Page 14 - An ultrasound-driven immune-boosting molecular machine for systemic tumor suppression

SCIENCE ADVANCES | RESEARCH ARTICLE

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rate of 1 ml/min, 0 to 10 min, 70 to 95% B; 10 to 15 min, 95 to 100% and C34 + US), US irradiation (1.064 MHz, 1.88 W/cm , 30 min)
B; and 15 to 25 min, 100% B. The injection volume was 20 l, and was conducted 4 hours after injection. Throughout the experiment,
the detection wavelength was 400 nm. all mice were kept out of the light. The weight and tumor volume of
the mice in each group were measured regularly. Three days after
Pharmacokinetic study the last treatment, the mice were anesthetized with an intraperitoneal
Sprague-Dawley rats weighing 220 to 250 g were obtained from injection of Avertin (tribromoethanol), and subcutaneous tumors
Liaoning Changsheng Biotechnology (Benxi, China). The animals were extracted and weighed. The in vivo antitumor experiment was
were fasted overnight with free access to water for at least 12 hours repeated four times in this model.
before administration. C34 was intravenously administered at a single On day 35 after the implantation of 4T1 cells, we performed a PET/
dose of 16 mg/kg body weight, and blood samples were collected CT measurement to evaluate lung metastasis. All scans were performed
from the suborbital vein into heparinized tubes at time intervals of with a Super Nova small-animal PET/CT scanner (PINGSENG
0.083, 0.25, 0.5, 1, 1.5, 2, 4, 6, 12, and 24 hours. Each collected blood Healthcare, Jiangsu, China). Mice with the orthotopic implantation
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sample was immediately centrifuged at 8000 rpm for 10 min. The of 4T1 cells to be monitored by F-FDG PET/CT were fasted for
plasma was separated and frozen below −80°C. The validated method 24 hours, with access to water only. Body weights were measured,
as mentioned above was applied to extraction and HPLC analysis of and mice were anesthetized by Avertin and injected using a tail-vein
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C34 in plasma. The concentration in plasma at different time points catheter with 10 MBq/0.1 to 0.2 ml of F-FDG. The lung was
was calculated from the plasma calibration curve. The relevant scanned by CT. PET data were acquired for 20 min following a de-
pharmacokinetic parameters were calculated by a drug and statistics lay of 40 min to allow for FDG uptake. In the end, lungs were col-
2.0 program (DAS 2.0, Mathematical Pharmacology Professional lected and fixed with Bouin’s solution. Lung micrometastases in five
Committee of China, Shanghai, China). lobes were counted directly through microscopic observation. Serum
was collected and analyzed by an enzyme-linked immunosorbent
Cross-priming assay in vitro assay (ELISA) kit for IFN-. For IFN- staining, frozen sections of
B16-OVA cells were incubated with NPe6 (25 M) or C34 (25 M) tumor tissues were fixed with Immunol Staining Fix Solution
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and then received irradiation (1.064 MHz, 3.21 W/cm ,10 min). (Beyotime, catalog number P0098) for 10 min at room tempera-
iBMDCs were added into each well and incubated for 2 days. The ture, washed three times with PBS, permeabilized with Triton X-100
+
OVAI-specific CD8 T cells were isolated from the splenocytes of solution (Beyotime, catalog number P0096) for 20 min, and blocked
+
OT-1 mice by using a CD8 T cell positive selection kit (STEM- with Immunol Staining Blocking Buffer (Beyotime, catalog number
CELL) and then added into each well. After incubating for 2 days, P0102) for 1 hour. The sections were then incubated with primary
all cells were collected and stained for flow cytometry analysis. antibodies including anti–IFN- antibody (1:200 dilution; BioLegend,
catalog number 507801) overnight at 4°C. Sections were rinsed, incu-
In vivo cytotoxicity assay bated with secondary antibodies (1:200 dilution; ProteinTech, cata-
Melanoma-bearing mice, which were subcutaneously inoculated log number SA00013-1) in the dark for 1 hour, and then stained
6
with 1 × 10 B16-OVA cells, received C34 + US treatment for two with DAPI (4′,6-diamidino-2-phenylindole). The images were acquired Downloaded from https://www.science.org at Dalian University of Technology on October 20, 2021
consecutive days. Seven days after the last treatment, splenocytes from using a Leica DMi8 microscope and the THUNDER Imaging System.
naive mice were pulsed with the OVA 257–264 peptide SIINFEKL and
b
the irrelevant lung carcinoma H-2K peptide Mut1 FEQNTAQP for Colon cancer with hepatic metastasis model
2 hours at 37°C. After washing three times with PBS, OVA 257–264 Here, we introduced a colorectal cancer hepatic metastasis model
peptide-pulsed and Mut1-pulsed splenocytes were incubated with using hemiplenectomy with little modification (65). BALB/c mice
high (4 M; CFSE high ) and low (0.5 M; CFSE low ) concentrations of were anesthetized with Avertin (240 mg/kg, intraperitoneally). A left
carboxyfluorescein diacetate succinimidyl ester (CFSE) (Invitrogen), flank incision was made to expose the spleen. The spleen was divided
respectively, for 15 min and inverted them once every 5 min. An into two hemispleens by using medium-sized Horizon surgical tita-
low
equal amount of CFSE high - and CFSE -labeled splenocytes was intra- nium clips, leaving the vascular pedicles intact. With a 27-gauge
5
venously injected into PBS (control mice)– or C34 + US–treated needle, 3 × 10 viable CT26 cells in 75 l of PBS were injected into
mice and naive mice as control. Splenocytes were collected after 16 hours, the spleen. Cells then flowed into the splenic and portal veins and
and residual CFSE high and CFSE low target cells remaining in the re- were deposited in the liver. The vascular pedicle draining the cancer-
cipients’ spleens were analyzed by flow cytometry. % Specific cyto- contaminated hemispleen was ligated with a small surgical clip. The
low
toxicity = [1 − (ratio of CFSE high /CFSE obtained from treated mice)/ CT26-contaminated hemispleen was then excised, leaving a functional
(ratio of CFSE high /CFSE low obtained from naive mice)] × 100% (64). hemispleen free of tumor cells. The following treatments were the
same as the aforementioned procedures. This experiment was repeated
Murine breast cancer model with lung metastases two times. At the end of the experiment, mice were sacrificed and
To establish breast cancer model with spontaneous lung metastasis, livers were excised, weighed, and photographed, and afterward,
5
4T1 cells (5 × 10 ) suspended in PBS were inoculated into the second they were studied by pathological analysis.
pair of mammary gland on BALB/c mice. Six days later, the tumors
3
were allowed to reach ~30 mm before the experiments. Mice were FACS analysis
randomly divided into five groups (n = 5), including (i) control, (ii) Tumor tissues, lymph nodes, and spleens were collected from mice
US alone, (iii) C34, (iv) NPe6 + US, and (v) C34 + US. PBS was after SDT and homogenized into single-cell suspensions following
intravenously injected into the control and US groups. The mice in the established protocol (66, 67). Then, the cell suspensions were
other groups were intravenously administered with NPe6 or C34 stained with different antibodies according to the standard proto-
(16 mg/kg). In the US irradiation groups (i.e., US alone, NPe6 + US, col. DCs in lymph nodes were stained with anti-CD11c–PE-Cy7

Wang et al., Sci. Adv. 2021; 7 : eabj4796 20 October 2021 13 of 15