Figure 1.
Therapeutic effects of M-HIFU on RM-9 tumors.
A, Ultrasound imaging guided M-HIFU treatment. Cavitation activities induced by M-HIFU within the tumor tissue are shown in regions with hyperechogenicity, indicated by arrows in the B-mode images (upper panel) and in the excised tumors (lower panel. Left: skin on top of the tumor was intact after M-HIFU treatment; middle: removed tumor showing intact exterior surface after M-HIFU treatment; right: bisected tumor showing mechanical lesions in the interior region after M-HIFU treatment. B, Tumor growth curve post rechallenge. 2×104 RM-9 cells were subcutaneously injected into the contralateral hind limb 30 days after M-HIFU treatment when the mice were fully recovered from surgical resection of the primary tumor. Volume of the rechallenged tumors was calculated based on caliper measurements of tumor size at 3-day intervals starting from day 7 following tumor rechallenge. *p<0.05, **p<0.01. C, Cumulative survival curve following tumor rechallenge. Mice reaching the humane endpoint were sacrificed. There were 10 mice in the surgery group and 12 in the HIFU + surgery group. P-value of 0.019 was determined by the log-rank test. Data were obtained from three independent experiments.
Figure 2.
STAT3 is constitutively activated in RM-9 cells and tumors, and the level of activation can be suppressed by M-HIFU.
A, Expression of p-STAT3 in RM-9 cells. RM-9 cells were subjected to IF staining for p-STAT3 and DAPI staining for nuclei. Under fluorescent microscopy, p-STAT3 was marked by red fluorescence while nuclei were labeled by blue signals. The p-STAT3-positive nuclei were indicated by merged pink signals. Solid arrows point to representative p-STAT3-positive nuclei whereas the dashed arrow marks a p-STAT3-negative nucleus. B, Expression pattern of p-STAT3 in vivo. RM-9 tumors were removed at three different developing time points as indicated and subjected to the IHC staining for p-STAT3. After counterstained by hematoxylin, p-STAT3-positive nuclei were indicated by dark brown signals (arrows) whereas p-STAT3-negative nuclei were in blue. C, Decreased p-STAT3 levels in RM-9 tumors after M-HIFU treatment. Two days after M-HIFU treatment, RM-9 tumors were removed and subjected to IHC staining for p-STAT3. Three tumors in each group were analyzed and the representative figures are shown. The IHC was independently performed thrice. Data was presented as Mean ± SD. The student’s t-test was conducted to determine p-values. *p<0.05. D, Expression down-regulation of three STAT3 downstream target genes by the M-HIFU treatment. Two days post M-HIFU treatment, RM-9 tumors was removed and mRNA was isolated. RT-PCR was then performed to detect levels of cyclin D1, Bcl-xL, IL-10 and β-actin. Band signals were quantified using the NIH ImageJ software. Representative images from three independent experiments are shown.
Figure 3.
Increased anti-tumor T cell responses after M-HIFU treatment.
A, 2 days after treatment, spleens from three mice treated with or without M-HIFU were collected and pooled. Proportion of splenic CD8+ T cells were determined by flow cytometry. Representative results from three independent experiments are shown. B, Statistical significance of the increase of the CD8+ cell population by combining HIFU with surgery. *p<0.05 C, Twelve days after surgery or M-HIFU plus surgery treatment, three mice from each group were sacrificed and their spleens were collected and pooled. IFN-γ ELISpot assay was performed with the pooled splenocytes. Mitomycin-C treated RM-9 cells were added as stimulators at the ratio of 1∶10 (RM9:splenocytes). EL-4 cells were used as an irrelevant cell control. Representative results from three independent experiments are shown. D, Enumeration of IFN-γ-positive cells and, and statistical analysis based on the ELISpot assay results. ***p<0.0001.
Figure 4.
Increased DC population after M-HIFU treatment.
Three mice from each group were sacrificed six days after surgery or HIFU plus surgery treatment. Single cell suspension was prepared from the pooled spleens or TDLNs; and CD11c+ cell population was determined by flow cytometry. Representative results from three independent experiments are shown. A, C, Representative flow patterns in detecting CD11c+ cells in spleen (A) and TDLNs (C). B, D, Statistical significance in the increase of the DC population in spleen (B) and TDLNs (D) based on the results of A and C, respectively. E, F, Evaluation of DC stimulation by detecting CD80-positive (E) and CD86-positive (F) CD11c+ cells in spleen and TDLNs. *p<0.05, ***p<0.0001.
Figure 5.
Decreased proportion of Treg after M-HIFU treatment.
Single cell suspension was prepared from the pooled spleens or TDLNs. Population of CD4+ Foxp3+ cells was thereafter determined as Treg by flow cytometry. Figures showed are the representatives from three independent staining and subsequent flow analysis. A, C, Representative flow patterns in detecting Tregs in spleen (A) and TDLNs (C). B, D, Proportion of the Treg population in spleen (B) and TDLN (D) based on the results shown in A and C, respectively. **p<0.01, ***p<0.0001.