
==== Front
Transl Oncol
Transl Oncol
Translational Oncology
1936-5233
Neoplasia Press

S1936-5233(24)00242-0
10.1016/j.tranon.2024.102115
102115
Original Research
Tumor perfusion enhancement by focus ultrasound-induced blood-brain barrier opening to potentiate anti-PD-1 immunotherapy of glioma
Shan Haiyan shanhaiyan99@qq.com
a1
Zheng Guangrong zhenggr1025@163.com
a1
Bao Shasha a
Yang Haiyan c
Shrestha Ujen Duwal a
Li Guochen a
Duan Xirui a
Du Xiaolan a
Ke Tengfei 250169995@qq.com
b⁎
Liao Chengde chengdeliao@qq.com
a⁎
a Department of Radiology, Yan 'an Hospital of Kunming City, Kunming, China
b Department of Radiology, The Third Affiliated Hospital of Kunming Medical University, Kunming, China
c Department of Ultrasound, Chongqing General Hospital, Chongqing University, Chongqing 401147, China
⁎ Corresponding authors. 250169995@qq.comchengdeliao@qq.com
1 These authors contributed equally to this work.

31 8 2024
11 2024
31 8 2024
49 10211511 4 2024
21 8 2024
27 8 2024
© 2024 Published by Elsevier Inc.
2024

https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Highlights

• FUS combined with microbubbles can promote the opening of BBB and increase the infiltration of T lymphocytes.

• The combination of FUS and PD-1 inhibitors significantly enhances tumor T-cell infiltration, delays tumor progression, and prolongs survival.

• Focused ultrasound therapy can promote the blood perfusion of the tumor, change the hypoxic environment of the tumor, and inhibit the growth of the tumor.

Objective

To demonstrate the feasibility of using focused ultrasound to enhance delivery of PD-1 inhibitors in glioma rats and determine if such an approach increases treatment efficacy.

Methods

C6 glioma in situ rat model was used in this study. Transcranial irradiation with FUS combined with microbubbles was administered to open the blood-brain barrier (BBB). The efficacy of BBB opening was evaluated in normal rats. The rats with glioma were grouped to evaluate the role of PD-1 inhibitors combined with FUS-induced immune responses in suppressing glioma when the BBB opens. Flow cytometry was used to examine the changes of immune cell populations of lymphocytes in peripheral blood, tumor tissue and spleen tissue of the rats. A section of rat brain tissue was also used for histological and immunohistochemical analysis. The survival of the rats was then monitored; the tumor progression and changes in blood perfusion of tumor were dynamically observed in vivo using multimodal MRI.

Results

FUS combined with microbubbles could enhance the blood perfusion of tumors by increasing the permeability of BBB (p < 0.0001), thus promoting the infiltration of CD4+ T lymphocytes (p < 0.01). Compared with the control group, the combination treatment group had increased in the infiltration number of CD4+(p < 0.05) and CD8+ T (p < 0.05); the tumor volume of the combined treatment group was smaller than that of the control group (p < 0.01) and the survival rate of the rats was prolonged (p < 0.05).

Conclusions

In this study, we demonstrated that the transient opening of the BBB induced by FUS enhanced tumor vascular perfusion and facilitated the delivery of PD-1 inhibitors, ultimately improving the therapeutic efficacy for glioblastoma.

Keywords

Glioma
Focused ultrasound
Immunotherapy
Programmed death receptor 1
Dynamic enhanced magnetic resonance imaging
==== Body
pmcIntroduction

