Page 12 - An ultrasound-driven immune-boosting molecular machine for systemic tumor suppression

SCIENCE ADVANCES | RESEARCH ARTICLE

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(Fig. 9, B and D). In addition, CD8 T cells primed by C34-mediated In summary, these satisfactory features of C34 allow a unimolecule-
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SDT exhibited a marked increase of an effector/memory CD8 subset mediated sono/immunosynergistic therapy to become more realistic.
expressing high levels of CD44 cell adhesion molecule (Fig. 9, B and E). C34-mediated SDT could be universally applied to different cancer
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Consistently, C34 + US induced higher differentiation of CD4 T cells types, offering a more facile treatment option to clinic.
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into the memory phenotypes (CD44 CD4 ) (Fig. 9, B and E), indi-
cating the potential of generating a long-lasting immune memory to
prevent tumor relapse (52). MATERIALS AND METHODS
Experimental design
In this study, a potent, biocompatible, and US-triggered multi-
DISCUSSION functional molecular machine, named C34, was designed and syn-
Emerging evidences have indicated that systemic tumor suppression thesized. The acoustic responsive activity of C34 was evaluated
requires combinational therapeutic strategies, which usually lead to in vitro. The antitumor efficacy of C34-mediated SDT was assessed
better clinical outcomes than monotherapies for cancer treatment in the models of breast cancer with lung metastasis and colon cancer
(53). However, combinational therapies may increase the complex- with liver metastasis. Immunological analysis was performed to test
ities of treatment and stress patients by bringing in multiple agents the intrinsic immune-boosting capacity of C34.
and regimens (54). Therefore, in this work, to address the pressing
clinical need for developing a simple yet effective therapeutic approach Materials
in regressing primary tumor and blocking metastasis simultaneously,
we explored a potent, biocompatible, and multifunctional molecu- Aldrich. Dimethyl sulfoxide and ethanol were purchased from
lar machine, C34, with preferential tumor accumulation capacity, Sinopharm Chemical Reagent Co. Antibodies against cell surface
US-activable cytotoxicity, and intrinsic immune-boosting effect. markers for flow cytometry [fluorescence-activated cell sorting
The unique chemical structure might be the key feature favoring (FACS)] assay were purchased from BioLegend.
the tumor accumulation of C34. There have been many studies sug-
gesting the tumor specificity of the chlorin-based sensitizers (55), Acoustic pressure amplitude measurements
largely due to their high plasma protein binding ability (56). C34, as The total acoustic power generated by the given source was measured
a derivative of CHC and an analog of hematoporphyrin, exhibited a with an ultrasound power meter (UPM) (UPM-DT100N, Ohmic
good affinity with the highly metabolic tumor tissue by being carried Instruments, USA). The acoustic wave intensity I SATA was calculated
by the plasma proteins to the tumor vascular system. Moreover, according to the formula
amphiphilicity has been reported as an important factor that affects P A
─
the tumor accumulation of porphyrin dyes as well (57, 58). The ex- I SATA = (1)
S
cellent permeability of the C34 molecule within the tumor cells was
attributed to the increased liposolubility by introducing the aromatic where P A is the total acoustic power measured by the UPM and S is
group to the CHC molecule. The amino acid residue of C34 served as a the active aperture of transducer.
hydrophilic group to obtain the hydrophilic-lipophilic balance. In A polyvinylidene difluoride (PVDF)–type hydrophone (HNR-0500;
addition, cancer cells have a higher magnitude of membrane potential calibration range, 250 kHz to 10 MHz) was placed vertically at a distance
than normal cells (59). C34 with positive charges at the carboxyl groups of 10 mm from the sensor surface to record the ultrasonic signals.
was preferentially prone to accumulate and stay within the tumors. The hydrophone was connected to a digital oscilloscope for the ob-
The intrinsic immune-boosting activity could be another one of servation, measurement, and processing of the spectrum. To take into
the most attractive features of the C34 molecule. Some literature has account pressure amplitude variations during the ultrasonic duration,
reported that chlorophyll a, a precursor molecule of C34, could the root mean square pressure value p RMS was calculated as (61)
modulate immunity through DCs; however, it lacks direct evidence
V RMS
(60). Our observations confirmed that C34 itself was able to trigger p RMS = ─ (2)

antitumor immunity by directly stimulating DCs, leading to the M
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generation of CD8 CTL responses. In addition, this effect could be where V RMS represents the root mean square voltage value at the
significantly amplified once being exposed under US irradiation hydrophone and M is the hydrophone sensitivity (V/Pa).
during SDT, which might be due to more extensive tumor antigen
2
exposure. Notably, 3 -aryl substituent was the only difference in the Determination of ROS production
chemical structures between Npe6 and C34, resulting in distinct A mixed solvent [ethanol/water, 1:1 (v/v)] containing C34 or NPe6
DPBF and DCFH diacetate (DCFH-DA) were purchased from Sigma- Downloaded from https://www.science.org at Dalian University of Technology on October 20, 2021
diversity in the property of molecular immunogenicity. This pheno- (10 M) and DPBF (20 M) was prepared. The solutions were then
menon prompted us to ask whether the mild immunogenicity of the irradiated by US for 10 min. Aliquots were taken at specific time
C34 molecule was associated with the introduction of the -extension points, and fluorescence at 460 nm (excitation = 417 nm) was re-
system to the macrocyclic structure of CHC, as we discussed previously corded with a fluorescence microplate reader. Control group in the
(25, 26). In addition, we have performed the reverse docking studies absence of any sonosensitizer (i.e., DPBF + US) was performed for
to look for the potential target proteins of C34 related to the DC comparative purposes.
stimulation. Further biological assays are needed to confirm the For the test of H 2O 2, the activated DCFH was prepared from
mechanism of action of the C34 molecule. Studies on the structure- DCFH-DA (62). The sonosensitizer was then added to the neutralized
activity relationship associated with the immune-boosting effect DCFH (20 M) solution in an equal volume of ethanol with a final
will be undertaken as well, which may provide new insights into the concentration of 10 M. The mixture solution was prepared just before
development of more potent sonosensitizers for cancer therapy. the beginning of US irradiation, which aimed to minimize the DCFH
Wang et al., Sci. Adv. 2021; 7 : eabj4796 20 October 2021 11 of 15