Original Article

Global Health & Medicine. 2024; 6(4):236-243.
DOI: 10.35772/ghm.2024.01037

Effects of low-frequency ultrasound combined with microbubbles onbreast cancer xenografts in nude mice§§

Xiaoli Peng1, , Lisha Li2,3,4, , Yingchun Liu1, Yuqing Guo1, Yun Pang1, Shengnan Ding1, Jing Zhou2,3,4, Ling Wang2,3,4,*,
Lin Chen1,*
1

Department of Ultrasound, Huadong Hospital, Fudan University, Shanghai, China;
Laboratory for Reproductive Immunology, Obstetrics and Gynecology Hospital of Fudan University, Shanghai, China;
3
The Academy of Integrative Medicine of Fudan University, Shanghai, China;
4
Shanghai Key Laboratory of Female Reproductive Endocrine-related Diseases, Shanghai, China.
2

Abstract: The aim of this study was to explore the effects of low-frequency ultrasound (US) combined withmicrobubbles (MBs) on breast cancer xenografts and explain its underlying mechanisms. A total of 20 xenografted
nude mice were randomly divided into four groups: a group treated with US plus MBs (the US + MBs group),a group treated with US alone (the US group), a group treated with MBs alone (the MBs group), and a control
group. In different groups, mice were treated with different US and injection regimens on an alternate day, threetimes in total. Histological changes, apoptosis of cells, microvascular changes, and the apoptosis index (AI) andmicrovascular density (MVD) of the breast cancer xenograft were analyzed after the mice were sacrificed. Results
indicated that the tumor volume in the US + MBs group was smaller than that in the other three groups (p < 0.001
for all). The rate of tumor growth inhibition in the US + MBs group was significantly higher than that in the US
and MBs groups (p < 0.001 for both). There were no significant differences in histological changes among the fourgroups. However, the AI was higher in the US + MBs group than that in the other three groups while the MVD waslower (p < 0.001 for all). All in all, low-frequency US combined with MBs can effectively slow down the growth ofbreast cancer in nude mice. In summary, low-frequency US combined with MBs has a significant effect on breast
cancer treatment. Cavitation, thermal effects, and mechanical effects all play a vital role in the inhibition of tumor
growth.
Keywords: low-frequency ultrasound irradiation treatment, nude mouse xenograft model of breast cancer, ultrasoniccavitation

Introduction
Worldwide, female breast cancer was the most common
cancer with around 2.3 million new cases (11.7%) andthe fifth leading cause of cancer death with around 0.7
million new deaths (1). Although more new cases werediagnosed in women over the age of 50, its incidence
in younger women is rising (2). In China, breast cancer
was the most common cause of cancer death (16.7%)
in women ages 15-44 (3). Screening technology is
becoming more mature and widespread, and morebreast cancers of a smaller volume are detected at aneven earlier stage (4). Therefore, early diagnosis andappropriate treatment affect patient prognosis. With the
spread of breast-conserving surgery and the widespreaduse of pre- and postoperative adjuvant therapy, local
treatment of breast cancer is developing rapidly (5).
According to different tumor types and stages, different
local treatments can be selected, such as radiotherapy(6), radiofrequency ablation (7), and microwave
ablation (8).
Ultrasound (US) is now one of the hot fields intreating solid tumors. Bioeffects of US mainly consistof thermal effects, cavitation, and mechanical effects.
High-intensity focused ultrasound (HIFU), as a therapy
with thermal effects, has been used clinically to treat
various solid tumors, like liver cancer and thyroid
cancer (9,10). However, HIFU is still not widelyused in breast cancer due to technical and equipment
limitations, and it has a variety of complications, suchas pain, skin burns, edema of the lungs, and major
pectoralis injury. As a non-thermal therapy, lowfrequency US has been widely studied in promoting
cell apoptosis in vitro and in vivo (11-14). Cavitation
plays an essential role in the inhibition of tumor growth
by low-frequency US. According to the state of MB
motion, cavitation is divided into stable cavitationand inertial cavitation; in the former, MBs move in(236)