Glioma is the most prevalent malignant tumor of central nervous system. The average time from diagnosis to death typically does not exceed 14.6 months, with a corresponding 5-years survival rate of only 9.8 % [1,2]. Current clinical practice for treating brain gliomas involves maximal surgical resection, followed by adjuvant chemotherapy and radiation therapy. However, the effectiveness of these treatment modalities in enhancing the prognosis and survival in glioma patients is limited because of the fact that the blood-brain/tumor barrier in central nervous system tumor is unique [3] and there is the presence of immunosuppressive tumor microenvironment [4]. Immune checkpoint inhibitors (ICI), such as programmed death receptor 1 (PD-1), represent a new generation of tumor therapy that activates the immune system to eliminate tumor cells. They have been proven effective in initiating anti-cancer immune responses in many types of tumors [[5], [6], [7]]. However, existing clinical data suggest poor efficacy of ICI in glioblastoma patients [7,8], and several reasons for this have been identified, with BBB being a major obstacle to the entry of large molecular antibodies like ICI into the brain [9]. Therefore, achieving efficient targeted delivery of ICI and other large molecular antibodies in vivo is crucial.

The BBB is a biological barrier that separates the brain's vascular system from neural tissues. It is primarily composed of brain endothelial cells and ventricular endothelial cells, and its main function is to filter and regulate the passage of substances. However, the BBB presents a challenge for large molecule antibodies, such as those targeting PD-1, as they have difficulty crossing it. This necessitates further research into combinatory immunotherapeutic approaches that can work in collaboration. The use of FUS in combination with microbubbles to open the BBB represents a novel approach for delivering intracranial chemotherapeutic drugs and treating gliomas. Numerous studies have demonstrated that FUS with microbubbles can safely and temporarily open the BBB in specific targeted areas [10,11]. This method effectively increased the permeability of larger molecules, thus enabling enhanced drug concentration in the brain parenchyma [12]. Hence, this technique overcomes the challenge of delivering large molecular drugs across the BBB and into the brain.

The aim of this study is to use FUS combined with microbubble to temporarily open the BBB; and to assess the synergistic effect of delivering PD-1 inhibitors concurrently to improve the immunosuppressive state of gliomas. Additionally, the study utilizes multimodal MRI to quantitatively evaluate and longitudinally monitor the opening of the BBB in vivo, as well as to observe the post-treatment effects on gliomas. This will help to assess the efficacy of new therapeutic approach in improving the prognosis in patients with gliomas and evaluate the metabolic state of the tumor post-treatment.

Materials and methods

Cells and animals

All animal experiments were approved by the Ethics Committee of Animal Experiments, Kunming Medical University, in adherence to the experimental animal care guidelines. Pathogen-free male Sprague–Dawley rats (100 ± 10 g) were purchased from the Department of Laboratory Animals, Kunming Medical University (Kunming, China). All rats were housed under specific pathogen-free condition. The C6 glioma cell line used in this experiment was obtained from the cell repository of the Kunming Institute of Zoology, Chinese Academy of Sciences.C6 cells were cultured in high-glucose DMEM (Invitrogen), supplemented with 10 % fetal bovine serum (Gibco) and 100 U/ml penicillin/streptomycin (Beyotime) at 37 °C under 5 % CO2.

Glioma model in situ

C6 glioma cells were harvested by the means of trypsinization and were diluted with phosphate-buffered saline into 1 × 108 cells/mL for implantation. A total of 10 μL of C6 glioma cells suspension were injected, at a depth of 4 mm from the brain surface. The injection was performed over a 10 min period, and the needle was withdrawn over another 5 min. At the very least 10 rats in each group were taken for efficacy and MRI study, and 3 rats in each group for immunohistochemical (IHC) study. A minimum of 7-8 rats in each group were alive at the end of treatment. Control rats were injected with C6 glioma cells, but received sham ultrasound procedure with no energy. A second group of rats was subjected to focused ultrasound at the selected pressure level (4 W) at day 11, day 13, day 15, and day 17 after tumor implantation. The third group of rats received a single dose PD-1 inhibitors (2 mg/kg) (MedChemExpress:HY-101097,PD-1-IN-17) via intraperitoneal injection (IP) at day 11, day 13, day 15, and day 17 after being injected with tumor cells. Fourth group of rats received a total of 4-dose PD-1 inhibitors (2 mg/kg/day) IP combined with 4 times of 4 W focused ultrasound on day 11, 13, 15 and 17.

