Access the full text.
Sign up today, get DeepDyve free for 14 days.
Background Hypoperfusion or resultant hypoxia in solid tumours is a main reason for therapeutic resistance. Augmenting the blood perfusion of hypovascular tumours might improve both hypoxia and drug delivery. Cavitation is known to result in microstreaming and sonoporation and to enhance drug diffusion into tumours. Here, we report the ability to enhance both tumour blood perfusion and doxorubicin (Dox) delivery using a new sononeoperfusion effect causing a cavitation effect on tumour perfusion in subcutaneous Walker-256 tumours of rats using ultrasound stimulated microbubble (USMB). Methods To induce the sononeoperfusion effect, USMB treatment was performed with a modified diagnostic ultrasound (DUS) system and SonoVue® microbubbles. The therapeutic pulse was operated with a peak negative pressure of 0.26 to 0.32 MPa and a pulse repetition frequency (PRF) of 50 Hz to 2 kHz. Contrast-enhanced ultrasound (CEUS) was used for tumour perfusion assessment. Results The USMB treatment of 0.26 MPa and 1 kHz could significantly enhance tumour perfusion with a 20.29% increase in the CEUS peak intensity and a 21.42% increment in the perfusion area for more than 4 hours (P < 0.05). The treatment also increased Dox delivery to tumours by approximately 3.12-fold more than that of the control (P < 0.05). Furthermore, ELISAs showed that vasodilators and inflammatory factors increased 4 hours after treatment ( P < 0.05), suggesting that the inflammatory response plays an important role in the sononeoperfusion effect. Conclusion The USMB-induced sononeoperfusion effect could significantly enhance the blood perfusion of Walker-256 tumours and promote drug delivery. It might be a novel physical method for overcoming the therapeutic resistance of hypoperfused or hypoxic tumours. Keywords Sononeoperfusion, Tumour, Diagnostic ultrasound, Microbubble, Doxorubicin Najiao Tang and Jiawei Tang contributed equally to the manuscript. *Correspondence: Yi Zhang email@example.com Zheng Liu firstname.lastname@example.org Department of Ultrasound, Xinqiao Hospital, Army Medical University, 83 Xinqiao Street, Chongqing 400037, PR China © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Tang et al. Cancer Imaging (2023) 23:29 Page 2 of 11 Background Therefore, the aim of this study was to investigate the Due to the upregulation of angiogenesis in solid cancers, proper parameters, Dox delivery and related mechanism tumour cells often grow more rapidly than the cells that for sononeperfusion. The parameters included the MI form blood capillaries [1, 2]. Proliferation and immaturity or acoustic pressure and the pulse repetition frequency force vessels away from tumour cells, leading to vascular (PRF). Cytokines such as vasodilators and inflammatory density reduction and a poorly organized vessel archi- factors were tested to explain the mechanism. tecture [3, 4], irregular tumour blood flow [ 5–8] and the compression of vessels by cancer cells [9, 10]. Therefore, Methods hypoperfusion or hypovascularization tends to develop DUS system and acoustic detection in solid tumours and has been considered a main fac- A commercial DUS system (VINNO70, VINNO Tech- tor for tumour hypoxia. Hypoperfusion and resultant nology Co. Ltd., Suzhou, China) connected to an X4-12 L hypoxia are strongly related to therapeutic resistance, linear array transducer was used for both therapeutic such as in pancreatic cancer and ovarian cancer [11–13]. US exposure and US imaging. The system was equipped Another important factor for the response to chemo- with contrast bubble imaging (CBI), integrated con- therapy in tumours is the distance from tumour vessels to trast-enhanced ultrasound (CEUS) imaging software tumour cells. At approximately 40–50 μm away from the and flash mode for microbubble destruction. The flash blood vessel, the doxorubicin (Dox) concentration drops mode was specifically modified to deliver customized to half of its perivascular concentration. Furthermore, the pulse sequences for regulating microbubble cavitation or average distance from tumour blood vessels to hypoxic so-called ultrasound stimulated microbubble (USMB), tissue is 90 to 140 μm . Therefore, tumour cells that named Vflash. The Vflash pulses can be operated with are distant from blood vessels might be exposed to low adjustable frequency, MI, pulse length (PL), PRF and concentrations of drug and a hypoxic state . destruction (on)/replenish (off) time as described pre - However, it is difficult to enhance tumour perfusion viously . In addition, the Vflash US beams can be because vasoactive drugs may create vascular steal, lead- weakly focused to a trapezoid region of interest (ROI) ing to a decrease in tumour