| PART I Parameters optimization of breast cancer xenografts in nude mice inhibition by exposure to low frequency ultrasoundObjective To explore the suitable parameters of breast cancer xenografts in nude mice treatment by exposure to low-frequency ultrasound.Methods The model of breast cancer was established by injection human breast cancer cells into the fat tissue under the right second breast of nude mouse. A total of 30 nude mice with breast cancer xenografts were randomly divided into 6 groups. Group 1, control group, was undergone empty exposure with ultrasound intensity 0 W/cm2 and duty cycle 0. Groupl～6 were exposure groups, and the ultrasound intensity and duty cycle were 0.5 W/cm2 and 20%,1 W/cm2 and 20%,1 W/cm2 and 50%,2 W/cm2 and 50%,2 W/cm2 and 100%, respectively. All the mice of exposure groups were undergone exposure by the scheduled parameters. After exposure or empty exposure 5min, the survival conditions and the tumor growth of all the mice were observed for 10 days. After the observing duration, the mice were sacrificed and the tumors were obtained. The histological changes of the tumors were analyzed with H-E staining. The apoptosis of tumor cells were detected with TUNEL staining and apoptotic indexes (AIs) were calculated.Results①After exposure or empty exposure, all the mice were survived except one of group 6. And there were no histological differences with H-E staining between the six groups’mice.②Compared pre-scarify with pre-exposure, the tumor volume of each group were significantly increased in some degree (P< 0.01). Before scarify, there were no significant difference in the tumor volumes of groupl-4 [(2.134± 0.248) cm3, (2.114±0.277) cm3, (cm3 2.063±0.192) cm3, (2.016±0.231) cm3] (P> 0.05), and there were also no significant difference in the tumor volumes of group 5 and group 6 [(1.704±0.209) cm3, (1.670±0.095) cm3] (P> 0.05), but the latter was significant less than the former(P< 0.01). ③The apoptosis of tumor cells was increased with rising of the exposure dose. The AI of the group 5 and group 6 [(17.23 ±0.59 highest)%, (17.92±0.67)%] were significantly hither than that of groupl-4 [(4.74±0.34)%, (7.03±0.69)%, (9.75±0.41)%, (11.83±0.66)%] (P< 0.01), but there no significant difference between the group 5 and group 6 (P> 0.05).Conclusion The parameter combination of ultrasound intensity 2 W/cm2, cycle duty 50% and exposure 5min is the suitable and safe for 1MHz low-frequency ultrasound exposure treatment mice with transplanting human breast cancer.PART II Microbubbles enhancement low-frequency ultrasound cavitation effect on inhibition of breast cancer xenografts in nude miceObjective To observe the curative effect of breast cancer xenografts in nude mice by cavitation of low-frequency ultrasound enhanced by microbubbles, and to explore the possible mechanism.Methods A total of 20 nude mice with breast cancer xenografts were randomly divided into 4 groups, control group, microbbubles(Mbs) group, ultrasound(US) group and US+Mbs group (n=5, each). US+Mbs group:during 0.2 ml microbubbles of SonoVue was slowly injected into the mice via the tail vein, the breast cancer xenografts were undergone exposure by 1MHz low-frequency ultrasound with the intensity of 2 W/cm and the duty cycle of 50%. US group:during 0.2 ml NS was slowly injected into the mice via the tail vein, the breast cancer xenografts were undergone exposure as the same as US+Mbs group. Mbs group:during 0.2 ml microbubbles of SonoVue was slowly injected into the mice via the tail vein, the breast cancer xenografts were undergone empty exposure. Control group:during 0.2 ml NS was slowly injected into the mice via the tail vein, the breast cancer xenografts were also undergone empty exposure. All the mice were undergone exposure or empty exposure one time every other day, a total of three times. After all the 3 times treatment, the mice were continually felled 6 days. The size of the breast cancer xenografts were measured by vernier caliper 6 times at each pre-treatment and every other day after the last treatment, and the volume of the breast cancer xenografts and the inhibition rate of tumor (IRT) of treatment groups were calculated. After the last measurement, the mice were sacrificed and the breast cancer xenografts were obtained. The histological changes, the apoptosis of cells and the microvascular changes of breast cancer xenograft were analyzed, and AIs and microvascular density (MDV) were calculated.Results ①The volume of breast cancer xenograft in US+Mbs group were inhibited dramatically. The volume of breast cancer xenograft of US+Mbs group [(0.864±0.493)cm3] was significantly less than that of US group [(1.666±0.131)cm3], Mbs group [(2.067±0.281)cm3] and control group [(1.841±0.248)cm3](P<0.01). However, the IRT of US+Mbs group (58.25%) was significantly larger than that of US group (19.45%) and Mbs group (10.98%) (P<0.01).