MRI scan and image measurement

Ten days after implantation, tumor sizes were measured using 3.0 Tesla MRI scanner (Ingenia Elition, Philips Healthcare). Tumor size was determined via longitudinal assessment by MRI at end of treatment to day 18. The rats were anesthetized with 2 % isoflurane throughout the MRI imaging process, placed in an 8 channel animal coil. The scan sequences included T2 plain scan, T1 enhanced and dynamic enhanced MR Images (DCE-MRI). Tumor size was quantified using T1-enhanced images with the following parameters: TR/TE = 510 ms/24 ms, matrix size = 168 × 162, FOV = 50 × 50 mm (NSA= 13). Tumor blood perfusion was evaluated by DCE-MRI pre- and post- treatment. The dynamic enhanced images were processed by Philips IntelliSpace Portal workstation MR Permeability data processing software. MRI perfusion parameters reflecting BBB permeability in each region of interest (ROI) were measured, including: Transport constant (Ktrans), rate constant (kep), extravascular extracellular space volume ratio (ve) and percentage of plasma contrast agent (vp); which were also used to generate false-color images of brain perfusion imaging parameters.

Treatment protocols

FUS treatment was performed with the Experimental apparatus for low power focused ultrasound (Chongqing Ronghai Ultrasonic Medical Engineering Research Center). The top of the cranium was first shaved with clippers, and a 1 ml syringe catheter was inserted into the tail vein for injections. The rat was then placed directly under a water sac with its head attached tightly to a thin-film, 1 × 1 cm2 window at the bottom of the water sac. Before FUS exposure, a 0.2 mL bolus of microbubbles (Sonovue, Shanghai Boleke Xinyi Pharmaceutical Co., LTD) mixed with 0.2 mL of saline was injected intravenously (IV), followed by flushing with 0.2 mL saline. During the experimental process, the optimal parameters for safe opening of BBB was determined by adjusting different energy and time settings.

Histological examination

To explore the changes of immune microenvironment in tumor after treatment. Rats were sacrificed after the second MRI scan. Paraformaldehyde-fixed and paraffin-embedded tumors were used to prepare 3 μm thick sections for immunohistochemistry (IHC) analysis. CD8+ marker (Abcam: ab33786) was employed to specifically bind to CTLs (CD3+/ CD8+ TILs); CD4+ marker (Biolegend:201520) was to precisely bind to CTLs (CD3+/CD4+ TILs). Tumor specimens were stained with hematoxylin and eosin for light microscopy.

Flow cytometry and antibodies

Anti CD4PerCP/Cyanine5.5(Biolegend:201520), anti CD3 APC(Biolegend:201414), anti CD8 PE(Biolegend:200608) and anti Foxp3 Alexa Fluor®(Biolegend:320012) were used for flow cytometry. Each sample tube was mixed with CD3, CD4 and CD8; incubated at room temperature and was kept away from light for 15 min, it washed twice with PBS, added with Cyto-Fast Fix/Perm Buffer cell fixing solution, then incubated at 4 °C for 20 min. Subsequently, the tube was rinsed twice with PBE, then Foxp3 was added, mixed well and incubated at room temperature and away from light for next 15 min. Ten thousand cells were collected from the stained samples by BD FACSCalibur flow meter; and each sample was repeated for 3 times. The results were obtained by Flowjo software, and the expressions of CD4+ T, CD8+ T and Treg cells were enumerated.

Statistical analysis

The flow cytometry results were expressed as the mean ± standard deviation. Tukey's multiple comparisons test was employed to statistically analyze the differences between the groups. Survival analysis was conducted using the Log-rank test. Additionally, one-way ANOVA was utilized to assess the differences in blood-brain barrier permeability parameters (Ktrans, kep, ve, vp) among the four groups, relative to tumor size expansion (tumor volume). All data calculations and statistical charts were generated using GraphPad Prism version 9.0 software. Differences were considered statistically significant when p < 0.05 (labeled as *; further labeled as ** when p < .001).

