Abstract
Introduction
Blood perfusion insufficiency and hypoxia are the main causes of drug resistance to chemotherapy in breast cancer. Increasing blood perfusion can improve drug delivery. This study aimed to investigate the effects of ultrasound-stimulated microbubbles (USMBs) on hemoperfusion in invasive breast cancer (IBC).
Methods
In this prospective clinical trial, 36 patients diagnosed with IBC were enrolled sequentially. The treatment group (n = 18, enrolled from June 2022 to April 2025) were treated with SonoVue® microbubbles (MBs) stimulated by ultrasound, with a mechanical index (MI) of 0.2–0.3: 1 mL of SonoVue® MBs was injected at 3.5-min intervals three times for a USMB treatment lasting 10 min. The control group (n = 18, enrolled from May to November 2025) received identical MB injections without ultrasound stimulation. Contrast-enhanced ultrasound (CEUS) was used to evaluate the changes in blood perfusion.
Results
In the treatment group, in comparison with the pre-treatment findings, the tumor perfusion area expanded (P < .001) and the time to peak (TTP) increased (P < .05) after USMB treatment. For regions exhibiting low enhancement inside the lesion on CEUS before USMB treatment, the area under the curve (AUC) (P < .001) and mean transit time (MTT) (P < .05) both increased following therapy. In the control group, none of the parameters showed statistically significant differences after the MB injections.
Conclusion
USMB treatment can improve blood perfusion in IBC, especially by enhancing the AUC and MTT in hypoperfused regions. These findings highlight the potential of USMB treatment as a noninvasive technique to enhance intratumoral drug delivery, although further validation of this approach is required.
Introduction
In 2020, breast cancer became the most frequently diagnosed malignancy, surpassing lung cancer for the first time, 1 and is now the leading cause of disability and death among women worldwide. 2 Neoadjuvant chemotherapy is an important part of the comprehensive treatment protocol for breast cancer, and patients who achieve pathological complete response (pCR) show improved survival, disease-free survival, and overall survival.3–5 However, neoadjuvant chemotherapy does not always yield ideal outcomes, with only 13%–22% of the patients showing pCR.4,6 Some studies have suggested that the accumulation of drugs in tumors is related to the degree of tumor vascularization. 7 The rapid growth of tumor cells and the heterogeneity of neovascularization within the tumor result in internal perfusion insufficiencies and create an internal hypoxic microenvironment. These factors reduce the deposited dose of chemotherapeutic drugs in tumors, which is one of the main reasons for drug resistance.8,9 Thus, promotion of vascularization, which can improve perfusion by increasing blood vessel density, is the latest vascular-regulation strategy. 10 Although this concept may initially appear paradoxical since angiogenesis can promote tumor growth,11,12 enhancing tumor vascularization and increasing blood perfusion can also ameliorate the hypoxic microenvironment within the tumor, alleviating drug resistance and enhancing anti-tumor efficacy.13,14 Thus, improving the degree of tumor vascularization and increasing intratumor blood perfusion can improve the effects of chemotherapy in preclinical breast cancer models. 15
The volume oscillation caused by ultrasonically excited microbubbles (MBs) is called cavitation. The cavitation effect is the physical basis for a series of biological effects. Under ultrasonic excitation, the MB generates cavitation resonance, and the consequent release of mechanical energy in the form of shockwaves and microjets results in tiny holes in the blood vessel wall or cell wall, which is called the acoustic hole effect. 16 These effects open the blood tumor barrier to enhance drug delivery.17,18 Many studies have shown that this technique is safe and effective.19,20 However, recent research on ultrasound-stimulated MBs (USMBs) has primarily focused on increasing drug extravasation in blood vessels and improving drug penetration in the extracellular matrix.21–23 USMBs have been shown to increase the blood perfusion of tumors, with studies showing increased blood perfusion in mice with PANC-1 pancreatic cancer, 24 MC38 colon cancer, 25 and breast cancer. 26 This phenomenon may be attributed to the fact that the cavitation resonance generated by USMBs causes mild damage to the tumor vascular endothelium, and a variety of factors released to repair this damage, such as vasodilators and inflammatory factors, cause vasodilation and hyperemia along with increased blood perfusion. 27 Thus, USMB treatment may be a new noninvasive method to stimulate enhanced blood perfusion in tumors.
To improve the effects of chemotherapy as well as the prognosis in patients with invasive breast cancer (IBC), we conducted a prospective clinical study using USMB technology to increase blood perfusion in IBC tumors. Since the initial single-arm phase (treatment group) had completed enrollment, a randomized design was not feasible. Therefore, we incorporated a subsequent control group, adopting a non-randomized design to isolate the specific effects of USMBs. To enhance the validity of our findings, the images were obtained by a single operator using a standardized imaging protocol, and an independent assessor subsequently performed blinded analysis of all images. This approach guaranteed both the consistency of data acquisition and the objectivity of the result interpretation.