perfusion . Previously, using the electronic focusing method (Fig. 1). The size of Xie and Lindner found that low intensity ultrasound (US) the ROI is also adjustable and can cover the tumour body, with a high mechanical index (MI) of 0.6–1.3 and micro- similar to a colour doppler sample volume. bubbles could stimulate and enhance myocardial or mus- The peak negative pressure (PNP) within a desig - cular perfusion, which was later called “sonoreperfusion” nated ROI of 1 × 1 cm was measured by a membrane [16–18]. Sonoreperfusion may be connected with the hydrophone (HMB-0500, ONDA Corp., Sunnyvale, CA, increase in ATP and the purinergic pathway . In our USA) positioned 2 cm away from the probe surface. The previous studies, we occasionally found that low MI diag- probe was placed above the hydrophone separated with nostic ultrasound (DUS) combined with microbubbles degassed water in a sink (AIMS III, ONDA Corp., Sunny- could enhance tumour blood perfusion and increase the vale, CA, USA). perfusion area of solid tumours, such as PANC-1 pancre- atic cancer and MC38 colon cancer in mice [20, 21]. This Microbubbles is a very interesting phenomenon, not only because the SonoVue® microbubbles (Bracco Sine Pharmaceutical DUS intensity for this treatment was extremely low and Corp., Ltd., Shanghai, China) are a commercially avail- 8 − 1 within FDA and IEC guidelines, but also because it only able US contrast agent that contains 10 mL micro- requires low MI emission under 0.5, which is much lower bubbles with a mean diameter range from 2.0 to 4.0 μm than that of the MI needed for muscular sonoreperfu- after preparation in 4.0 mL of saline solution. The Son - sion . This US-stimulated perfusion enhancement oVue® suspension was then used in both CEUS as an US effect of solid tumours might be a novel and noninvasive contrast agent and in therapeutic US as cavitation nuclei. method to overcome tumour hypoxia, a major obstacle to CEUS is a reliable method to assess tumour perfusion. therapy. According to the EFSUMB guidelines, the mean time- Considering the low intensity, low MI and tumour per- intensity curves within the tumours after bolus injection fusion response features of the treatment, we named the of a contrast agent were qualified to reflect microbubble effect “sononeoperfusion”, representing US, neoplasm wash-in and washout, thus representing the condition of and perfusion improvement. Sonoreperfusion usually tumour perfusion . refers to US-stimulated myocardial or muscular blood reperfusion effects. However, tumour vascular construc - Animal model and experimental design tion is chaotic, disorganized, immature, dysfunctional A total of 81 Sprague‒Dawley (SD) rats bearing subcu- and mechanically vulnerable, which is quite different taneous Walker-256 tumours were used. The tumour from normal developed vessels [22, 23]. model was made by injecting 0.2 mL of Walker-256 cell Tang et al. Cancer Imaging (2023) 23:29 Page 3 of 11 Fig. 1 Schematic illustration of the USMB treatment in rats. A: Tumour perfusion enhancement is related to vasodilator release and promotes Dox uptake. B: Treatment plan 1: 56 SD rats received USMB treatment for 10 minutes and CEUS imaging at baseline, immediately after and 4 hours later. The control group received sham DUS. C: Treatment plan 2: Another 25 rats were enrolled for the Dox delivery study. The animals were treated with the selected USMB parameters and sham. After treatment, Dox solution was injected at approximately 3 hours 20 minutes. Table 1 The treatment parameters of all groups. parameter combinations and one control group. The Groups US parameters Dox treatment parameters of all groups are illustrated in injection(mg/kg) Frequen- PNP PL PRF Table 1. All of the treatments were performed with an cy (MHz) (MPa) (cycles) (Hz) X4-12 L linear array transducer operating at a central Control - - - - 10 (Plan 2) frequency of 4 MHz. Two PNP outputs of 0.26 MPa and Group A 4 0.26 10.5 50 - 0.32 MPa (equal to MI values of 0.13 and 0.16, respec- Group B 4 0.26 10.5 1 k 10 (Plan 2) tively), which were measured by the hydrophone, were Group C 4 0.26 10.5 2 k - selected to test the PNP variable in the experimental Group D 4 0.32 10.5 50 - groups. Under the fixed PL of 10.5 cycles, we selected Group E 4 0.32 10.5 1 k - Group F 4 0.32 10.5 2 k - 50 Hz, 1 and 2 kHz as three PRF variables (Table 1). In treatment plan 2 (Fig. 1C), Dox served as a chemo- suspension (approximately 1 × 10 /mL) into the inner therapeutic agent because it was detectable by fluores - thigh of the rat. Then, the model was included in the cent imaging and quantified by high-performance liquid study when the tumour size reached approximately 1 cm chromatography (HPLC). For Dox delivery, another 25 in diameter. All