Results

Parameter screening of FUS

Parameters of FUS determine its biological effect, the key lies in selecting appropriate parameters. Thus, careful selection of FUS parameters is critical for ensuring safe and effective treatment. In our experiment, we administered ultrasound contrast microbubbles via intravenously in rats, while simultaneously targeting the rat's head with a low-power focused ultrasound experimental device for FUS irradiation. During the experiment, we adjusted different irradiation energies, irradiation times, and volumes of injected microbubbles to obtain optimal parameter combination for the opening of BBB, which was observed by Evans blue dye staining. The safe FUS irradiation parameters were ultimately determined to be 4 W for 200 s (with a pulse duration of 2 s and a duty cycle of 20 %). Additionally, we confirmed through enhanced magnetic resonance imaging that the range and depth of BBB opening under these irradiation conditions met the therapeutic requirements (Fig. 2).

The effect of tumor perfusion by FUS induced

Under the action of ultrasound contrast agents, the cavitation effect produced by FUS can increase the permeability of vascular wall, thereby affecting blood perfusion of tumor. The changes in MRI signal intensity helps to detect whether the BBB is open during ultrasound treatment and the potential changes occurring in surrounding tissues. In this experiment, we used dynamic contrast-enhanced MRI to assess the changes in tumor blood perfusion pre- and post- treatment. Our study results showed that FUS treatment significantly enhanced tumor blood perfusion, with the Ktrans value in the FUS group being much higher than that in control group (p < .001), and the kep value (p < .01) and vp value (p < .01) were also higher than those in control group (Fig. 3) (Fig. 4).

Changes of tumor microenvironment induced by FUS therapy

Increased amount of tumor blood perfusion can alleviate immunosuppression in the tumor microenvironment, thereby promoting the infiltration of CD8+ T cells and improving the efficacy of PD-1 inhibitor therapy (Fig. 1). In this experiment, the tumor blood perfusion in the FUS treatment group was significantly higher than that in control group, and the perfusion in the combined therapy group was next in line. The results of HE staining demonstrated that the number of tumor-infiltrating lymphocytes in both single treatment group and combined treatment group was significantly increased in comparison to control group. The immunohistochemistry results demonstrated a significant increase in the infiltration of CD4+ and CD8+ T lymphocytes in both the FUS alone group and the combined treatment group, compared to the tumor control group (Fig. 5), and these infiltrating lymphocytes were mainly concentrated around the tumor periphery. To further validate the results of our experiment, we conducted flow cytometric analysis of immune cells; which also demonstrated that FUS treatment and combined therapy enhances the infiltration of T lymphocytes in tumors which was consistent with the immunohistochemistry results. In addition, flow cytometry ascertained that the percentage of Tregs in the FUS group and combined therapy group was lower than the other groups (Figs. 6, 8).Fig. 1 Diagram of FUS-induced blood-brain barrier opening to enhance delivery of PD-1 inhibitors in glioma therapy (By Figdraw).

Fig 1

Fig. 2 Determining the irradiation parameters and experimental timings for the safe opening of the BBB using FUS. (A) The area indicated by the arrow on the left of Figure A is the EB staining under the condition of FUS power of 4W, irradiation time of 200S, and no MBs injection. No obvious blue-stained area is seen by the naked eye. The right brain tissue is 3W, irradiation time of 200S plus MBs EB staining. In Figure B, the staining of the left brain tissue was 4W under irradiation time of 200S plus MBs, and the staining of the right brain tissue was 5W under irradiation time of 200S. The brown bleeding area was visible to the naked eye. Figure C is the MRI T1 enhanced scan image, and the high signal on the left is the BBB leakage range under the condition of 4W, 200S plus MBs. The high signal on the right is a brain tumor. (D) Experimental timeline.

