Page 13 - An ultrasound-driven immune-boosting molecular machine for systemic tumor suppression

SCIENCE ADVANCES | RESEARCH ARTICLE

self-oxidation process. Under free-field US condition, DCFH im- with PBS before placing into 96-well plates and stained with the
mediately reacted with formed H 2O 2, oxidized to DCF, a highly fluo- Annexin V–FITC/PI Apoptosis Detection Kit (KeyGEN BioTECH).
rescent species with an emission peak at 525 nm (excitation = 488 nm). Spheroids were monitored in real time; bright-field and fluorescence
Control group without adding any sonosensitizer was also performed images were taken using the Cytation 5 MultiMode Microplate
for comparative purposes. Reader (BioTek, USA) every 20 min.
Cell culture and animals In vitro DC stimulation assay
MCF-7 human breast cancer cells and 4T1 murine breast cancer cells BMDCs were generated from the bone marrow of male C57BL/6
were obtained from the National Infrastructure of Cell Line Resource mice according to a typical method (63). The immature DCs were
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(Beijing, China). CT26 murine colon cancer cells were purchased seeded in a 96-well plate at a density of 8 × 10 cells per well and
from KeyGEN BioTECH Corp. Ltd. (Nanjing, China). These cells incubated with C34, NPe6, or CHC in the dark. After 6 hours, the
were incubated on the cell culture plate in Dulbecco’s modified Eagle’s cells were washed and replaced with fresh medium. The cells were
medium (DMEM) (HyClone) supplemented with 10% fetal bovine further incubated for 24 hours. Then, the DCs were harvested and
serum (PAN-Seratech) and 1% penicillin-streptomycin (HyClone) stained with anti-CD11c–phycoerythrin (PE)–Cy7 (BioLegend, catalog
at 37°C in a humidified, 5% CO 2 atmosphere. Male C57BL/6 mice number 117318), anti-CD80–fluorescein isothiocyanate (FITC)
(6 to 8 weeks, 18 to 20 g), female BALB/c mice (6 to 8 weeks, 18 to (BioLegend, catalog number 104706), and anti-CD86–allophycocyanin
20 g), and male ICR mice (6 to 8 weeks, 25 to 30 g) were purchased (BioLegend, catalog number 105012) antibodies and analyzed by
from Liaoning Changsheng Biotechnology (Benxi, China). B16- flow cytometry (FCM) (Thermo Fisher Scientific, Attune NxT).
OVA (OVA-transfected B16F10) cells and the splenocytes of OT-1
mice containing transgenic insert for mouse Tcra-V2 and Tcrb-V5 Detection of ICD in vitro
genes were gifts from H. Gao of Sichuan University, China. All animal 4T1 cells in the exponential phase were resuspended with serum-
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experimental protocols in this study were performed after being free medium at cell densities of 5.4 × 10 cells/ml. C34 was added to
approved by the Animal Research Ethics Committee of Dalian DMEM and irradiated by US for 10 min. Following a further incu-
University of Technology. bation of 6 hours, 4T1 cells in each group were harvested and lysed.
The extracted total proteins were resolved by gel electrophoresis,
Cellar uptake which was followed by electroblotting onto a PVDF membrane
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MCF-7 cells were resuspended at densities of 5.4 × 10 cells/ml in a (Millipore, Billerica, MA, USA). The PVDF membrane was blocked
2-ml culture tube. The cells were then exposed to a 25 M solution with 5% skim milk in TBST (tris-buffered saline–Tween 20) and
of different sonosensitizers in serum-free medium for 0.5 hours in immunoblotted with anti-HSP70 antibody and anti-GAPDH
the dark. After incubation, the cells were washed two times with (glyceraldehyde phosphate dehydrogenase) antibody (1:1500; Cell
1 ml of PBS and finally lysed with 1 ml of methanol to release the Signaling Technology). After further incubation with a horseradish
intracellular sonosensitizers. Quantitative concentration was calcu- peroxidase–conjugated secondary antibody (1:2000; Univ-Bio), the
lated from the standard curve of concentration-dependent fluores- specific protein bands were visualized with an ECL detection sys- Downloaded from https://www.science.org at Dalian University of Technology on October 20, 2021
cence intensity. tem (Bio-Rad, USA).
The extracellular ATP in conditioned medium that was secreted
In vitro sonocytotoxicity assay from treated cells was determined by using an enhanced ATP bio-
MCF-7 cells in the exponential phase were resuspended with serum- luminescent assay kit (Beyotime Biotechnology, catalog number
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free medium at cell densities of 5.4 × 10 cells/ml. Then, all samples S0027) according to the manufacturer’s instructions.
were randomly divided into four groups: control, C34 alone (C34),
US alone (US), and C34 + US. For C34 and C34 + US groups, the In vivo biodistribution
cells were incubated in the medium containing C34 solution in the ICR male mice were used to establish the xenograft H22 tumor
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dark. After 0.5 hours, the cells were harvested and washed twice by model. H22 mouse hepatoma cells (1 × 10 in 50 l of PBS) were
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PBS. MCF-7 cells were resuspended at a concentration of 3 × 10 cells/ml injected subcutaneously into the right side of the mouse back. Mice
in DMEM (serum free) and transferred into a cylindrical polystyrene were intravenously administered C34 at a dose of 16 mg/kg body,
tissue culture tube for US irradiation. US and C34 + US groups were and in vivo fluorescence signals of tumors were monitored at differ-
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then treated with free-field US (1.064 MHz, 3.21 W/cm ) for 10 min. ent post-injection time points using a small animal imaging system
After various treatments, the cells were placed into 96-well plates (NightOWL II LB983). Tumor-bearing mice were sacrificed after
and grew for 24 hours. Cell viability was estimated by the standard administration at different time points, and tumor and major tissues
MTT assay. were taken for ex vivo HPLC analysis.
Compared to 2D cell cultures, the 3D tumor spheroids can pro- C34 was extracted from tissues by a protein precipitation and
vide a more accurate tumor-mimicking microenvironment. First, liquid-liquid extraction method. Methanol was used as the protein-
agarose [1.5% (w/v)] was dissolved in DMEM and casted in 96-well precipitating agent, and chloroform was used to extract C34. HPLC
plates to form molds with homogeneous circular recesses. Next, analysis was performed using a Waters 2695 system including
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MCF-7 cell suspension (100 l; 3 × 10 cells/ml), corresponding to Waters 2695 Separations Module and Waters 2489 UV-Vis Detector
3000 total cells per well, was incubated at 37°C for 24 hours. Afterward, with an autosampler. Chromatographic separation was performed
DMEM containing 50 M C34 was added to each well and incubated on a Unitary C18 column (4.6 mm by 250 mm, 5 m) and main-
in the dark at 37°C for another 24 hours. Following incubation, the tained at a temperature of 40°C. The mobile phase consisted of 5 mM
spheroids were transferred to a cylindrical polystyrene tissue culture tetrabutylammonium phosphate monobasic solution (buffer A) and
tube for exposure to US. After exposure, the spheroids were washed methanol (buffer B). A gradient elution was performed at a flow

Wang et al., Sci. Adv. 2021; 7 : eabj4796 20 October 2021 12 of 15