of the animal experimental procedures rats were randomly divided into one experimental group were approved by the Institutional Animal Care and Use (n = 14) and one control group (n = 11). Committee of the university. Among the 81 SD rats, 56 rats were randomly Experimental procedures divided into seven groups, including six experimen- The animals were anaesthetized by intraperitoneal injec - tal groups (A-F) according to different treatment tion of 2% pentobarbital sodium at 2 ml/kg, and the Tang et al. Cancer Imaging (2023) 23:29 Page 4 of 11 tumour surface was shaved and depilated. A catheter of carbon dioxide with exposure to 100% CO at a filling connected to a 22G needle was inserted into the caudal rate of 20% cv/min. The tumours from Groups B and E vein to establish the channel for intravenous injection. and the control were harvested. The tumour tissues were High-resolution two-dimensional (2-D) DUS was per- minced into small pieces and homogenized. Then, the formed with the same VINNO70 system and the X4-12 L homogenates were centrifuged to obtain the supernatant transducer to find the maximal dimension of the tumour for enzyme-linked immunosorbent assays (ELISAs). section (Fig. 1). Then, a standard CEUS was conducted The contents of eNOS, PGE2, PGD2, PGF2, PGI2, C3a, staying on the section using low MI contrast mode and C5a, LTC4 and TNF-α in tumour tissues were deter- an intravenous bolus injection of 0.15 mL SonoVue®. Ten mined by the Rat eNOS-3 ELISA Kit, Rat PGE2 ELISA minutes after the CEUS study, the hand-held transducer Kit, Rat PGD2 ELISA Kit, Rat PGF2α ELISA Kit, Rat was placed in contact with the tumour surface but sep- PGI2 ELISA Kit, Rat C3a ELISA Kit, Rat C5a ELISA Kit, arated with a 2-cm-thick gel pad while the Vflash treat - Rat LTC4 ELISA Kit and Rat TNF-α ELISA Kit, respec- ment was turned on for 10 minutes. The parameters for tively (MEIMIAN Industrial Co., Ltd., Jiangsu, China). USMB treatment were different in each group (Table 1). The absorbance optical density (OD) of each well was During the USMB treatment, 0.4 mL of SonoVue® sus- measured at 450 nm. The levels of ATP, NO and ROS in pension was slowly and constantly injected into the cau- tumour tissues were determined by an ATP assay kit, NO dal vein during the treatment. After treatment, CEUS assay kit and reactive oxygen species assay kit (Nanjing performance was repeated twice on the same 2-D sec- Jiancheng Bioengineering Institute, China), and the OD tion, immediately and 4 hours later (Fig. 1B). The con - values were measured by Microplate Reader according to trol group received only sham US exposure without MB the instructions. injection. For the Dox study, the experimental animals were Dox concentration treated with PNP of 0.26 MPa and PRF of 1.0 kHz combi- For the quantification of the Dox concentration, the nation based on previous results of the best tumour per- rats in the treated group (n = 14) and the control group fusion enhancement, while the control received sham US (n = 11) were sacrificed 40 minutes after Dox infusion. exposure. Three hours and 20 minutes after treatment, Approximately half of the tumour bulk tissues were 10 mg/kg Dox solution (Meilun, Dalian, China) was taken, and the Dox content was determined by HPLC. injected through the tail vein (Fig. 1C). Another half of the tumour sample was frozen and sliced, the nuclei were stained with DAPI, and the sec- Tumour perfusion quantitation tions were examined under a fluorescence microscope The dynamic video clips of CEUS before treatment, (Nikon Eclipse C1, Nikon, Japan). Dox can spontaneously immediately after treatment and 4 hours after treat- emit red light, while the nuclei appeared blue under UV ment were analysed by the perfusion parametric imag- excitation. ing software of the machine. After manual drawing of the tumour borderline, the machine could automatically Histological examination generate a time-intensity curve (TIC) of tumour contrast One tumour sample from the treated group or the con- intensity, including the peak intensity (PI) and area under trol was stained with haematoxylin and eosin (H&E) for curve (AUC) data. The PI is the peak value of the TIC, morphological observation. Under a light microscope, and the AUC is integrated by the area under the TIC tumour cells are surrounded by connective tissue in a within 60 s starting from TIC elevation. disordered arrangement. For the calculation of the tumour perfusion area rate, the images of the largest tumour contrast perfusion area Statistical analysis in the clip were intercepted. Then, the tumour perfu - SPSS 25.0 software was used for statistical analysis. Mul- sion area was manually delineated using Adobe Photo- tifactor repeated-measures ANOVA was used to deter- shop