Fig 2

Anti-tumor enhancement induced by combination therapy

To evaluate the effectiveness of treatment on glioma, we used MRI to longitudinally assess the progression of gliomas in each experimental group. Fig. 3 shows the typical T1-weighted enhanced images used for quantifying tumor volume. The tumor progression rate was analyzed on the 10th and 18th day after tumor implantation, as shown in Fig. 7. Compared to control group, the combined therapy group had slower tumor progression and smaller tumor volume (p < 0.001). The survival rates of rats in each group are shown in the figure; compared to the single-treatment and control groups, the survival time of the rats in the combined therapy group was significantly extended (p < 0.05). Overall, our data suggest that the combined FUS treatment with PD-1 inhibitors enhances the anti-PD-1-mediated anti-tumor activity.Fig. 3 MRI of the tumor growth process and images of the brain tissue sampling. (A) MRI T1-weighted enhanced images on the 10th and 18th day after tumor implantation; (B) Comparison of tumor volumes among different groups on the 18th day after tumor implantation; (C) Images of brain tissue sampling from rats after treatment completion, with the tumor primarily located in the anterior part of the right brain. **P < 0.01 indicates statistical significance.

Fig 3

Fig. 4 Images from dynamic contrast-enhanced magnetic resonance imaging (DCE-MRI). (A) Pseudocolor images of perfusion parameters from DCE-MRI; (B) Comparison of perfusion parameters between different groups. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 indicate levels of statistical significance.

Fig 4

Fig. 5 H&E staining images and Immunohistochemistry. (A) Representative images of H&E staining of tumor tissues from different groups, with the yellow triangles pointing to areas representing tumor-infiltrating lymphocytes; (B) Comparison of the number of CD4+ cells from immunohistochemistry; (C) Representative images of immunohistochemical staining of tumor tissues from different groups; (D) Comparison of the number of CD8+ cells from immunohistochemistry; *P < 0.05, **P < 0.01, ****P < 0.0001 indicate levels of statistical significance.

Fig 5

Fig. 6 Representative flow cytometry staining analysis charts. Comparison of cellular populations of CD3+/CD4+, CD3+/CD8+, and CD4+/Foxp3+ T lymphocytes in glioma animal tumor tissues: control group, FUS only group, PD-1 inhibitor only group, and the combined PD-1 inhibitor + FUS group.

Fig 6

Fig. 7 Tumor progression and survival analysis. (A) MRI was used to measure tumor volume on Day 10 and Day 18 post-tumor implantation; (B) Survival of the four groups of rats was tracked post-tumor implantation, with the control group (n = 12), FUS group (n = 12), PD-1 inhibitor group (n = 12), and PD-1 inhibitor + FUS group (n = 12); *P < 0.05, **P < 0.01 indicate levels of statistical significance.

Fig 7

Fig. 8 Flow analysis compared the expression of immune cells in tumor tissue, peripheral blood and spleen tissue. Changes of CD3+/CD4+, CD3+/CD8+, CD4+/Foxp3+ T lymphocyte populations in tumor tissues, peripheral blood and spleen tissues in different groups (control group, FUS group, PD-1 inhibitor group and FUS/PD-1 combination group). In tumor tissues, compared with the control group, the number of Th cells in the FUS treatment group and the combination treatment group was relatively increased, the number of CTL cells in the combination treatment group was increased, and the number of Tregs cells in the PD-inhibitor group was higher than that in other treatment groups. In peripheral blood, the number of Th and CTL cells in FUS group was lower than that in other groups. In spleen tissue, Th, CTL and Treg cells in FUS group were lower than those in other groups. *P < 0.05, **P < 0.01, *** P < 0.001, **** P < 0.0001.

