation along with injection of saline containing 1% alcohol (v:v), BAL-ATO-treated group: injection of freshly prepared BAL-ATO solution, and RT + BAL-ATO group: combined total body RT and BAL-ATO-treatment. The dose of BAL-ATO was indicated by the quality of ATO contained in the prepared mixture. The mice were injected (i.p.) daily with saline or BAL-ATO with a dose equivalent to 30 mg/kg ATO for five days within a week.
An RS 2000 biological system X-ray irradiator (dose rate, 234 cGy/min) was used to irradiate xenografted animals. RT and RT + BAL-ATO groups of mice received 1 Gy of total body irradiation 2-4 hours after injection on Monday, Thursday and Friday followed by a second treatment within a week consisting of only BAL-ATO. The same treatment used in the first week was repeated in the third week.
Survival observation was carried out from the beginning of treatment to time when all of the animals without intervention died. Tumor sizes were measured every 3 days, and volumes (V) were calculated with the following equation: V = L × W2 × π/6, where L was a tumor length and W was the width. Measurements were performed from the first to the 37th treatment day.
Live imaging, hypoxia analysis and sample collection
Pancreatic cancer xenograft animal models were established using the methods described above, and 6 mice were included in each group. The treatment schedule was the same as the that described above for the tumor growth analysis.
Three mice in each group bearing tumors were imaged using an IVIS Lumina II system (Caliper Life Sciences, Hopkinton MA, USA) equipped with a charge-coupled device camera at 2 days after the last radiation treatment. The mice received i.v. administration of 100 μL of Annexin-vivo 750 (catalog no. NEV11053, PerkinElmer, Inc) and then were anesthetized with 2% isoflurane in 100% O2. Images were acquired by recording the bioluminescent signals and were analyzed with Living Image software (Version 4.2, Caliper Life Sciences). After 3 days, the corresponding animals received BAL-ATO treatment 2 hours before HP-RedAPC-MAb (catalog no. HP8-x, Hypoxyprobe, Inc) was administered. The mice were sacrificed on the 26th treatment day. The tumors were then frozen for tissue sections and the frozen tissues sections were subjected to hypoxia analysis by confocal laser scanning microscopy (Leica TCS SP8, Leica Microsystems Inc, Wetzlar, Germany).
The remaining mice (n = 3) in each group were also sacrificed on the 26th day of the treatment, and blood samples for analysis with a blood & gas analysis system (KT-6300, Pioway, Nanjing, China) were drawn from the orbits of the animals before euthanasia. The tumors were harvested and photographed. The removed tumors were separated and then kept for western blot, IHC, TUNEL and H&E staining assays. Besides, the animal organs including brains, livers as well as kidneys were removed for H&E staining.
H&E staining IHC and TUNEL assays
Paraffin-embedded tumor or organic sample slides were dewaxed, rehydrated, pretreated with hydrogen peroxide and washed with PBS. The samples were then stained with hematoxylin and eosin (H&E), followed by rinsing, and the coverslips were mounted onto slides with Permount (Fisher Scientific, San Francisco, CA). For IHC staining, endogenous peroxidase was blocked with 3% hydrogen peroxide, and the tissue samples on slides were then incubated with primary anti-human antibodies, including anti-HIF-1ɑ (catalog no. ab5168, Abcam), anti-CD24 (catalog no. ab31622, Abcam, Cambridge, UK), and anti-CD44 (catalog no. ab189524, Abcam, Cambridge, UK). Then color visualization was done with SPlink Detection Kits (catalog no. SP-9000, ZSGB-BIO) in accordance with the manufacture’s instruction. TUNEL assay was performed with an in situ cell death detection kit (catalog no. 11684817910, Roche Applied Science) referring to the protocol supplied together with the product. All slides were analyzed and photographed by an experienced pathologist.
Western blot analysis
Tumor tissues were washed with cooled PBS, and then samples with sizes of approximately 0.5 cm × 0.5 cm × 0.5 cm were placed in EP tubes containing 0.5 ml of lysate solution and kept on ice. The samples were cut into pieces, followed by grinding with an electric grinder and a 30-minute incubation on ice. Protein was extracted with centrifugation and quantified with a BCA protein assay kit (Cell Signaling Technology, Inc. Danvers, MA, USA). Protein samples (30 μg) were separated with SDS‑PAGE and transferred onto PVDF membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked for 1 hour with 5% non‑fat milk solution at room temperature and then incubated with buffers containing primary antibodies, including anti-HIF-1ɑ (catalog no. ab51608, Abcam), anti‑Nestin (catalog no. ab22035, Abcam), anti‑Gli1 (catalog no. sc-515781, Santa Cruz Biotechnolog), and anti‑ɑ-tubulin (catalog no. sc-134237, Santa Cruz Biotechnology, Santa Cruz, CA, USA), overnight at 4 °C. Then corresponding Peroxidase-labeled anti-mouse (catalog no. P0217, DAKO) or anti-rabbit (catalog no. P0260, DAKO) IgG secondary antibody were employed to incubate the membranes for 1 hour. The western blot gel image was produced by an Minichemi 610 chemiluminescent imager (Sagecreation, Beijing, China).
Each calculated value representing a result was expressed as the mean ± standard error of the mean. Differences between groups were analyzed by one-way ANOVA analysis with PRISM software (GrafPad Software, Inc., San Diego, CA, USA). Significant differences between groups were set at P < 0.05.