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periodic, nonlinear oscillations under the action of
periodically varying US. In the latter, MBs periodically
expand and contract until they burst, releasing large
amounts of energy. Low-frequency US has not beenstudied in depth in terms of its efficacy and mechanismof action in breast cancer. In our previous work, wefound that the most suitable exposure parameters werea frequency of 1 MHz, an intensity of 2 W/cm2, a duty
cycle of 50%, and an exposure time of 5 min. The aimof the current study was to explore the effects of lowfrequency US combined with MBs and explain its
underlying mechanisms.
Materials and Methods
Cell culture
MDA-MB-231 cells (Shanghai Cancer Institute) were
cultured in a RPMI-1640 medium (Gibco, America)
supplemented with 10% of FBS (HyClone, US) at
37°C in an atmosphere of humidified 5% CO2. All
experiments were performed in accordance with therequirements of the Guidelines for the Care and Use ofLaboratory Animals and were approved by the Animal
Experiment Ethics Committee of Shanghai Medical
College of Fudan University.
Xenograft model establishment and sample collection
Female Balb/c nude mice (4-5 weeks old, 18-24 g) wereacquired from Shanghai Xipur-Bikai Experimental
Animal Co. Mice were bred under special pathogen-free(SPF) conditions.
When MDA-MB-231 cells reached the logarithmic
phase, they were digested with a 0.25% EDTA trypsin
digestive solution (Gibco, America), suspended, andharvested by centrifugation at 1,200 rpm for 5 min. A
total of 1 × 106 cells suspended in 100 μL of PBS were
injected subcutaneously under the second pair of rightmammary fat pads of each nude mouse. After injection,
the tumor growth was observed, and on the 10th day,the tumor grew to 10 mm, and the xenograft model was
successfully established.
US treatment procedures
The US750 low-frequency ultrasonic therapeutic
instrument (ITO Co. Ltd., Japan) was used in this study.
US exposure was as follows: a frequency of 1 MHz, an
intensity of 2 W/cm2, a duty cycle of 50%, and exposure
time of 5 min. SonoVue (Bracco, Milan, Italy), whichcontained sulfur hexafluoride gas and had a phospholipid
monolayer shell, was shaken with 5 mL of normal saline
into a MB suspension, and 0.2 mL of the MB suspension
was injected into the mouse via the tail vein.
A total of 20 xenografted nude mice were randomly
divided into four groups. The US plus MBs (US + MBs)

group was treated with MBs followed by US. The MBs
group was treated with MBs combined with empty
exposure. The US group was treated with saline followed
by US. The control group was treated with saline
combined with empty exposure. All of the mice had
undergone US or empty irradiation on an alternate day,three times in total. After the third treatment, all mice
were kept under SPF conditions for 6 days. Afterwards,
all xenografted mice were sacrificed. The tumors were
removed for observation and fixed in formalin for further
study.
Measurement of tumor growth
The tumor volume was calculated with the following
formula: Volume (V) = (π × a × b2)/6. The length (a)and width (b) of tumors were measured before each
irradiation and every other day after the last irradiation,
six times in total. The following formula was used to
calculate the rate of tumor growth inhibition (TGI):TGI (%) = (1 - V/V0) × 100%. (V represented the final
volume in treatment groups, and V0 represented the final
volume in the control group)
Hematoxylin and eosin (H&E) staining
The harvested tumor specimens were fixed in a 4%
formalin solution for 24 h. The tissues were embedded
in paraffin and then cut into 4-μm-thick sections.
The sections were stained with H&E for pathologicalexamination. A microscope was used to photograph thesections at ×400 magnification to observe the tumor
tissue pathology and structural changes.
Terminal deoxynucleotidyl transferase dUTP nick-end
labeling (TUNEL) assay
Using the In Situ Cell Death Detection Kit (POD,
Roche company, Germany), apoptosis of the tumor
cells was determined with TUNEL. Apoptotic cellswith DNA fragmentation stained brown. Slides werecounterstained with hematoxylin, and the apoptotic cells
stained brownish-yellow. The total number of tumor cell
nuclei and TUNEL-positive cell nuclei were counted(magnification, ×400). Eight high-magnification fields
were analyzed in each section. The positive cells andtotal cells in every field were counted and then theapoptosis index (AI) was calculated using the following
formula: AI = (Number of positive cells/Number ofwhole cells) × 100%. The AI of all eight fields wasaveraged as the AI for the section.
CD34 immunohistochemical staining
The paraffin sections were dewaxed. The tissue sections
were incubated with 3% methanol hydrogen peroxidefor 5 min at room temperature, washed with PBS(237)

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three times to block endogenous peroxidases, and thenblocked with dilute goat serum at room temperature for
20 min to block nonspecific antigens. Then, the tissues
were continuously incubated with CD34 antibodies
overnight, secondary antibodies for 20 min, and thechromogenic agent for 20 min, followed by hematoxylin
counterstaining, dehydration, and mounting. The nuclei
and cytoplasm of vascular endothelial cells stainedbrownish-yellow. The sections were observed under 40×
magnification to determine areas of high blood vessel
density, i.e., hot spots. The number of vessels in hot spots
was counted under 400× magnification. After observing
five hot spots, the microvascular density (MVD) was
calculated.
Statistics
Quantitative data are expressed as the mean ± standard
deviation. Differences between or within groups were
tested using one-way analysis of variance (ANOVA), anddifferences in ratios were tested using a chi-squared test.
A P value﹤0.05 indicated statistical significance, and ap value﹤0.001 indicated marked statistical significance.
Statistical analysis was performed using the software
SPSS 13.0.