Fig 8

Discussion

This study explored the therapeutic effects of combining FUS with PD-1 blockade immunotherapy as a novel approach for treating glioma. We hypothesized that FUS binding with PD-1 blockers could synergistically enhance anti-tumor response. Previous experiments have demonstrated that in the presence of microbubbles, FUS can significantly increase vascular permeability [13].These changes in capillary permeability across various organs and tumor tissues, can affect the TME and are beneficial in triggering anti-cancer immune responses [[14], [15], [16], [17], [18]]. The clinical efficacy of immunotherapy in patients with “cold” tumors is not satisfactory, often characterized by a low level of neoantigens, a lack of tumor-infiltrating cytotoxic T cells, and the presence of an immunosuppressive microenvironment [19,20]. One of the most prominent and earliest reported immune-related effects of brain glioma, is severe systemic T cell exhaustion [4], and the immunosuppressive nature of its immune microenvironment has led to the current lack of effective treatments for brain glioma. PD-1 is a critical immune checkpoint that transmits an inhibitory signal to T cells upon binding with its two ligands, PD-L1 and PD-L2. This interaction induces T cells to enter a state of rest, decreases T cell proliferation, hinders their ability to recognize and eliminate cancer cells, and leads to reduced self-proliferation or apoptosis of the T cells; ultimately effectively attenuating the body's immune response [21]. In a variety of solid tumors, tumor cells and their microenvironment upregulate the expression of PD-L1 persistently and bind to PD-1 on the surface of tumor-specific CD8+ T cells, inhibiting the function of effector T cells [22], inducing immune evasion by the cancer cells, thereby achieving "immune escape". Fortunately, blocking the PD-L1/PD-1 pathway can enhance anti-tumor effects. Currently, PD-L1 monoclonal antibodies in combination with other tumor treatment methods have become part of the third-line treatment regimen for various cancers, including advanced lung cancer, which can prolong patient survival and improve prognosis [23]. Among various pathological types, it has been demonstrated that patients with glioblastoma exhibit a significantly heightened expression of PD-1, which is associated with a poor prognosis [24]. In patients with glioma, the expression levels of PD-1 on the surface of CD4+ and CD8+ T cells in peripheral blood are significantly higher than in healthy individuals; and the ratio of CD4+/CD8+ is inverted. Single-agent PD-1 inhibitor therapy for brain glioma has been trialed in clinical settings, yet it has not effectively improved patient prognosis or extended survival times [[25], [26], [27]]. In this experiment, we employed focused ultrasound as a novel method to augment the effectiveness of PD-1 inhibitor therapy in the treatment of brain glioma, a technique not previously explored in existing literatures. Our findings revealed that, compared to the administration of PD-1 inhibitor therapy alone, the combination approach significantly enhanced the infiltration of lymphocytes within the tumor, including both CD4+ and CD8+ T cells, and ameliorated the issue of poor T-cell penetration in the tumor area.