CC (Adobe), and the rate of tumour perfusion area mine the influence of different groups on the blood was calculated by the perfusion area/entire tumour area perfusion of Walker-256 tumours at different time points ×100%. The increment of the tumour perfusion area rate for the PI, AUC and tumour perfusion area of CEUS. If was calculated by the percentage of perfusion rate after there was an interaction, it was necessary to test the sep- treatment minus the percentage of perfusion rate before arate effects, and the Bonferroni method was used for treatment. pairwise comparison. The contents of ATP, eNOS, PGF2, PGI2, LTC4, TNF-α and ROS in tumour tissues were Vasodilators and inflammatory factors analysed by one-way ANOVA with a completely ran- Immediately after the experimental procedures, 56 ani- dom design, and the LSD method was used for further mals in the perfusion study were sacrificed by inhalation comparison between groups. The variance of NO, PGE2, Tang et al. Cancer Imaging (2023) 23:29 Page 5 of 11 Table 2 PI and AUC values of CEUS before and after treatment ( ) x ¯ ± s Group PI (dB) AUC (dB•s) Pre-treatment Post-treatment 4 h later Pre-treatment Post-treatment 4 h later b ab Control 127.1 ± 8.4 131.2 ± 12.3 106.9 ± 14.2 7262.6 ± 505.5 7543.0 ± 740.3 6094.4 ± 779.4 A 114.4 ± 11.6 119.0 ± 8.0 119.4 ± 23.3 6481.5 ± 646.3 6633.8 ± 439.8 6811.7 ± 1260.8 a ac a ac B 120.9 ± 14.1 136.5 ± 23.3 145.0 ± 14.1 6892.3 ± 817.1 7690.6 ± 1280.8 8131.1 ± 858.5 C 124.7 ± 22.4 132.9 ± 19.7 125.2 ± 19.2 7050.0 ± 1256.6 7508.1 ± 1105.7 6972.5 ± 998.8 D 113.2 ± 11.2 118.7 ± 16.3 117.2 ± 14.6 6384.1 ± 703.2 6556.5 ± 669.2 6446.1 ± 810.2 a a E 119.7 ± 17.0 127.6 ± 15.1 119.0 ± 14.8 6730.4 ± 1049.6 7178.7 ± 899.9 6743.5 ± 807.9 F 118.1 ± 6.3 124.7 ± 9.6 118.4 ± 10.6 6632.6 ± 427.4 6805.3 ± 652.6 6665.1 ± 617.6 a b Compared with the same group before treatment, P < 0.05; compared with the same group immediately after treatment, P < 0.05; compared with the control at the same time, P < 0.05 Table 3 The percentages of perfusion area before and after treatment ( ) x ¯ ± s Groups Pre-treatment(%) Post-treatment(%) 4 h later(%) Control 67.12 ± 10.07 71.18 ± 9.76 50.42 ± 14.89 Group A 60.88 ± 15.46 66.91 ± 13.91 63.98 ± 22.82 a ac Group B 69.94 ± 15.67 81.42 ± 18.26 91.36 ± 10.59 Group C 67.41 ± 12.84 75.82 ± 13.05 73.50 ± 17.16 Group D 58.08 ± 17.39 62.65 ± 19.12 56.79 ± 20.30 Group E 59.22 ± 14.10 69.75 ± 18.46 65.94 ± 17.81 Group F 68.94 ± 15.48 76.34 ± 13.36 65.51 ± 19.21 a b Compared with the same group before treatment, P < 0.05; compared with the same group immediately after treatment, P < 0.05; compared with the control at the same time, P < 0.05 PGD2, C3a and C5a in tumour tissues was uneven. The Dox concentration independent sample Kruskal‒Wallis rank sum test was Four hours after treatment, we applied HPLC and fluo - used, and Bonferroni correction was used for further rescence microscopy to evaluate the Dox concentration comparison between groups. The concentration of Dox within the tumour tissues. HPLC showed that the Dox in tumour tissues was determined by an independent concentration in the control group was 1152.71 ± 369.83 sample T test. A p value less than 0.05 was considered ng/g and that in the treated group (PNP 0.26 MPa, PRF statistically significant. 1.0 kHz) was 3246.59 ± 1301.85 ng/g. The Dox concentra - tion in the treated tumours was 2.82-fold higher than that Results of the control (Fig. 4D). Fluorescence microscopy showed Tumour blood perfusion that Dox fluorescence intensity in the treated group (PNP The results showed that the sononeoperfusion effects of 0.26 MPa, PRF 1.0 kHz) was significantly higher with a Groups B and E (PRF 1.0 kHz) were significant immedi - wider distribution compared with that of the control ately after treatment (P < 0.05) (Tables 2 and 3). Immedi- (Fig. 4A). The average Dox fluorescence intensity of the ately after treatment, the PI increased by an average of treated group was 3.12-fold greater than that of the con- 12.39% in Group B and an average of 7.17% in Group E trol (Fig. 4B). (P < 0.05). The AUC increased by an average of 11.11% in Group B and an average of 7.34% in Group E (P < 0.05). Cytokine detection and histological examination The average incremental perfusion area rate was 11.84% ELISAs showed that cytokines, including vasodilators in Group B and 10.53% in Group E (P < 0.05). However, and inflammatory factors, increased 4 hours after treat - for the other groups (A, C, D, F and the control), the PI ment. ATP, eNOS, NO, PGF2, PGI2, C5a, LTC4, TNF-α elevations ranged from 3.12 to 7.28%, the AUC from 2.81 and ROS in Group B (PNP 0.26 MPa, PRF 1.0 kHz) were to 7.33%, and the incremental perfusion area from 4.06 higher than those of the control (P < 