Materials and Methods
Study Participants
This prospective clinical trial (Clinical trial registration number: NCT06158217) received approval from the Biomedicine Ethics Committee. All participants provided written informed consent. The reporting of this study conforms to the CONSORT statements and its extension guidelines for reporting non-randomized studies.28,29
A total of 36 patients with pathologically confirmed IBC were enrolled between June 2022 and November 2025. The first 18 consecutive patients (June 2022 to April 2025) constituted the treatment group and received the USMB intervention. The subsequent 18 patients (May to November 2025) were assigned to the control group, which received identical MB injections without ultrasound treatment. The intervention was conducted within 24 h before the scheduled tumor-removal surgery. The basic characteristics of the patients in both groups are shown in Table 1.
Baseline Characteristics of the Patients.
The inclusion criteria were as follows: (1) patients diagnosed with IBC; (2) patients scheduled to undergo surgical resection; (3) maximum lesion diameter < 4 cm; and (4) age over 18 years. On the other hand, we excluded patients with allergies to SonoVue® or severe cardiopulmonary insufficiency, patients who had already received neoadjuvant chemotherapy, cases in which breast cancer boundaries could not be identified on ultrasound, pregnant women, individuals with mental illness, and patients who were unable to cooperate for other reasons.
Ultrasound Scanning and Cavitation Equipment
The VINNO G86 (VINNO Technology Co., LD, Suzhou, China) color Doppler ultrasound diagnostic instrument with the L4-16 high-frequency linear array probe was utilized for this study. Since stable cavitation is expected to occur throughout the treatment when the mechanical index (MI) is set to 0.2,30,31 we adjusted the other parameters as follows to achieve an MI as close to 0.2 as possible: duty cycle, 0.4%; frequency, 3 MHz (the lowest frequency allowed by the equipment); sound power, 30%; pulse repetition frequency, 1500 Hz; pulse length, 8 cycles; pulse time, 3 s; and pulse interval time, 1.0 s. The treatment duration was 10 min. The depth at which the lesion is located can influence MI; thus, the MI range was maintained at 0.2–0.3. During the ultrasound treatment process, the ultrasound probe was moved uniformly over the surface of the tumor to enable homogenous treatment of the whole tumor. This procedure was performed only in the treatment group by Physician A, who had over 10 years of experience in breast diagnosis.
Tumor Perfusion Assessment with Contrast-Enhanced Ultrasound
Contrast-enhanced ultrasound (CEUS) examinations of the tumors were conducted to evaluate blood perfusion before the treatment. SonoVue® (Bracco, Milan, Italy) was used as the contrast agent, and an intravenous bolus was administered through a median cubital vein. We used 2.4 mL of SonoVue®, completing the injection within 2 s and subsequently flushing the catheter with 5 mL of saline solution. Imaging was performed using CEUS imaging software for a duration of 1.5 min, and video images were stored dynamically. The CEUS examinations were performed by Physician B, who has extensive CEUS experience and was blinded to the patient grouping.
In the visual evaluation of the degree of tumor enhancement, tumors were categorized as having high enhancement when they exhibited a higher degree of enhancement than the surrounding normal breast tissue. Conversely, tumors were classified as showing equal enhancement when the degree of enhancement was similar to that of the surrounding normal breast tissue, and as showing low enhancement when the degree of enhancement was lower than that of the surrounding normal breast tissue. The obtained images were analyzed using the perfusion parametric image (PPI) software package. To ensure blinded analysis, all 72 dynamic CEUS video images from the 36 patients were anonymized, randomly sorted, and assigned consecutive identification numbers. These images were then independently analyzed by Physician C, who has extensive CEUS experience (over 5 years specifically in breast CEUS). This physician was blinded to the patient group assignment (treatment or control), the timing of the image (pre- or post-intervention), and the overall study design and objectives.
The region of interest (ROI) within the lesion was selected, and the time-intensity curve (TIC) was then drawn to obtain quantitative parameters for blood perfusion, including peak intensity (PI), area under the curve (AUC), time to peak (TTP), arrival time (AT), acceleration time (ATT), mean transit time (MTT), and descending time/2 (DT/2) (Figure 1). Five minutes after completion of the treatment, CEUS examinations were repeated to assess changes in tumor blood perfusion. The gain, dynamic range, and size of the ROI rectangle were consistent in both pre-and post-treatment assessments.