The BBB represents a major obstacle in the treatment of the central nervous system. It is composed of an unusually abundant and structurally unique set of tight junctions between vascular endothelial cells and a thick basement membrane. The barrier is maintained through the regulation by astrocytes and pericytes, which prevents the passage of vast majority of therapeutic drugs [[28], [29], [30]]. Under the irradiation of FUS with appropriate parameters, when microbubbles pass through the sound beam, the cavitation effect of the ultrasound causes the gas within the microbubbles to alternately expand and compress the microbubbles until they burst. This results in the loosening of the tight junctions between endothelial cells that constitute the BBB, thereby opening the BBB [31]. In this study, after the treatment with FUS combined with microbubbles, we observed the opening of the BBB using Evan's blue staining and MRI enhancement. Moreover, with the increase of ultrasound power, the range and degree of the BBB opening were broader and deeper, respectively. However, exceeding a certain limit would lead to cerebral parenchymal hemorrhage. Chen et al. [3] reported a preclinical feasibility study on the disruption of the BBB induced by FUS to enhance the delivery of IL-12 in a C6 glioma rat model. The study indicated that the FUS-induced opening of the BBB did not have a significant impact on the number of T lymphocytes in normal rats, while there was a change in the quantity of the tumor-infiltrating lymphocytes (TILs) populations in the glioma-bearing rats. Our study results confirmed that in the presence of microbubbles, FUS treatment significantly increased the permeability of the BBB and the blood perfusion of the tumor. Likewise, FUS treatment also promoted and enhanced the immune anti-tumor response. In this study, FUS treatment increased the number of CD4+T cells infiltrated, while the number of CD4+ and CD8+T cells infiltrated increased in the combined treatment group, and the survival time of the combined treatment group was the longest. Therefore, we believe that both CD4+T and CD8+T cells play an anti-tumor immune effect during treatment, but due to the dual action of FUS and PD-1 inhibitors in this study, we are still unclear about the specific immune effect process. Chen's [32] research suggests that CD4+ T cells play a dominant role in inhibiting GBM compared to CD8+ T cells. Ausejo-Mauleon et al. [33] believe that CD8+ T cells play the most significant role in the treatment of gliomas with TIM-3 inhibitors. The reason for these different experimental results may be that both CD4+ and CD8+T cells are activated under the action of multiple combined immunotherapies, and different treatments have different effects on immune cell populations.

Tumor cells can induce dysfunction in T cells and NK cells and promote the expansion of regulatory T cells and myeloid-derived suppressor cells. As a result, within the tumor tissue, the number of cytotoxic T lymphocytes, represented by CD8+ T cells, tends to be lower than in peripheral tissues; while the number of Treg cells tends to be relatively higher. In many cases, an increase in frequency of regulatory T cells is associated with a poorer prognosis. Treg cells can suppress the activation of effector T cells through variety of mechanisms [4], and their presence is considered to be a contributing factor to immunosuppression and decrease in anti-tumor immunity in affected patients [34]. Fecci et al. [35] also noted in a previous study that the proportion of circulating inhibitory Treg cells (CD4+/Foxp3+) population in GBM patients was abnormally high (2.5fold increase) compared to normal individuals. In this study, we also observed that the proportion of suppressive Treg cells (CD4+/Foxp3+) within the tumor was higher in control group and the group treated only with PD-1 inhibitors as compared to the group treated solely with FUS and the combination therapy group. We speculated that this may be due to the promotion of immune anti-tumor responses after FUS and combined therapy, leading to a decrease in the number of Treg cells within the tumor and alleviation of the tumor's immune suppression state.

Tumor hypoxia is related to the invasiveness of glioblastoma multiforme (GBM). The presence of hypoxia in tumor activates hypoxia inducing factors (hfs) mediated hypoxia signaling pathway, thereby activating biological mechanisms. It ensures GBM's adaptation and survival under conditions of hypoxia and limited nutrient supply [36]. Hypoxia is still neglected in the clinical treatment of GBM, and previous studies have confirmed that hypoxia in tumor tissues overlaps with inflammation, which is conducive to the proliferation of immunosuppressive cells and the development of cytotoxic T cells, which is not conducive to the treatment of glioblastoma [37]. Therefore, in this study, FUS combined with microvesicles was used to promote blood perfusion in the tumor area and quantitative analysis of intratumor blood perfusion was performed by DCE-MRI [[38], [39], [40]]. The results showed that Ktrans value, kep value and vp value in FUS treatment group were significantly higher than those in control group, and higher Ktrans value indicated increased vascular permeability and abundant blood supply. A higher kep value indicates that the contrast agent returns to the blood vessel more quickly, which may be related to increased vascular permeability and decreased lymphatic excretion. Higher ve values indicate increased blood vessel density, which may be related to the formation of new blood vessels. Therefore, we conclude that after FUS treatment, blood perfusion in the tumor area is increased, vascular permeability is increased, and oxygen depletion in the tumor area is reduced [41,42]. In addition, the number of intratumoral CD4+ and CD8+T cells infiltrated increased after FUS and combination therapy. It has been confirmed in the literature that VEGF is a major regulator of immunosuppression, which can reduce the infiltration of T effector cells, activate antigen-specific regulatory T cells (Tregs), and inhibit the maturation of dendritic cells (DCs) by down-regulating intercellular adhesion molecule 1 (ICAM1) and vascular cell adhesion molecule 1 (VCAM1) [43]. Based on this, we speculated that focused ultrasound treatment reduced the hypoxia in the tumor area, inhibited the increase of HIF-1α, promoted the decrease of VEGF expression, and increased the infiltration of effector T cells. Previous studies have demonstrated the important value of DCE-MRI in the grading of glioblastoma, but few studies have used DCE-MRI to longitudinally evaluate the prognosis and treatment outcomes of glioblastoma. Our study demonstrates that, compared to conventional MRI sequences, DCE-MRI can provide more accurate quantitative parameters for evaluating the prognosis and treatment outcomes of glioblastoma.

