High intensity focused ultrasound enhances anti-tumor immunity in melanoma through promoting CD4 Th1 effector T cell responses

Background: Melanoma accounts for more than 80% of deaths from all dermatologic cancers, mainly due to its widespread metastasis. High intensity focused ultrasound (HIFU) is a promising technique for cancer therapy. Here, we investigated the efficacy of HIFU against melanoma and the underlying mechanisms. Methods: A melanoma allograft mouse model was established to examine the tumor progression and survival rate. Anti-tumor immunity was determined by measuring cytokines, regulatory T cells (Tregs), Th17 cells and CD8+ effector T cells. Western blot, qPCR, RNAi and luciferase assay were performed to confirm the expression and regulation of microRNA (miR)-9-5p and transforming growth factor beta (TGF-β). Results: HIFU exposure significantly suppressed melanoma growth and metastasis by activating interferon gamma (IFN-γ) secretion, inhibiting Tregs and Th17 cells, and stimulating CD8+ effector T cells. TGF-β was a direct target of miR-9-5p. The anti-tumor effect of HIFU might be mediated through the miR-9-5p/TGF-β pathway. Conclusion: HIFU activates anti-tumor response and alters tumor microenvironment, which may serve as a potential therapeutic strategy for melanoma treatment.


Introduction
Melanoma develops from the melanocytes and is the most malignant type of skin cancer. Although melanoma accounts for only 4% of skin cancers, it causes more than 80% of deaths from all dermatologic cancers (1,2). In melanoma patients, the major cause of death is widespread metastasis (3). Melanoma can spread through the lymphatic and/or vascular system to the liver, brain, lung, bone, breast, colon and subcutaneous tissue even at the early stages (3,4). Therefore, investigation and the development of effective therapy to inhibit the growth and metastasis have crucial importance in melanoma treatment.
High intensity focused ultrasound (HIFU), also known as focused ultrasound surgery, is a non-invasive therapeutic technique for localized treatment of tumors, which exhibits thermal and mechanical effects: the former one induces cancer cell destruction through coagulation necrosis, while the latter one generates radiation force, cavitation and micro-streaming on tumor tissues (5,6). HIFU has been used to treat various tumors and improve the prognosis of cancer patients (6)(7)(8)(9)(10)(11). Uchida et al. reported that the HIFU therapy improved prostate cancer outcomes, and the 10-year survival rate reached 89.6% among 918 patients (11).Breast cancer is an ideal target for HIFU treatment due to its superficial position.The complete ablation rate of HIFU therapy reached up to 71% in 173 breast cancer patients (10). HIFU treatment also improved the survival outcome of unresectable liver cancer patients, where out of 49 hepatocellular carcinoma (HCC) patients, 39 (79.5%) had primary effectiveness and only 4 (8.1%) had complications (9). In addition, HIFU exposure was also employed for treatment of kidney cancer, and applied as the palliative treatment for pancreatic cancer (7,8). All these reports have indicated that HIFU is a promising strategy for the treatment of various cancers. Although HIFU exposure has been used for the treatment of multiple cancers, the underlying molecular mechanisms are not yet well understood. Cumulative evidence has suggested that cell death and tissue damage during HIFU are closely linked to immune response, which is induced by the cell debris after cancer cell destruction, as well as tumor microenvironment alteration such as the changes of T cell subsets (12)(13)(14). Xia et al. reported the anti-tumor immune responses of HIFU exposure in HCC mouse model, the authors demonstrated that 14 days of treatment could significantly promote the secretion of interferon gamma (IFN-γ) and tumor necrosis factor alpha (TNF-α), enhance the cytotoxicity of cytotoxic T lymphocytes (CTL), and increase the number of CD8+ effector T cells (15). The dendritic cells (DC) were also found activated after HIFU exposure in a colorectal cancer mouse model: there was about 4-fold increase in CD11c+ cells and more than 5-fold CFSE+ DC accumulation in lymph nodes after HIFU exposure, accompanying with enhanced CTL activity and increased IFN-γ production (12). However, the therapeutic effect of HIFU exposure on melanoma treatment and the underlying mechanism is poorly defined.
In the current study, we demonstrated that HIFU exposure could significantly suppress melanoma growth and metastasis through activating the anti-tumor immunity and altering the tumor microenvironment in mouse and cell models. We further revealed that HIFU-induced decrease in regulatory T cells (Tregs) and Th17 cells was regulated by the microRNA (miR)-9-5p/transforming growth factor beta (TGF-β) pathway. Notably, HIFU exposure also increased the number of CD8+ effector T cells in melanoma tissues. This study may pave the way for the clinical application of HIFU in melanoma treatment.