Time-intensity curve (TIC) of region of interest (ROI) generation in the lesion. PI: peak intensity, AUC: area under the curve, TTP: time to peak, AT: arrival time, ATT: acceleration time, MTT: mean transit time, DT/2: descending time/2.
Assessments of Perfusion Area
The dynamic CEUS images were reviewed, and the image with the largest perfusion area was selected. The tumor perfusion boundary was manually traced, and the tumor perfusion area was calculated using the area calculation software provided with the device. The perfusion area was measured at the time point showing the highest degree of enhancement and the time points 5 s before and after the highest degree of enhancement; the average value of these three measurements was recorded as the final value for perfusion area.
Procedures Using Microbubbles
Clinical trials on safe doses of SonoVue® have shown that a single intravenous dose can be as high as 0.3 mL/kg,32,33 while the cumulative dose with multiple doses can be as high as 0.6 mL/kg. 34 The maximum single dose and the cumulative dose used in this trial were 2.4 mL and 7.8 mL, respectively, both of which were within the safe range. Since nonlinear imaging generated by 1 mL of MBs has been shown to be detectable, 35 and the life expectancy of 1 mL of MBs in vivo is approximately 4–5 min, 31 we injected 1 mL MBs/3.5 min to ensure the presence of MBs throughout the treatment cycle, and injected 5 mL of normal saline to rinse the catheter after each injection of MBs. This procedure was repeated three times consecutively. The same MB injection protocol was employed in both treatment and control groups. The trial process is illustrated in Figure 2.
Experimental flow chart. US: ultrasound; CEUS: contrast-enhanced ultrasound; MB: microbubble; USMB: ultrasound-stimulated microbubble.
Statistical Analysis
The sample size was determined on the basis of previous studies that investigated the use of ultrasound combined with MBs in breast cancer. On the basis of the effect sizes reported by Zhou et al 26 for changes in perfusion parameters (PI, AUC), a sample-size estimation was performed using PASS 15 software (power = 80%, α = 0.05, two-sided), which yielded required sizes of 12–21 patients per group. Considering the exploratory nature of this study and practical constraints, a total of 36 patients were enrolled and assigned consecutively, with 18 patients each allocated to the treatment and control groups.
Continuous variables showing a normal distribution were expressed as mean ± standard deviation (SD). Categorical variables were presented as percentages (%), and comparisons were performed using the Chi-square test or Fisher's exact probability test, as appropriate. Statistical analysis was performed using Graph Pad Prism 8.0 (Graph Pad Software, San Diego, USA). The comparison between tumor blood perfusion before and after the treatment was assessed using a paired t-test or Wilcoxon matched-pair signed-rank teat, and statistical significance was determined at P < .05.
Results
A total of 36 patients were included in this study, of which 18 each were allocated to the treatment and control groups. The treatment group contained 17 female patients and one male patient, while the control group only had female patients. In terms of pathological classification, the treatment group included 16 and two cases of invasive ductal carcinoma and invasive lobular carcinoma, respectively, whereas the corresponding values in the control group were 17 cases and one case, respectively. The two groups showed no significant differences in baseline characteristics. The baseline characteristics of the patients are shown in Table 1.
Tumor Perfusion Assessment
In the treatment group, the perfusion area after USMB treatment was larger than that before treatment (2.64 [1.74, 4.70] vs 2.18 [1.40, 3.89]; P < .001; Figure 3A-B). In addition, the TTP after USMB treatment was significantly longer than that before treatment (32.51 ± 14.57 vs 27.43 ± 9.93; P < .05; Figure 3C-D), while other parameters showed no significant differences (P > .05; Table 2).
Quantitative analysis of the contrast-enhanced ultrasound (CEUS) findings. Figures A-D present the entire lesion as an area of interest. A and B show that the perfusion area of the lesion increased significantly after USMB treatment. A shows the changes in the perfusion area before and after the trial, with a straight line connecting the perfusion area values (represented on the ordinate axis by each dot) of the same patient before and after the trial. B shows the mean value and standard deviation of the perfusion area before and after the trial (mean ± SD). C and D show that the TTP increased significantly after USMB treatment. The low-enhancement area inside the lesion was considered the area of interest in Figure E-H, which showed that the AUC increased and MTT increased after USMB treatment in comparison with that before treatment.
Changes in Blood Perfusion Parameters Before and After USMB Treatment.
PI: peak intensity; SD, standard deviation; AUC: area under the curve; TTP: time to peak; AT: arrival time; ATT: acceleration time; MTT: mean transit time; DT/2: descending time/2.