Results
Low-frequency US combined with MBs inhibited tumor
growthTumor growth was significantly inhibited in the US +MBs group. The tumor volume in the US+MBs group(0.961 ± 0.490 mm3, p < 0.001) was smaller than thatin the control (2.067 ± 0.281 mm3, p < 0.001), MBs(1.949 ± 0.250 mm3, p < 0.001), and US groups (1.542± 0.133 mm3, p < 0.001), and the TGI in the US + MBs
group (53.51%, p < 0.001) was greater than that in theMBs (10.98%, p < 0.001) and US groups (25.40%, p <0.001). Compared to the control and MBs groups, thetumor volume in the US group was markedly smaller (p< 0.05 for both), and the TGI in the US group was higher
than that in the MBs group (p < 0.001). There were no
significant differences in the tumor volume in the control
and MBs groups (p > 0.05). The trends in tumor growthin the four groups are shown in Figure 1. Tumor volumes
and TGIs are shown in Table 1.
Low-frequency US combined with MBs reduced
erythrocyte-filled vessels without other pathologicalchangesFigure 1. Growth trends for breast cancer xenografts. Tumor growth in the US + MBs group slowed down significantly andthe slope was relatively slight. Tumor growth in the US group slowed down, and the slope was steeper than that in the US + MBs
group. Trends in tumor growth were similar in the MBs and control groups, and the slopes were steep.
Table 1. Comparison of tumor growth in each group after treatment
Group

n

Tumor volume before treatment (mm3)Tumor volume before execution (mm3)

TGI (%)

Control
MBs
US
US + MBs

55
5
50.434 ± 0.065
0.437 ± 0.062
0.454 ± 0.062
0.431 ± 0.025

2.067 ± 0.281
1.949 ± 0.250
1.542 ± 0.133a
0.961 ± 0.490b

10.98
25.40c53.51d(a) p < 0.05, US vs. Control, MBs, respectively; p < 0.001, US vs. US + MBs. (b) p < 0.001, US + MBs vs. Control, MBs, US, respectively. (c)p < 0.001, US vs. MBs. (d) p < 0.001, US + MBs vs. MBs, US, respectively. US: ultrasound; MBs: microbubbles; TGI: rate of tumor growth
inhibition.

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Pathological changes in tumor tissues are shown in
Figure 2. H&E staining did not reveal noticeable
histological differences in each group, and no apparent
necrosis was observed. Under microscopic observation,
the number of erythrocyte-filled vessels decreasedin the US + MBs group compared to the other threegroups. Erythrocyte leakage was seen in the interstitium
in some areas in the US + MBs group.
Low-frequency US combined with MBs increased
apoptosis
As shown in the TUNEL assay, the distribution of
apoptotic cells in each group are shown in Figure 3. A
few scattered stained apoptotic cells were seen in thecontrol and MBs groups. In the US group, the number
of apoptotic cells increased, and they were scattered.
In the US + MBs group, apoptotic cells increased
significantly, and they were present in sheets, andespecially around blood vessels. The AI in the US +MBs group (49.02 ± 2.85%) was significantly higher
than that in the other three groups (US: 11.04 ± 0.34%;
MBs: 6.15 ± 0.29%; control: 4.68 ± 0.22%; p < 0.001
for all). The AI in the US group was markedly higher
than that in the control and MBs groups (p < 0.001 forboth). There were no significant differences in the AI in
the MBs and control groups (p > 0.05). The AIs in thefour group are shown in Table 2.
Low-frequency US combined with MBs reduced themicrovascular density
After CD34 staining, the microvascular endothelium
stained brownish-yellow, as shown in Figure 4. In thecontrol and MBs groups, several microvessels wereobserved in areas where the tumor thrived. However,the number of microvessels decreased in the US group.
In the US + MBs group, the number of microvessels
decreased significantly, and the residual lumens wereTable 2. Comparison of AI and MVD after treatment
among groups

Figure 2. H&E staining of breast cancer xenografts (×400).
(A) In the control group, donor blood vessels are visible at theedge of the tumor, and the lumen is filled with erythrocytes(black arrow). (B) In the MBs group, a few vessels are seen in
the tumor, and a few erythrocytes are seen in the lumen (dottedarrow). (C) There are few vessels in the US group. (D) In the
US + MBs group, erythrocytes leaked into the interstitium(white arrow). H&E: hematoxylin and eosin.