Animals
6-8 weeks old C57BL/6J mice were employed in the current study, which were ordered from Shanghai Laboratory Animal Center (China). The mice were housed under standard breading conditions with 12 light/12 dark cycle, at 23ºC with 40-60% humidity, standard chow diet feeding, with water accessible at all times. All mouse experiments have been approved by the Shanghai Jiaotong University Affiliated Sixth People's Hospital East Campus.

Melanoma xenograft model
200 μL single-cell suspension of B16-F10 melanoma cells (3 × 10 5 ) was implanted subcutaneously into the left flank of mice. Tumor size (width × length; mm 2 ) was determined with a caliper every 2 days. Tumor volume was calculated as previously described (16).

HIFU treatment
All experimental mice were randomly divided into sham-HIFU and HIFU groups when the diameter of larger tumor reached 7 to 8 mm. The HY2900 HIFU tumor therapy system (Haying Tech., Wuxi, China) was used for HIFU ablation in this study. The mice were anaesthetized using ketamine (2 mL/kg) through intravenous injection. After anesthesia, the skin on top of the tumor nodule area was shaved and layed with ultrasound transmission gel. The mice of HIFU group were treated with 4.5 W and 9.2 MHz ultrasound. Treatment was performed point by point, and started at the center of the nodule with 6 mm therapy depth. Each point was treated for 1 min, with 1 sec pulse duration, and 5 sec exposure separation. The procedures of sham-HIFU group were similar to HIFU group with no HIFU exposure.

Pulmonary metastasis assay
The mice bearing primary tumors were intravenously injected with B16-F10 cells at 7 days after HIFU treatment and monitored until death. The volume of melanoma tumor, number of pulmonary metastasis and cumulative survival rate were measured and recorded. The health mice without melanoma xenograft were used as the normal control group in this study.

Histological analysis
The tumor modules were excised from flank skin and fixed in 4% paraformaldehyde overnight and then paraffin embedding, sectioning, and hematoxylin eosin (H&E) staining were performed as previously described (17).

Enzyme-linked immunosorbent assay (ELISA)
The blood was collected from normal, sham-HIFU and HIFU mice after 14 days of treatment, centrifuged at 5000 g for 6 min. The plasma concentrations of IFN-γ, TNF-α, interleukin (IL)-6, and TGF-β were determined by using the Beyotime Biotechnology (Shanghai, China) specific ELISA kits: mouse IFNγ ELISA kit (PI508), mouse TNF-α ELISA kit (PT512), mouse IL-6 ELISA kit (PI326), and mouse/rat TGF-β ELISA kit (PT878), following the PerkinElmer, Singapore). The cell viability of each well was normalized to that of the control. All cytotoxicity assays were conducted in triplicates (six wells per sample for each time point). Viability of the NSCs after treatments of various CM was determined using the MTT assay as well as LIVE/DEAD viability/cytotoxicity kit (Invitrogen, USA).
Immunoblotting analysis B 1 6 -F 1 0 c e l l s o r f r o z e n m e l a n o m a t u m o r tissues were homogenized and lysed using the radioimmunoprecipitation assay buffer (Thermo Fisher Scientific, Waltham, USA). Samples were detected using Western blotting as described previously (18). The primary antibodies of TGF-β (ab92486, 1:1000 dilution) and GAPDH (EPR16891, 1:2000 dilution) were ordered from Abcam (Cambridge, MA, USA).

Luciferase reporter assay
B16-F10 cells were cultured in 12-well plates at the density of 6 × 10 4 cells/per well 24 h before transfection. The TGF-β 3'UTR-WT or TGFβ 3'UTR-mut reporter was co-transfected with pRLSV40 into the B16-F10 cells. Luciferase activity was measured by using the Dual-Luciferase Reporter Assay System (Promega, Madison, USA). The results were normalized by the value of Renilla activity and presented as fold change to the control group.

Statistical analysis
Statistical analysis was performed using Prism 8 software. Student's t test, one-way or two-way analysis of variance (ANOVA) were used to analyze the differences between groups. Data were shown as mean ± standard deviation (SD).