Analysis of the low-enhancement area inside the lesion showed that in comparison with the values before USMB treatment, AUC (583.27 [361.10, 994.95] vs 396.86 [178.52, 655.83], P < .001) and MTT (56.14 ± 18.63 vs 45.78 ± 16.54, P < .05) both increased after treatment (Figures 3E-H and 4), while the other parameters showed no statistically significant differences (all P > .05; Table 2). However, analysis of the high-enhancement area inside the lesions showed no significant differences for all parameters (P > .05; Table 2).
Changes in blood perfusion in the hypoperfusion area before and after USMB treatment. Panels A and B show the same lesion before (A) and after (B) treatment: the hypoperfused region (outlined by the curve) is markedly samller in B than in A, and the tumor-feeding vessels (indicated by short and long arrows) show increased perfusion. Panels C and D represent another lesion before (C) and after (D) treatment: the hypoperfused area is significantly diminished in D, and the feeding artery (marked by the long arrow) indicates that images C and D were acquired at comparable anatomical levels.
The control group showed no significant changes between the pre- and post-intervention assessments for any of the perfusion parameters (all P > .05) (Table 2).
Safety Assessment
All participants underwent active and continuous adverse-event monitoring. The following potential adverse events were recorded: 1. pain (actively inquired using a Numerical Rating Scale at the end of the treatment, where 0 indicated no pain and 10 indicated severe pain); 2. skin reactions (visual inspection of the treatment area for erythema, rash, or injury); 3. allergic reactions (inquiring about and observing symptoms such as urticaria or dyspnea); and 4. thrombosis-related symptoms (inquiring about and examining the injected arm for swelling, pain, or increased skin temperature). All monitoring results were documented in a predefined adverse-event log.
All patients in this study tolerated the USMB treatment well. No treatment-related pain, skin reactions, allergic reactions, or thrombotic events were reported or observed. In conclusion, under the conditions of this study, a single session of USMB treatment did not induce any serious adverse events.
Discussion
In this prospective clinical study, we aimed to investigate the effect of USMB treatment on breast cancer blood perfusion. USMB treatment significantly expanded the tumor perfusion area (2.64 [1.74, 4.70] vs 2.18 [1.40, 3.89], P < .001) and significantly prolonged the TTP (32.51 ± 14.57 vs 27.43 ± 9.93, P < .05). In the control group, neither parameter showed significant changes before and after the procedure. Given the abundant heterogeneous vasculature surrounding breast cancer tumors, 36 the observed post-treatment increase in the tumor perfusion area may result from the perfusion-enhancing effects of USMBs in the peripheral heterogeneous vasculature. The TTP reflects the period from the beginning of MB injection to the moment when MBs reach the PI. The presence of immature tumor vessels with leaky arrangement of endothelial cells, absence of pericyte coverage, and incomplete basement membrane contribute to structural and functional abnormalities of the vascular network. Consequently, during CEUS examinations, malignant tumors typically exhibit faster enhancement than surrounding normal tissue, which is reflected by a lower TTP. 37 Yang et al 38 reported that multiple USMBs could induce tumor microvessel dilation and promote normalization of heterogeneous vasculature. Vascular normalization is characterized by tighter endothelial cell junctions, increased pericyte coverage, and a higher proportion of functional blood vessels. 38 This shift toward normalization reduces vascular leakage and promotes more orderly blood perfusion within the tumor. Consequently, the rate of contrast agent accumulation and the speed at which it reaches PI in the tumor decrease, which manifests as an increase in TTP. This enhancement pattern is more closely aligned with that of normal tissue. This study showed that the TTP of tumors after USMB treatment was significantly longer than the pre-treatment levels, indicating potential modulation of tumor vascular function. However, the underlying mechanisms for this functional improvement, including the possibility that USMB treatment induces tumor vascular normalization, require further investigation and validation.