Figure 3. Tumor apoptosis after treatment in four groups(×400). (A) and (B) There were a few scattered apoptotic cells
in the control and MBs groups. (C) Apoptotic cells increased
and were scattered in the US group. (D) Apoptotic cells
significantly increased and were present in sheets in the US +MBs group.

Group

n

AI (%)MVD

Control
MBs
US
US + MBs

55
5
5

4.68 ± 0.22
6.15 ± 0.29
11.04 ± 0.34a
49.02 ± 2.85b

10.93 ± 0.37
10.48 ± 0.44
6.59 ± 0.24a
3.75 ± 0.36b(a) p < 0.001, US vs. Control, MBs, US + MBs, respectively. (b)p < 0.001, US + MBs vs. Control, MBs, US, respectively. MVD:
microvascular density; AI: apoptosis index; US: ultrasound; MBs:
microbubbles.

Figure 4. MVD after treatment in four groups (×400). (A)and (B) There are many microvessels in the control and MBs
group. (C) Microvessels decreased slightly in the US group.
(D) Microvessels decreased significantly in the US + MBs
group, and the residual lumens are mostly closed. MVD:
microvascular density.

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mostly closed. MVD in the US group was lower
than that in the control and MBs groups (p < 0.001).
The MVD in the US + MBs group (3.75 ± 0.36) was
markedly lower than that in the other three groups (US:11.04 ± 0.34; MBs: 6.15 ± 0.29; and control: 4.68 ± 0.22;
p < 0.001 for all). There were no significant differencesin the MVD in the control and MBs groups (p > 0.05).
The MVDs in the four groups are shown in Table 2.
Discussion
US is widely investigated and has developed indiagnostic and therapeutic fields, and low-frequency
US is currently a topic of therapeutic study. Comparedto high-frequency US, low-frequency US produces lessheat and more cavitation. The ability of low-frequency
US to inhibit tumor proliferation had been provedin several studies. Jang et al. observed cell death inmelanoma cells mixed with Optison MBs after lowintensity US (15). Cao et al. found that low-intensity
US suppressed the ability to proliferate, form colonies,
and invade AsPC-1 cells (11). However, its clinicaluse in breast cancer in vivo is still unclear. The current
study established a breast cancer model in nude miceand then subjected breast cancer xenografts to lowfrequency US treatment combined with MBs. This
study tried to clarify its clinical effects and discussed
the underlying mechanisms.
Different US regimens can lead to different celloutcomes. When the frequency is fixed, total cell deathand the ratio of necrosis increases with increasing
intensity (16). In our previous study, we used different
parameters for US (1 MHz) to treat breast cancer
xenografts in nude mice. US at a frequency of 1
MHz, an intensity of 2 W/cm2, and a duty cycle of
50% maximally inhibited tumor growth without thedeath of nude mice. With the current treatment, lowfrequency US (a frequency of 1 MHz, an intensity of
2 W/cm2, a duty cycle of 50%) significantly inhibitedbreast cancer xenografts, as evinced by the inhibition oftumor growth and abundant apoptotic cells, especiallyin the US + MBs group. Interestingly, there was noevidence of necrosis during the whole treatment. Whentumors were treated with HIFU, significant coagulation
necrosis may occur (9,17). The current study indicatedthat tumor destruction induced by low-frequency US
was due to cell apoptosis, rather than cell necrosis.
When US irradiation at a specific frequency andintensity is applied to a liquid, cavitation nuclei willbe created, followed by a change in volume, collapse,
and a burst of the nuclei. Transient cavitation bubbles
oscillate strongly and exist for only a few acoustic
cycles, eventually collapsing, producing a large
number of free radicals and a high local temperatureand pressure (18,19). Oscillating bubbles also create
microstreaming, which can induce shear stress on
nearby cells or vessel endothelium (20). In addition, theforce of radiation pushes bubbles towards the directionof wave propagation, which may have some impact on
the endothelium. Actions on endothelium cause various
stimuli, which are related to cell apoptosis alone or
together, including damage to nuclear DNA (21),reactive oxygen species (22), a change in membrane
permeability (23), disturbance of the calcium balance(24,25), and stimulation of the endoplasmic reticulum(26).
All of the above proved that cavitation-related
effects lead to apoptosis. However, inducing cavitationin whole blood is not easy, probably because the bodycontinuously filters impurities, including cavitation
nuclei (27). SonoVue MBs were used as artificial
cavitation nuclei, which markedly lowered thecavitation threshold and amplified the effect of US.
Results indicated significantly better efficacy in the
US + MBs group than that in the US group, which was
confirmed in several other studies. Shen et al. used US
at a frequency of 21 kHz on rabbit VX2 liver tumors,and the TGI was the highest in the US + MB group(28). Cao et al. indicated that US at a frequency of 45
kHz had the most efficient effect on decreasing cell
viability and suppressing the ability of AsPC-1 cells to
proliferate and form colonies in the US + MB group(11). However, comparing the efficacy of US acrossstudies is not easy because different investigators useddifferent US parameters and treatment conditions andacted on different targets.
In addition to cavitation, thermal effect is one of
the essential bioeffects of low-frequency US. Mild
hyperthermia can induce apoptosis in tumor cells andact synergistically with other therapies since it leads toseveral significant physiological changes by increasing
the tumor blood flow and the perfused fraction of thetumor (29-31). When modulated electro-hyperthermia
treatment was applied to the BALB/c mouse isograftmodel, the elevated expression of heat shock protein(HSP) 70 indicated heat shock-related cell stress, andthe treated tumor showed significant signs of apoptosis
and upregulation of caspase-3 (32). The stress protein
response to US irradiation produced a large amount of
HSPs, and elevated levels of HSP can trigger apoptosis(32). All of the above indicate that hyperthermia
plays a certain role in oncotherapy. It has rarely beenused clinically alone or with other therapies, so more
research needs to be conducted in the future.
Neovascularization is associated with thereproduction, invasion, and metastasis of tumors. The
destruction of supply vessels that provide sufficient
oxygen and other nutrients to tumor cells can inhibit
the growth of a tumor. The current study noted obvious
vessel damage and erythrocyte leakage in the US +MBs group, which might be related to the cavitationand thermal effects of low-frequency US. Shock waves
generated by MBs collapsing near the vessel wall
may create liquid jets that damage endothelium and(240)