HIFU exposure suppresses melanoma tumor progression
The melanoma tumor bearing mice were administrated with sham-HIFU or HIFU, respectively, H&E staining result showed that the cell proliferation of melanoma in HIFU exposure mice was slowed down compared to that in the sham group mice ( Figure 1A). Notably, HIFU exposure significantly inhibited the primary tumor growth. 22 days after B16-F10 cell subcutaneous injection, tumor volume in mice of the sham-HIFU group were 2-4-fold larger than that in the HIFU-treated mice ( Figure 1B). We further investigated the pulmonary metastasis in mice with primary tumors after B16-F10 cell intravenous injection, as shown in Figure 1C, HIFU exposure markedly reduced the number of tumor nodules in the lung compared to the sham-HIFU treatment (19 vs 34, P < 0.01). Moreover, the cumulative survival rate showed that HIFU exposure mice had much longer survival rate than mice in the sham control group (Figure 1D, P < 0.05). These results indicated that HIFU exposure displayed promising antitumor effects on inhibiting tumor growth, attenuating pulmonary metastasis and improving host survival in mouse melanoma model.

HIFU exposure improves the anti-tumor immune response
Immune response was detected in HIFU and sham-HIFU treated mice by measuring serum concentrations of tumor microenvironment-associated cytokines, including IFN-γ, TNF-α, IL-6 and TGF-β. Pleiotropic molecule IFN-γ has been reported to possess the anti-proliferative, proapoptotic and anti-tumor abilities (19). As shown in Figure 2A, the concentration of IFN-γ in the serum of HIFU exposure mice was 2-fold higher than that in the sham-HIFU mice (68.3 pg/mL vs 32.7 pg/mL). Two proinflammatory cytokines, TNF-α and IL-6, were relatively higher in melanoma mice compared to normal mice. However, there was no difference between HIFU and sham-HIFU mice (Figure 2B and 2C). The regulatory cytokine TGF-β is an important enforcer of immune tolerance, and tumors that secrete high levels of TGF-β may escape from immune surveillance (20). Here, we found that the TGF-β serum level was significantly decreased in HIFU treated mice compared to the sham control mice (Figure 2D). These data suggested the HIFU exposure might promote anti-cancer immunity via modulating cytokine secretion.

HIFU exposure inhibits Tregs and Th17 cells, and activates CD8+ tumor infiltrating lymphocytes (TILs)
To further investigate the anti-tumor immunity improvements after HIFU exposure, we detected the subset of CD4+ and CD8+ T cells using flow cytometric analysis. Tregs (Foxp3+CD4+/CD45+), the immunosuppressive subset of CD4+ T cells, was significantly increased in melanoma mice compared to normal control (1.19% vs 3.23%). Noticeably, in comparison with mice with sham-HIFU treatment, Treg population in HIFUtreated mice was significantly decreased (Figure 3). Th17 cells also increase tumor progression by activating angiogenesis and immunosuppressive activities (21). In line with the results of Tregs, the Th17 cell population (CD4+/IL-17+) in HIFU exposure mice was 2.49%, which was markedly lower than that in sham-HIFU mice (4.47%, Figure 4). CD8+ TILs have critical tumor suppressive roles. As shown in Figure 5, there was almost 3-fold increase in the CD8+ (CD45+/CD8+) population in HIFU exposure mice compared to sham-HIFU mice (17.59% vs 6.18%). All these flow cytometric data indicated that HIFU exposure suppressed the immune tolerance and improved tumoricidal effector response in tumor tissues.

HIFU exposure promotes anti-tumor immunity through attenuating TGF-β expression in melanoma tissues
Since TGF-β plays a very important role in promoting the generation and differentiation of Tregs and Th17 subsets from naïve CD4+ T cells (22). We searched the candidate regulators of TGF-β in StarBase/ENCORI database and identified miR-9-5p, which was inhibited in melanoma tumor tissues and restored by HIFU treatment (Figure 6A). Then we tested the TGF-β expression in mouse melanoma samples with or without HIFU exposure. Both the mRNA and protein levels of TGF-β were significantly increased in melanoma tissues, notably, the up-regulated TGF-β expression was markedly inhibited by HIFU treatment (Figure 6B-6D). These results suggested that the antitumor effect of HIFU exposure might be mediated by the miR-9-5p/TGF-β pathway.