0.05). There were no to 8.41%, and none of there were significant ( P > 0.05). significant changes in PGD2, PGE2, or C3a among the Four hours after treatment, the effect was further groups (P > 0.05) (Fig. 5). enhanced in Group B (PNP 0.26 MPa) with an increase Light microscopy revealed that microvascular hyper- of 20.29% in PI, 18.22% in AUC and 21.42% in incre- aemia and inflammatory cell infiltration were obvious mental perfusion area when compared with the baseline (Fig. 4C) 4 hours after treatment in the treated tumours (P < 0.05). There was no significant difference in Group E (PNP 0.26 MPa, PRF 1.0 kHz) (P < 0.05), while there was (PNP = 0.32 MPa) and the other groups 4 h later (P > 0.05) no significant difference in the control ( P > 0.05). (Tables 2 and 3) (Figs. 2 and 3). Tang et al. Cancer Imaging (2023) 23:29 Page 6 of 11 Fig. 2 B-Mode and CEUS images of tumours in the five groups. Compared with Pre-treatment and Post-treatment, tumour perfusion increased signifi - cantly in Groups B and E and further increased after 4 hours in Group B. No significant perfusion change was found in the other groups Discussion the Vflash mode can regulate cavitation by changing the In this study, we comprehensively investigated the proper MI, PL, PRF and destruction/replenishment time . acoustic parameters, duration time, perfusion area, Dox Additionally, different from some previous studies using delivery, cytokines and related pathological changes B-mode  or conventional flash mode [ 27], the Vflash associated with the sononeoperfusion effect. For the mode is not only able to provide sufficient microbubbles first time, we proposed the new term “sononeoperfu - as cavitation nuclei during replenishment but can also sion” to represent this tumour perfusion enhancement weakly focus the cavitation activities to a designated ROI effect induced by DUS and microbubbles. The effect had and regulate the cavitation intensity for microbubble previously appeared in immunotherapy of MC38 colon vibration or destruction. All US emissions were confined cancer and chemotherapy of PANC-1 pancreatic cancer to FDA and IEC guidelines. in mice [20, 21]. This study further explored the effect Second, the sononeoperfusion effect was remarkable from many aspects mentioned above to obtain a better and repeatable under proper USMB treatment, i.e., only understanding. in Groups B and E. The best tumour perfusion improve - First, therapeutic US was generated from a modi- ment was observed in Group B (PNP 0.26 MPa and PRF fied DUS system (VINNO 70) as previously described 1.0 kHz) with a 20.29% increase in PI, an 18.22% increase . The system was modified with a new Vflash mode in AUC and a 21.42% increment in the perfusion area rate based on its conventional flash mode, that is, a micro - (Tables 2 and 3). This effect lasted for 4 hours. Previous bubble destruction mode during CEUS. The acoustic studies determined that sonoporation had a significant emission can be weakly focused to a ROI by using elec- therapeutic effect when using a long PL, specifically 40-µs tronic phased-focus technology, unlike the small and pulses [28, 29]. To explore the variations in PNP and PRF, strong focus of high-intensity focused US. Furthermore, we selected a burst of 10.5 cycles as the PL within the Tang et al. Cancer Imaging (2023) 23:29 Page 7 of 11 Fig. 3 A: The percentage of tumour perfusion increased 4 hours after treatment compared with pre-treatment. In Group B, the incremental perfusion area was 21.42%. **P < 0.01. B, C, D: The variations in the perfusion area rate and the PI and AUC values of tumours in the control group and Groups B and E. *P < 0.05, **P < 0.01 limitation of regulation instead of 1–2 cycles of conven- low PNP means less cavitation bioeffects or less risk in tional DUS. Obviously, 1.0 kHz was the best PRF in this clinical translation. study to stimulate tumour perfusion (Fig. 2), and 50 Hz Third, US-mediated drug delivery has been well docu - and 2.0 kHz might be either too low or too high in acous- mented in many studies . The USMB treatment com - tic intensity, thereby failing to induce the effect. PNP is bination of 0.26 MPa and 1.0 kHz demonstrated not only regarded as the most related parameter in cavitation , the best perfusion effect but also resulted in good Dox and a low PNP amplitude of 0.26 MPa was preferable to delivery. HPLC and fluorescence microscopy showed that acquire the effect (Tables 2 and 3). The 0.32 MPa PNP the Dox concentration of the treated tumours was up to seemed to be less effective. The incremental percent - 3.12-fold higher than that of the control (Fig. 4B and D). age of the tumour perfusion area rate, which might be This means that the simple combination of usual DUS, the most convincing evidence