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cause holes in the vessel wall, activating coagulationand causing thrombosis (33). Shen et al. found thatsonication with low-frequency US at 20 kHz and MBsof the rabbit carotid artery formed holes, local vascularwall defects, and arterial elastic membrane separation(34). Shen et al. indicated that diffused interstitialhemorrhages and vascular thrombi were observed after
US treatment with MBs in rabbit VX2 hepatic tumors(28). The above studies indicated that low-frequency
US combined with MBs can disrupt supply vessels,
which may explain the inhibition of tumor growth.
Compared to vessels of normal tissue, tumor vessels
are immature, abnormal, and highly permeable. Somelarge tumor vessels lack smooth muscle and are only
consist of endothelium and a basement membrane.
The endothelium in tumors is also structurally
defective; it is discontinuous and full of gaps, causing
hemorrhaging and facilitating permeability. Poorly
differentiated cell contacts, abnormal cell-celljunctions, and exaggerated leakiness cause defects inendothelial cell barrier function (35). Tumor vessel
density is very heterogeneous, and the arrangement ofvessels is chaotic, especially in the center (35). Due toeither increased endothelial permeability or tortuous
vessels, the blood flow rate in tumor sites decreases,
and MBs tend to be trapped at tumor sites rather
than normal tissue sites. DeOre et al. found that thelonger the residence time of MBs, the more sufficient
thermal effects of damaging the endothelium (36).
The difference in temperature enables the selective
disruption of tumor blood vessels without causing asignificant adverse effect on normal blood vessels (37).
In our study and other studies, low-frequency US
has displayed a certain level of efficacy on solid tumors
and no skin damage, death of mice, or metastasis wereobserved in our study, there are still concerns about thenegative impact of using low-frequency US. For thisreason, some researchers have discussed the possible
negative effects of low-frequency US irradiation. Yanget al. found that after exposure to 0.6W/cm 2 for 15
minutes, epidermal and dermal necrosis, excoriation,and inflammatory cell infiltration were seen in skintissue (38). While after exposure to 0.35W/cm 2 for
15 min, mouse skin displayed no obvious changes(38). Hence, at certain frequencies and intensities, US
irradiation caused no significant damage to the skinof mice. Similarly, when controlled within a certain
threshold, the effect of US irradiation on human skinis negligible (39). Although whether low-frequency
US irradiation does harm to the surrounding normal
tissues was not considered in our study, it has been
determined by other researchers. Wang et al. treatedmouse pancreatic cancer cells and normal pancreatic
ductal epithelial cells with low-frequency US; with
MB concentrations under 15%, the viability of normal
pancreatic cells was not affected, while with MB
concentrations under 30%, the rate of inhibition of