Discussion
HIFU is a promising method for the non-invasive ablation of various tumors (6,13). In this study, we applied the HIFU technique for melanoma treatment and demonstrated that HIFU exposure suppressed the proliferation and growth of the primary tumors, inhibited pulmonary metastasis, and improved the cumulative survival rate of melanoma mice. HIFU treatment not only activates immune response including IFN-γ upregulation and TGF-β down-regulation but also changes T cell subsets in tumor microenvironments, such as suppression of Tregs and Th17 cells and activation of CD8+ effector T cells. In line with our findings, Liu et al. reported that HIFU treatment significantly stimulated DC maturation and tumor infiltration surrounding the thermal lesion compared to the control in the MC-38 and B16 tumor models (23). Moreover, the enhanced cytotoxic T lymphocyte activity and elevated IFN-γ secretion accompanying the expansion of DCs in lymph nodes were observed by another research group who performed HIFU   treatment on colon adenocarcinoma tumors (12). Based on the above findings, the activation of anti-tumor immunity might be one of the major advantages of HIFU treatment. The T cell subset alteration after HIFU exposure is another important beneficial effect of HIFU treatment. CD8+ T cells are able to differentiate into cytotoxic T lymphocytes upon the activation of antigen-presenting cells, which then exert an efficient anti-tumor attack (24). However, Tregs play essential roles in maintaining the immune system self-tolerance and immunosuppression, thus promote tumor development and progression (25). In addition, Th17 cells are a double-edged sword in tumor immunity: on the one hand Th17 cells promote tumor progression through increasing angiogenesis and immunosuppression, while on the other hand Th17 cells induce the anti-tumor immune response by improving effector T cell filtration and IFN-γ secretion (22). In the current study, we found that HIFU exposure significantly reduced the population of Tregs and Th17 cells, while increased CD8+ effector T cells in melanoma tumor tissues. Meanwhile, alteration in other subsets of immune cells has been observed by previous studies. Fifteen pancreatic cancer patients were treated by HIFU exposure in a clinical trial, and the authors found the CD4+, CD4+/CD8+ and natural killer (NK) cells were increased in the serum of 10 patients, and NK cell activity was dramatically increased after HIFU treatment (26). Similarly, in a HCC cell vaccine study, the researchers found the cytotoxicity of cytotoxic T lymphocytes was highly increased after HIFU exposure, and these vaccines displayed stronger anti-tumor ability compared to non-HIFU ones (27). All these results suggest that HIFU treatment could induce systemic change in the tumor microenvironment, which consequently improves anti-tumor immunity.
MiRNAs play important roles in T lymphocyte differentiation and maturation, as well as immune response and immune tolerance (28). TGF-β promotes the CD4+ derived Treg differentiation, which mediates immunosuppression in various tumors (25,29). Here, we have found that TGF-β is a target of miR-9-5p, the expression of which is significantly up-regulated by HIFU exposure in melanoma tissues. Luciferase reporter assay has confirmed that miR-9-5p is able to bind to TGF-β 3'UTR directly. Overexpression of miR-9-5p could inhibit TGF-β expression on both mRNA and protein levels in melanoma cells. A recent study showed that miR-134 targeted CD86, a T lymphocyte activation antigen, in mouse melanoma cells. HIFU exposure dramatically inhibited miR-134, then enhanced CD86 expression, which could promote the secretion of IFN-γ and TNF-α, and improve anti-tumor immunity in the melanoma allograft mouse model (16). In line with these results, Li et al. examined the therapeutic effect of HIFU on colorectal cancer metastasis and found that HIFU exposure increased the expression of miR-124, which targeted STAT3, a transcription factor overexpressed and activated in various cancer cells and tumor-associated immune cells, and then inhibited invasion and metastasis of colon cancer cells (30). The in vitro regulation of miR-9-5p/TGF-β pathway in melanoma cells has been demonstrated by this study, but whether they also play key regulatory roles in antitumor immunity in vivo is still unknown. Future work will further validate the in vivo regulation of HIFU-mediated miR-9-5p/TGF-β pathway in melanoma treatment.
In the current study, we have demonstrated that HIFU  exposure could suppress melanoma growth and metastasis in mouse and cell models. HIFU treatment activates antitumor immune response, inhibits Tregs and Th17 cells, and increases CD8+ effector T cells in melanoma. We also reveal that TGF-β is a direct target of miR-9-5p. Mechanistically, the anti-tumor effect of HIFU might be mediated by up-regulating miR-9-5p and then suppressing the expression of TGF-β in melanoma cells.