②There were no significant difference in histological changes among the four groups. However, the AI of US+Mbs group [(49.02±2.85)%] was significantly larger than that of US group [(11.04±0.34)%], Mbs group [(6.15±0.29)%] and control group [(4.68±0.22)%](P< 0.01). And the MDV of US+Mbs group (3.75±0.36) was significantly less than that of US group (6.59±0.24), Mbs group (10.48±0.44) and control group (10.93±0.37) (P <0.01).Conclusion Exposure of 1 MHz low frequency ultrasound combined with microbubbles can effectively inhibit the growth of breast cancer xenografts in nude mice. The possible mechanism of this inhibition is direct destruction of neovascularization in the tumors by ultrasonic cavitation, and which lead to the apoptosis of the tumor cells. The histological changes, the apoptosis of cells and the microvascular changes of breast cancer xenograft were analyzed, and AIs and microvascular density (MDV) were calculated.PART ⅢMultimodal ultrasound evaluation of curative effect of breast cancer xenografts in nude mice exposure to low-frequency ultrasound with microbubblesObjective To explore the assessment effect of multi-mode ultrasound (including 2DUS, CDFI, RTE,VTQ, ABVS and CEUS) in evaluation of curative effect of breast cancer xenografts in nude mice exposure to low-frequency ultrasound with microbubble.Methods A total of 20 nude mice with breast cancer xenografts were randomly divided into 2 groups, control group and treatment group (n=10, each).The treatment group and the control group were processed as the same as the US+Mbs group and control group in the Part Ⅱ respectively. After all the times treatment finished, the mice were continually felled 6 days. Then, the mice were scarified and the samples of the breast cancer xenografts were obtained. All the mice were underwent multi-mode ultrasound examination two times at pre-treatment and pre-sacrifice. The size of breast cancer xenografts were measured by these two modes and the volume of breast cancer xenografts were calculated according to the measurement of the two modes. According to the real-time elastosonography, the stiffness of the breast cancer xenografts were divided into 1-5 score from low to high. The transverse shear wave velocity (SWV) of the breast cancer xenografts were measured by VTQ. According to the color images of CDFI, the blood flow of the breast cancer xenografts were divided into 0～4 grade from less to more. The blood perfusion of the breast cancer xenografts were observed by real-time CEUS images, and the parameters of intensity of peak (Peak), time to intensity of peak (Tp), area under the curves (AUC) and mean time of transit (MTT) were analyzed from the time-intensity curves of CEUS. For the samples, the size of the breast cancer xenografts were measured by vernier caliper, and the volume of breast cancer xenografts were calculated. The histological changes, the apoptosis of cells and the microvascular changes of breast cancer xenograft were analyzed, and the AIs and the MDVs were calculated. The orphological characteristics of breast cancer xenografts were observed by 2DUS and ABVS. All the parameters detected at pre-sacrifice were compared with that detected at pre-treatment, and the parameters detected at pre-sacrifice in treatment group were compared with that in control group. Moreover, the volumes according to the measurement of 2DUS, ABVS and the vernier caliper were compared with each other.Results ①There were no significant difference in histological changes between the treatment group and the control group, but the AI of treatment group [(48.71±2.38)%] was dramatically higher than that of control group [(4.77±0.26)%] (P<0.01), and the MDV of treatment group (3.82±0.31) was significantly lower than that of control group(10.59±0.64) (P<0.01). ②The growth of breast cancer xenografts was inhibited obviously in treatment group. The volume of the treatment group [(0.507±0.095)cm3] was significantly smaller than that of the control group [(0.802±0.109)cm3] (P<0.01), but the differences of the volume of breast cancer xenografts which measured by 2DUS and ABVS pre-sacrifice compared with that of the samples measured by vernier caliper post-sacrifice was not significant (P>0.05). ③Most of the breast cancer xenografts were score 4～5 in real-time elastosonography at pre-treatment and pre-sacrifice in the two groups. There were no significant differences in the real-time elastosonography score among treatment group at pre-sacrifice and pre-treatment and control group at pre-sacrifice (P>0.05). However, SWV detected at pre-sacrifice [(2.08±0.24)m/s] by VTQ in the treatment group was significantly lower than that detected at pre-treatment [(2.61±0.21)m/s] and that detected at pre-sacrifice in the control group [(2.75±0.27)m/s] (P<0.05).④Most of the breast cancer xenografts were grade 0～II in color images of CDFI at pre-treatment and pre-sacrifice in the two groups. There were no significant differences in the blood grade among treatment group at pre-sacrifice and pre-treatment and control group at pre-sacrifice (P>0.05). However, the perfusion of the treatment group at pre-sacrifice was dramatically decreased on the real-time images of CEUS. Analyzing of the time-intensity curves, Peak and AUC of treatment group at pre-sacrifice [(28.70±6.75)%, (1564.20±449.22)%s] was significantly less than that at pre-treatment [(54.20±6.18)%, (2954.00±702.66)%s] and that of control group at pre-sacrifice[(56.70±8.55)%, (2978.80±633.75)%s] (P<0.01, respectively). However, there were no significant difference of Tp and MTT among the treatment group at pre-sacrifice and at pre-treatment and the control group at pre-sacrifice (P> 0.05).Conclusion Multi-mode ultrasound, including 2DUS, ABVS, VTQ, CEUS and its time-intensity curve, can effectively assess the curative effect of breast cancer xenografts in nude mice exposure to low-frequency ultrasound with microbubbles in the aspects of morphology, tissue stiffness and blood perfusion. |
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