pancreatic cancer cells increased progressively (40).
The difference between normal cells and cancer cells
when subjected to low-frequency US irradiation isclosely related to apoptotic factors. For example,
survivin is a member of the inhibitor of apoptosis
protein family, which are selectively expressed invarious malignant tumors but which are not expressedor which are expressed at lower levels in normal tissues(41). Under the action of low-frequency US, the upand down-regulation of pro- and anti- apoptotic factors
jointly leads to the apoptosis of cancer cells, thereby
inhibiting the proliferation of cancer cells. Studies haveshown that low-frequency US irradiation can inhibit
the migration of cancer cells, thereby reducing thepossibility of metastasis. In a study by Wei et al., lowfrequency US was applied to prostate cancer cells, andcancer cell migration in the experimental group was
significantly inhibited compared to that in the control
group (42). In a study by Wang et al., low-frequency
US irradiation slightly decreased the migration of
pancreatic cancer cells (40). When cells have stem
cell-like features, they were considered to be the maincause of metastasis and drug resistance (43). Yang et al.
found that low-intensity US suppressed the migrationof ovarian cancer stem cells by inducing morphologicalchanges, F-actin formation, and increasing membrane
stiffness (44). These findings indicate that underappropriate conditions, low-frequency US irradiation
will not damage the skin of the experimental subject oreven the patient, its effect on cancer cells and normal
cells differs significantly, and it inhibits the metastasisof cancer cells to a certain degree.
There were several limitations in this study. First,
this study was based on a single type of breast cancer
cell, whereas there are multiple types of breast cancer
cells in clinical practice. More experiments need to be
designed to verify whether low-frequency US combinedwith MBs will still work on the other types of breast
cancer. Second, this study did not explore underlying
mechanisms. The continued exploration of cavitation,
its molecular mechanisms, and the dose-response
relationship between US parameters and anticancer
efficiency need to be further studied. Third, this study
only noted an inhibitory effect of low-frequency US
irradiation on breast cancer cells, but the treatment
failed to completely destroy cancer cells, so whether it
can be used alone in clinical settings or how it can be
combined with other treatments, such as chemotherapy
and radiotherapy, needs to be verified further.
In conclusion, the results of this study indicated thatcombining low-frequency US with MBs can suppress
tumor growth by inducing apoptosis and blocking theblood supply. The optimization of low-frequency US
treatment should enable it to be an essential tool in thetreatment of breast cancer.
Funding: This work was supported by grants from a(241)Global Health & Medicine. 2024; 6(4):236-243.

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project for the Science and Technology Innovation
Action Plan-Medical Innovation Research Programof the Shanghai Municipal Science and Technology
Commission (grant no. 22Y11911600 to L Chen) andprojects under the National Natural Science Foundationof China (grant no. 82374243 to L Wang and grant no.
82304906 to LS Li).
Conflict of Interest: The authors have no conflicts ofinterest to disclose.

15.

16.

17.

References
1.

2.

3.
4.
5.
6.
7.

8.

9.
10.

11.

12.

13.

14.

Sung H, Ferlay J, Siegel RL, Laversanne M,
Soerjomataram I, Jemal A, Bray F. Global cancer
statistics 2020: GLOBOCAN estimates of incidence andmortality worldwide for 36 cancers in 185 countries. CA
Cancer J Clin. 2021; 71:209-249.
Carioli G, Malvezzi M, Rodriguez T, Bertuccio P, NegriE, La Vecchia C. Trends and predictions to 2020 inbreast cancer mortality in Europe. Breast. 2017; 36:8995.
Zheng R, Zhang S, Zeng H, Wang S, Sun K, Chen R, Li L,
Wei W, He J. Cancer incidence and mortality in China,
2016. J Natl Cancer Center. 2022; 2:1-9.
Loibl S, Poortmans P, Morrow M, Denkert C, Curigliano
G. Breast cancer. Lancet. 2021; 397:1750-1769.
Waks AG, Winer EP. Breast cancer treatment: A review.
JAMA. 2019; 321:288-300.
Bradley JA, Mendenhall NP. Novel radiotherapy
techniques for breast cancer. Annu Rev Med. 2018;69:277-288.
Xia LY, Hu QL, Xu WY. Efficacy and safety ofradiofrequency ablation for breast cancer smaller than 2
cm: A systematic review and meta-analysis. Front Oncol.
2021; 11:651646.
Yu M, Pan H, Che N, et al. Microwave ablation ofprimary breast cancer inhibits metastatic progression
in model mice via activation of natural killer cells. Cell
Mol Immunol. 2021; 18:2153-2164.
Feril LB, Fernan RL, Tachibana K. High-intensity
focused ultrasound in the treatment of breast cancer. Curr
Med Chem. 2021; 28:5179-5188.
Peek MC, Ahmed M, Napoli A, ten Haken B,
McWilliams S, Usiskin SI, Pinder SE, van Hemelrijck M,
Douek M. Systematic review of high-intensity focusedultrasound ablation in the treatment of breast cancer. Br J
Surg. 2015; 102:873-882; discussion 882.
Cao J, Hu C, Zhou H, Qiu F, Chen J, Zhang J, Huang P.
Microbubble-mediated cavitation promotes apoptosis
and suppresses invasion in AsPC-1 cells. Ultrasound
Med Biol. 2021; 47:323-333.
Wu Y, Liu X, Qin Z, Hu L, Wang X. Low-frequency
ultrasound enhances chemotherapy sensitivity andinduces autophagy in PTX-resistant PC-3 cells via theendoplasmic reticulum stress-mediated PI3K/Akt/mTOR
signaling pathway. Onco Targets Ther. 2018; 11:56215630.
Ji Y, Han Z, Shao L, Zhao Y. Evaluation of in vivo
antitumor effects of low-frequency ultrasound-mediatedmiRNA-133a microbubble delivery in breast cancer.
Cancer Med. 2016; 5:2534-2543.
Zhang B, Zhou H, Cheng Q, Lei L, Hu B. Low-