of the sononeoperfusion intravenous administration of Dox and SonoVue® micro- effect, was 11.48% immediately after 0.26 MPa USMB bubbles may provide a convenient way to gain a better treatment and continually rose to 21.42% four hours later. chemotherapeutic effect, as in a clinical pancreatic can - For 0.32 MPa USMB, the increment was 10.53% imme- cer study . Previous studies have always attributed diately after but dropped to 6.72% four hours later. This USMB-enhanced drug delivery to sonoporation [28, result indicated that the effect required low PNP under 33], a process in which US activates microbubbles and 0.3 MPa and a proper PRF of 1.0 kHz. Stable cavitation increases the permeability of biological barriers . usually dominates under 0.4 MPa PNP . Therefore, However, the sononeoperfusion effect might be another the sononeoperfusion effect was likely to be linked with effect existing in USMB-enhanced drug delivery, which microbubble stable cavitation. These results were con - has been ignored by other related studies. We use the sistent with some previous studies on US drug delivery designation USMBs here instead of ultrasound-targeted using low pressure below 0.4 MPa . It is obvious that Tang et al. Cancer Imaging (2023) 23:29 Page 8 of 11 Fig. 4 A, The Dox concentration in the control and treated groups was observed under a fluorescence microscope 4 hours after treatment. B, The mean fluorescence intensity of Dox was significantly higher in the treated group than in the control group. C, Four hours after treatment, HE sections showed microvascular hyperaemia (white arrow) and inflammatory cell infiltration (yellow arrow) in the treated tumours but not in the control tumours. D, The Dox concentration was significantly higher in the treated group than in the control by HPLC. * P < 0.05 microbubble destruction (UTMD), a term strongly con- permeability through the release of cytokines. ELISAs nected to inertial cavitation. showed that cytokines were significantly increased, such It is well known that solid tumours always develop as vasodilators, ATP, eNOS, NO, PGF2, and PGI2, as hypoperfused and hypoxic areas, resulting in chemo- well as inflammatory factors, including C5a, LTC4, and therapy, radiotherapy and immunotherapy resistance. TNF-α (Fig. 5). Light microscopic manifestation also This hypoxic area lies between the perfused tumour and supported the inflammatory response in that microvas - necrotic tumour [35, 36]. We consider that the sononeo- cular hyperaemia and inflammatory cell infiltration were perfusion effect might stimulate and recover the blood observed in the USMB-treated tumour (Fig. 4C). Since perfusion of the area, thus increasing the tumour perfu- the USMB at a stable cavitation level can only produce sion area and improving drug delivery. minor mechanical injury to the vessel wall, it cannot Finally, we tried to explain the mechanism of sononeo- cause significant changes in microscopic tumour mor - perfusion by detecting related cytokines within tumour phology. Furthermore, ROS, which are oxygen-contain- tissues. The mechanical effect of stable cavitation under ing molecules with high reactivity, can reduce multidrug 0.3 MPa  may release microstreaming and shear resistance and initiate oxidative stress-induced tumour force. These mechanical effects permeabilize the vascu - cell death . ROS were overproduced 4 hours after lar wall, called sonoporation, but they also cause slight USMB treatment. injury to the wall. The injury could trigger an inflamma - Sonoreperfusion effects have been discovered in skel - tory response and the repair process. The inflammatory etal muscle in recent years and may be a promising response triggers vasodilation and an increase in vascular solution for peripheral vascular diseases or muscular Tang et al. Cancer Imaging (2023) 23:29 Page 9 of 11 Fig. 5 A-L, The contents of ATP, eNOs, NO, PGF2, PGI2, PGD2, PGE2, C5a, C3a, LTC4, ROS and TNF-α in tumour tissues of the control, Group B and Group E 4 hours after treatment. *P < 0.05, **P < 0.01 Tang et al. Cancer Imaging (2023) 23:29 Page 10 of 11 ischaemia . Since sonoreperfusion can only be stimu- Supplementary Information The online version contains supplementary material available at https://doi. lated under microbubble inertial cavitation with a high org/10.1186/s40644-023-00545-y. PNP of 0.9–1.7 MPa , the sononeoperfusion effect is likely to be induced only under stable cavitation with Supplementary Material 1 PNP ranging from 0.26 to 0.32 MPa. The noninvasive Supplementary Material 2 sononeoperfusion effect, operating within the diagnostic intensity, might be a novel physical method to overcome Acknowledgements hypoperfused or hypoxic conditions of solid tumours Not applicable. that are confirmed