Furthermore, for the area with low perfusion inside the lesion, the results showed that the AUC (583.27 [361.10, 994.95] vs 396.86 [178.52, 655.83], P < .001) and MTT (56.14 ± 18.63 vs 45.78 ± 16.54, P < .05) after USMB treatment were higher than those before treatment, consistent with the results of previous studies.25–27 The intensity of the CEUS signal in the tumor is directly proportional to the degree of vascularization. 7 Breast cancer tumors with low enhancement exhibit a low degree of vascularization, high cell density, and close intercellular connections. Consequently, these tumors are more susceptible to hypoperfusion and hypoxia, which adversely affect drug penetration and cause resistance against chemotherapy drugs. 39 An increase in the AUC indicates an elevation in blood volume within low-perfusion areas, potentially alleviating the intratumoral hypoxic microenvironment and promoting drug delivery. Tang et al 27 showed that the USMB-induced sononeoperfusion effect could significantly enhance blood perfusion in Walker-256 tumors and promote drug delivery. In comparison with the findings for the same group before treatment, the AUC after USMB treatment increased by an average of 18.22%, enhancing doxorubicin delivery to tumors to approximately 3.12 times that in the control (P < .05). In this study, the MTT was significantly higher after USMB treatment than before treatment (56.14 ± 18.63 vs 45.78 ± 16.54, P < .05). A prolonged MTT indicates an extended microbubble retention time, which may enhance drug retention. 26
In the present study, USMBs did not alter blood perfusion within the hyper-enhanced region of the tumor. TIC analysis showed no significant changes in blood flow in the high-enhancement area within the lesion before and after USMB treatment, which may be related to the heterogeneity within the tumor. Different tumor types show different responses in this technique, and different effects may occur even within the same tumor.40–43 These variations in results despite using the same ultrasound parameters can be attributed to the vascular system within the tumor. Each tumor possesses unique vascular characteristics, such as vascular density, perfusion, and maturity, 10 which determine the number of MBs entering the tumor. The cavitation effect is related to the concentration of MBs.44,45 In addition, different breast cancer cell line phenotypes show different optimal combination parameters of USMBs, 44 and no standardized optimal parameter combinations have been recommended for USMB studies involving human breast cancer. While minor damage to the blood vessel wall can trigger the release of inflammatory factors and vasodilators, leading to vasodilation and increased perfusion, 27 severe damage can cause rupture of the blood vessel wall, thrombosis, lumen obstruction, and reduced blood perfusion.46–48 Thus, the technical parameters of ultrasound and their effects on each other and the mechanisms underlying perfusion are complex, requiring further investigation. 49
Clinical Translation and Future Directions
As a preliminary investigation, this study provides initial validation of the immediate effects of USMBs in improving blood perfusion in IBC, offering a theoretical basis and preliminary clinical evidence for subsequent research combining USMBs with neoadjuvant chemotherapy. Its definitive clinical utility, however, requires confirmation through prospective randomized controlled trials that integrate USMBs into neoadjuvant chemotherapy regimens, with pathological complete response (pCR) rates and patient survival as the primary endpoints.
The study had several limitations that require consideration. Although the acoustic parameters and microbubble concentration used in this study were based on existing protocols, the limitations imposed by tumor heterogeneity imply that individualized treatment protocols still require optimization. Furthermore, the analysis relied solely on hemodynamic parameters; therefore, direct evidence to confirm that USMBs induce vascular normalization or promote neovascularization is currently lacking; future validation through immunohistochemistry, RNA sequencing, and molecular assays of markers such as vascular endothelial growth factor (VEGF) and hypoxia-inducible factor (HIF)-1α on postoperative specimens is warranted. Finally, this is a preliminary exploratory study requiring validation in larger cohorts.
Conclusion: This study confirmed that USMB therapy can improve tumor blood perfusion in IBC (increasing the perfusion area of IBC tumors and prolonging TTP). Specifically, for the hypoperfused regions within tumors, USMBs significantly increased the blood flow (elevated AUC) and extended the microbubble mean transit time (elevated MTT). However, due to the heterogeneity in tumor perfusion, USMBs showed no significant effect on blood-flow parameters in the original hyperperfused regions within tumors. These findings suggest that USMB treatment is a promising noninvasive technique, potentially enhancing chemotherapy drug delivery by improving tumor blood perfusion, particularly in hypoperfused regions. Given the complex structure of human breast tissue and the high heterogeneity of breast cancer tumors, future studies should aim to further explore and optimize USMB treatment parameters to achieve personalized application.
Footnotes
Abbreviations
Acknowledgements
The authors thank all team members and colleagues in the Medical Center of Ultrasound, Beijing Friendship Hospital, Capital Medical University for their helpful cooperation and all the study participants for their patience and support. The authors would like to sincerely thank Dr Xinyu Zhao from the Clinical Epidemiology and Evidence-Based Medicine Center of Beijing Friendship Hospital, Capital Medical University, for his expert guidance on the statistical analysis for this study.
Ethics Approval and Consent to Participate
This study was approved by the Bioethics Committee of Beijing Friendship Hospital affiliated to Capital Medical University (approval number: 2022-P2-126-01). All patients signed informed consent forms. This study was retrospectively registered in ClinicalTrials.gov with the trial registration number NCT06158217. Our study adhered to the provisions of the 1975 Declaration of Helsinki, as revised in 2024.
Funding
The authors received no financial support for the research, authorship, and/or publication of this article.
Declaration of Conflicting Interests
The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.