18.

19.
20.
21.

22.

23.
24.

25.

26.

27.
28.

29.
30.

(242)

frequency low energy ultrasound combined withmicrobubbles induces distinct apoptosis of A7r5 cells.
Mol Med Rep. 2014; 10:3282-3288.
Jang KW, Seol D, Ding L, Lim TH, Frank JA, Martin
JA. Ultrasound-mediated microbubble destruction
suppresses melanoma tumor growth. Ultrasound Med
Biol. 2018; 44:831-839.
Feng Y, Tian Z, Wan M. Bioeffects of low-intensity
ultrasound in vitro: Apoptosis, protein profile alteration,and potential molecular mechanism. J Ultrasound Med.
2010; 29:963-974.
Guan L, Xu G. Damage effect of high-intensity
focused ultrasound on breast cancer tissues and theirvascularities. World J Surg Oncol. 2016; 14:153.
Merouani S, Hamdaoui O, Rezgui Y, Guemini M.
Theoretical estimation of the temperature and pressure
within collapsing acoustical bubbles. Ultrason
Sonochem. 2014; 21:53-59.
Stricker L, Lohse D. Radical production inside an
acoustically driven microbubble. Ultrason Sonochem.
2014; 21:336-345.
Nejad SM, Hosseini H, Akiyama H, Tachibana K.
Reparable cell sonoporation in suspension: Theranostic
potential of microbubble. Theranostics. 2016; 6:446-455.
Zuo K, Xu Q, Wang Y, Sui Y, Niu Y, Liu Z, Liu M, Liu
X, Liu D, Sun W, Wang Z, Liu X, Liu J. L-ascorbic acid2-phosphate attenuates methylmercury-induced apoptosis
by inhibiting reactive oxygen species accumulation and
DNA damage in human SH-SY5Y cells. Toxics. 2023;
11.
Fu Y, Ye Y, Zhu G, Xu Y, Sun J, Wu H, Feng F, Wen
Z, Jiang S, Li Y, Zhang Q. Resveratrol induces humancolorectal cancer cell apoptosis by activating themitochondrial pathway via increasing reactive oxygen
species. Mol Med Rep. 2021; 23.
Wang LY, Zheng SS. Advances in low-frequency
ultrasound combined with microbubbles in targeted
tumor therapy. J Zhejiang Univ Sci B. 2019; 20:291-299.
Tijore A, Yao M, Wang YH, Hariharan A, Nematbakhsh
Y, Lee Doss B, Lim CT, Sheetz M. Selective killing oftransformed cells by mechanical stretch. Biomaterials.
2021; 275:120866.
Romero-Garcia S, Prado-Garcia H. Mitochondrial
calcium: Transport and modulation of cellular processes
in homeostasis and cancer (Review). Int J Oncol. 2019;54:1155-1167.
Zheng P, Chen Q, Tian X, Qian N, Chai P, Liu B, Hu
J, Blackstone C, Zhu D, Teng J, Chen J. DNA damage
triggers tubular endoplasmic reticulum extension topromote apoptosis by facilitating ER-mitochondria
signaling. Cell Res. 2018; 28:833-854.
ter Haar G. Safety and bio-effects of ultrasound contrast
agents. Med Biol Eng Comput. 2009; 47:893-900.
Shen ZY, Liu C, Wu MF, Shi HF, Zhou YF, ZhuangW, Xia GL. Spiral computed tomography evaluationof rabbit VX2 hepatic tumors treated with 20 kHz
ultrasound and microbubbles. Oncol Lett. 2017; 14:31243130.
Dunne M, Regenold M, Allen C. Hyperthermia can alter
tumor physiology and improve chemo- and radio-therapy
efficacy. Adv Drug Deliv Rev. 2020; 163-164:98-124.
Liao S, Hu X, Liu Z, Lin Y, Liang R, Zhang Y, Li Q, Li Y,
Liao X. Synergistic action of microwave-induced mild
hyperthermia and paclitaxel in inducing apoptosis in thehuman breast cancer cell line MCF-7. Oncol Lett. 2019;