to have therapeutic resistance. Authors’ contributions Another possible application of sononeoperfusion would Najiao Tang: Validation, Formal analysis, Investigation, Resources, Data be a quick prediction of therapeutic response once it is Curation, Visualization. confirmed to be connected with hypoxic tumours. The Jiawei Tang: Conceptualization, Methodology, Validation. Junhui Tang: Software, Visualization. only potential risk of this effect would be tumour metas - Qiong Zhu: Methodology, Investigation. tasis, and the risk has been proven negative in our pre- Xiaoixiao Dong: Investigation. vious study . The effect might have existed in many Yi Zhang: Investigation, Data Curation, Writing-Original Draft, Writing-Review & Editing, Visualization. previous related studies but was neglected [26, 32]. Ningshan Li: Investigation. This is a preliminary experimental study. We did not Zheng Liu: Writing-Review & Editing, Supervision, Project administration, test more parameters or measure the cavitation magni- Funding acquisition. tude for the sononeoperfusion effect, considering the Funding complexity of cavitation. Proper acoustic parameter Open Access funding enabled and organized by Projekt DEAL combinations, including the microbubble concentra- This work was supported by the National Natural Science Foundation of China (Nos. 82127804, 82102075, and 82102077), the National Key Research and tion, may greatly influence the effect. This study did not Development Program of China (No. 2017YFC0107300), and the Chongqing prove the improvement in the hypoxic microenviron- Talent Project and Chongqing Chief Expert Program in Medicine. ment of solid tumours. In addition, the mechanistic study Data availability of the sononeoperfusion effect was only limited to the All data generated or analysed during this study are included in this published inflammatory response. Further signalling molecules article. and pathways related to the effect should be taken into consideration. Declarations Ethics approval and consent to participate Conclusion All of the animal experimental procedures were approved by the Institutional In this work, we demonstrated that modified DUS com - Animal Care and Use Committee of Army Medical University. bined with microbubbles enhances blood perfusion of Consent for publication rat Walker-256 tumours, which was named the sononeo- Not applicable. perfusion effect, thus promoting chemotherapeutic drug (Dox) delivery by up to 3.12-fold. This study also demon - Competing interests The authors declare that they have no competing interests. strated that the sononeoperfusion effect might be related to the inflammatory response by the release of vasodila - Received: 3 September 2022 / Accepted: 12 March 2023 tors and inflammatory factors. Abbreviations AUC area under curve CEUS contrast-enhanced ultrasound Dox doxorubicin References DUS diagnostic ultrasound 1. Denekamp J, Hobson B. Endothelial-cell proliferation in experimental ELISA immunosorbent assay tumours. Br J Cancer. 1982;46(5):711–20. H&E haematoxylin and eosin 2. Tannock IF, Hayashi S. The proliferation of capillary endothelial cells. Cancer HPLC high-performance liquid chromatography Res. 1972;32(1):77–82. MI mechanic index 3. Less JR, Skalak TC, Sevick EM, Jain RK. Microvascular architecture in a mam- OD optical density mary carcinoma: branching patterns and vessel dimensions. Cancer Res. PI peak intensity 1991;51(1):265–73. PL pulse length 4. Brown JM, Giaccia AJ. The unique physiology of solid tumors: opportunities PNP peak negative pressure (and problems) for cancer therapy. Cancer Res. 1998;58(7):1408–16. RF pulse repetition frequency 5. Intaglietta M, Myers RR, Gross JF, Reinhold HS. Dynamics of microvascular ROI region of interest flow in implanted mouse mammary tumours.Bibl Anat, 1977(15 Pt 1):273–6. SD Sprague‒Dawley 6. Chaplin DJ, Olive PL, Durand RE. Intermittent blood flow in a murine tumor: TIC time-intensity curve radiobiological effects. Cancer Res. 1987;47(2):597–601. US ultrasound USMB ultrasound stimulated microbubble UTMD ultrasound-targeted microbubble destruction Tang et al. Cancer Imaging (2023) 23:29 Page 11 of 11 7. Chaplin DJ, Trotter MJ, Durand RE, Olive PL, Minchinton AI. Evidence for inter- 24. Zhang Y, et al. Eec ff t of diagnostic ultrasound and microbubble- mittent radiobiological hypoxia in experimental tumour systems. Biomed enhanced chemotherapy on metastasis of rabbit VX2 tumor. Med Phys. Biochim Acta. 1989;48(2–3):255–9. 2021;48(7):3927–35. 8. Dewhirst MW, Braun RD, Lanzen JL. Temporal changes in PO2 of R3230AC 25. Sidhu PS, et al. The EFSUMB Guidelines and Recommendations for the clinical tumors in Fischer-344 rats. Int J Radiat Oncol Biol Phys. 1998;42(4):723–6. practice of contrast-enhanced Ultrasound (CEUS) in non-hepatic applica- 9. Padera TP, et al. Pathology: cancer cells compress intratumour vessels. Nature. tions: Update 2017 (Long Version). Ultraschall Med. 2018;39(2):e2–e44. 