Global Health & Medicine. 2024; 6(4):236-243.

www.globalhealthmedicine.com

17:603-615.
31. Qu Y, Li J, Ren J, Leng J, Lin C, Shi D. Enhanced
magnetic fluid hyperthermia by micellar magnetic
nanoclusters composed of Mn(x)Zn(1-x)Fe(2)O(4)nanoparticles for induced tumor cell apoptosis. ACS
Appl Mater Interfaces. 2014; 6:16867-16879.
32. Danics L, Schvarcz CA, Viana P, Vancsik T, Krenacs T,
Benyo Z, Kaucsar T, Hamar P. Exhaustion of protective
heat shock response induces significant tumor damage
by apoptosis after modulated electro-hyperthermia
treatment of triple negative breast cancer isografts inmice. Cancers (Basel). 2020; 12.
33. Chen H, Brayman AA, Kreider W, Bailey MR, Matula
TJ. Observations of translation and jetting of ultrasoundactivated microbubbles in mesenteric microvessels.
Ultrasound Med Biol. 2011; 37:2139-2148.
34. Shen ZY, Jiang YM, Zhou YF, Si HF, Wang L. Highspeed photographic observation of the sonication of a
rabbit carotid artery filled with microbubbles by 20-kHz
low frequency ultrasound. Ultrason Sonochem. 2018;
40:980-987.
35. Ribatti D, Nico B, Crivellato E, Vacca A. The structureof the vascular network of tumors. Cancer Lett. 2007;
248:18-23.
36. DeOre BJ, Galie PA, Sehgal CM. Fluid flow rate dictates
the efficacy of low-intensity anti-vascular ultrasoundtherapy in a microfluidic model. Microcirculation. 2019;26:e12576.
37. Levenback BJ, Sehgal CM, Wood AK. Modeling ofthermal effects in antivascular ultrasound therapy. J
Acoust Soc Am. 2012; 131:540-549.
38. Yang M, Xie S, Adhikari VP, Dong Y, Du Y, Li D.
The synergistic fungicidal effect of low-frequency andlow-intensity ultrasound with amphotericin B-loaded
nanoparticles on C. albicans in vitro. Int J Pharm. 2018;
542:232-241.
39. Ahmadi F, McLoughlin IV, Chauhan S, ter-Haar G.
Bio-effects and safety of low-intensity, low-frequency

40.

41.
42.
43.

44.

ultrasonic exposure. Prog Biophys Mol Biol. 2012;
108:119-138.
Wang H, Ding W, Shi H, Bao H, Lu Y, Jiang TA.
Combination therapy with low-frequency ultrasoundirradiation and radiofrequency ablation as a synergistic
treatment for pancreatic cancer. Bioengineered. 2021;
12:9832-9846.
Westphal S, Kalthoff H. Apoptosis: Targets in pancreaticcancer. Mol Cancer. 2003; 2:6.
Wei C, Bai WK, Wang Y, Hu B. Combined treatment of
PC-3 cells with ultrasound and microbubbles suppresses
invasion and migration. Oncol Lett. 2014; 8:1372-1376.
Huang T, Song X, Xu D, Tiek D, Goenka A, Wu B,
Sastry N, Hu B, Cheng SY. Stem cell programs incancer initiation, progression, and therapy resistance.
Theranostics. 2020; 10:8721-8743.
Yang Y, Du M, Yu J, Chen Z. Biomechanical response ofcancer stem cells to low-intensity ultrasound. J BiomechEng. 2023; 145.

---Received June 8, 2024; Revised August 16, 2024; Accepted
August 20, 2024.
Released online in J-STAGE as advance publication August24, 2024.
§

These authors contributed equally to this work.
*Address correspondence to:
Lin Chen, Department of Ultrasound, Huadong Hospital,
Fudan University, 221 West Yan'an Road Shanghai, 200040,
China.
E-mail: cl_point@126.com
Ling Wang, Laboratory for Reproductive Immunology,
Obstetrics and Gynecology Hospital of Fudan University, 419
Fangxie Road, 200011 Shanghai, China.
E-mail: Dr.wangling@fudan.edu.cn(243)