2004;427(6976):695. 26. Kotopoulis S, Dimcevski G, Gilja OH, Hoem D, Postema M. Treatment of 10. Minchinton AI, Tannock IF. Drug penetration in solid tumours. Nat Rev Cancer. human pancreatic cancer using combined ultrasound, microbubbles, and 2006;6(8):583–92. gemcitabine: a clinical case study. Med Phys. 2013;40(7):072902. 11. Pereira M, Matuszewska K, Jamieson C, Petrik J. Characterizing endocrine 27. Eisenbrey JR, et al. US-triggered Microbubble Destruction for Augmenting status, Tumor Hypoxia and Immunogenicity for Therapy Success in Epithelial Hepatocellular Carcinoma response to Transarterial Radioembolization: a Ovarian Cancer. Front Endocrinol (Lausanne). 2021;12:772349. Randomized Pilot Clinical Trial. Radiology. 2021;298(2):450–7. 12. Ryan DP, Hong TS, Bardeesy N. Pancreatic adenocarcinoma. N Engl J Med. 28. Kotopoulis S, et al. Sonoporation-enhanced chemotherapy significantly 2014;371(11):1039–49. reduces primary tumour burden in an orthotopic pancreatic cancer xeno- 13. Grkovski M, et al. Multiparametric imaging of Tumor Hypoxia and Perfusion graft. Mol Imaging Biol. 2014;16(1):53–62. with (18)F-Fluoromisonidazole dynamic PET in Head and Neck Cancer. J Nucl 29. Delalande A, Kotopoulis S, Postema M, Midoux P, Pichon C. Sonoporation: Med. 2017;58(7):1072–80. mechanistic insights and ongoing challenges for gene transfer. Gene. 14. Primeau AJ, Rendon A, Hedley D, Lilge L, Tannock IF. The distribution of the 2013;525(2):191–9. anticancer drug Doxorubicin in relation to blood vessels in solid tumors. Clin 30. Ferrara K, Pollard R, Borden M. Ultrasound microbubble contrast agents: Cancer Res. 2005;11(24 Pt 1):8782–8. fundamentals and application to gene and drug delivery. Annu Rev Biomed 15. Dewhirst MW, Navia IC, Brizel DM, Willett C, Secomb TW. Multiple etiologies Eng. 2007;9:415–47. of tumor hypoxia require multifaceted solutions. Clin Cancer Res. 2007;13(2 31. Lammertink BH, et al. Sonochemotherapy: from bench to bedside. Front Pt 1):375–7. Pharmacol. 2015;6:138. 16. Xie F, et al. Treatment of acute intravascular thrombi with diagnostic 32. Dimcevski G, et al. A human clinical trial using ultrasound and microbubbles ultrasound and intravenous microbubbles. JACC Cardiovasc Imaging. to enhance gemcitabine treatment of inoperable pancreatic cancer. J Control 2009;2(4):511–8. Release. 2016;243:172–81. 17. Istvanic F, et al. Sonoreperfusion therapy for microvascular obstruction: a step 33. Lentacker I, De Cock I, Deckers R, De Smedt SC, Moonen CT. Understanding toward clinical translation. Ultrasound Med Biol. 2020;46(3):712–20. ultrasound induced sonoporation: definitions and underlying mechanisms. 18. Belcik JT et al. Augmentation of limb perfusion and reversal of tissue ischemia Adv Drug Deliv Rev. 2014;72:49–64. produced by ultrasound-mediated microbubble cavitation.Circ Cardiovasc 34. Bouakaz A, Zeghimi A, Doinikov AA. Sonoporation: Concept and Mecha- Imaging, 2015. 8(4). nisms. Adv Exp Med Biol. 2016;880:175–89. 19. Belcik JT, et al. Augmentation of muscle blood Flow by Ultrasound 35. Jing X, et al. Role of hypoxia in cancer therapy by regulating the tumor micro- Cavitation is mediated by ATP and Purinergic Signaling. Circulation. environment. Mol Cancer. 2019;18(1):157. 2017;135(13):1240–52. 36. Graham K, Unger E. Overcoming tumor hypoxia as a barrier to radiotherapy, 20. Feng S, et al. Chemotherapy augmentation using low-intensity Ultrasound chemotherapy and immunotherapy in cancer treatment. Int J Nanomedicine. Combined with Microbubbles with different mechanical indexes in a pancre - 2018;13:6049–58. atic Cancer model. Ultrasound Med Biol. 2021;47(11):3221–30. 37. Perillo B, et al. ROS in cancer therapy: the bright side of the moon. Exp Mol 21. Li N, et al. Tumor perfusion enhancement by ultrasound stimulated micro- Med. 2020;52(2):192–203. bubbles potentiates PD-L1 blockade of MC38 colon cancer in mice. Cancer Lett. 2021;498:121–9. 22. Viallard C, Larrivée B. Tumor angiogenesis and vascular normalization: alterna- Publisher’s Note tive therapeutic targets. Angiogenesis. 2017;20(4):409–26. Springer Nature remains neutral with regard to jurisdictional claims in 23. Liu Z, et al. Disruption of tumor neovasculature by microbubble enhanced published maps and institutional affiliations. ultrasound: a potential new physical therapy of anti-angiogenesis. Ultra- sound Med Biol. 2012;38(2):253–61.
Cancer Imaging – Springer Journals
Published: Mar 23, 2023
Keywords: Sononeoperfusion; Tumour; Diagnostic ultrasound; Microbubble; Doxorubicin
Access the full text.
Sign up today, get DeepDyve free for 14 days.