Our study has limitations to keep in mind [1]. In this experiment, the rats underwent a relatively long MRI scan, which requires precise control of anesthesia dosage and duration. Excessive anesthesia dosage can lead to rat mortality, while insufficient dosage can affect the quality of the images, which can have adverse effects on the experiment [2]. After the experimental treatment, we did not promptly conduct a quantitative analysis of PD-1 expression in the collected tissue, resulting in the lack of data on PD-1 expression within the tumor post-treatment. In future experiments, we will perform a quantitative analysis of PD-1 expression in tumor tissue following treatment to more objectively clarify the changes in the tumor microenvironment.

In summary, the combination FUS with microbubbles technology can facilitate the entry of PD-1 inhibitors into the brain, increasing the infiltration of active T lymphocytes and improving the immune suppression within glioblastoma. Additionally, the combination of immunotherapy is expected to be a future research direction for immunotherapy in glioblastoma.

Conclusions

Our study demonstrated that FUS can combined microbubbles can enhance the tumor perfusion by open the BBB transiently, thereby potentiate the anti-PD-1 treatment in generating antitumor activity. In addition, DCE-MRI offers a non-invasive approach to monitor tumor blood perfusion and presents a novel method for evaluating the effectiveness of tumor treatments.

Funding

This study was supported by the 10.13039/501100001809 National Natural Science Foundation of China (82160340 ), the Scientific Research Fund Project of Education Department of Yunnan Province (2023Y0818), the Yunnan Talents Support Program (Grant No. XDYC-MY-2022-0064) and the Postgraduate Education Innovation Fund of Kunming Medical University (2024B013 and 2024S122).

CRediT authorship contribution statement

Haiyan Shan: Writing – original draft, Software, Methodology, Investigation, Formal analysis, Data curation, Conceptualization. Guangrong Zheng: Supervision. Shasha Bao: Investigation. Haiyan Yang: Methodology. Ujen Duwal Shrestha: Writing – review & editing. Guochen Li: Data curation. Xirui Duan: Methodology. Xiaolan Du: Investigation. Tengfei Ke: Supervision. Chengde Liao: Writing – review & editing, Supervision, Funding acquisition.

Declaration of competing interest

The authors declare that there are no conflicts of interest regarding the publication of this article.
==== Refs
References

1 Sheehan K. Sheehan D. Sulaiman M. Padilla F. Moore D. Sheehan J. Investigation of the tumoricidal effects of sonodynamic therapy in malignant glioblastoma brain tumors J. Neurooncol. 148 1 2020 9 16 10.1007/s11060-020-03504-w Epub 2020/05/04 32361864
2 Stupp R. Hegi M.E. Mason W.P. van den Bent M.J. Taphoorn M.J. Janzer R.C. Effects of radiotherapy with concomitant and adjuvant temozolomide versus radiotherapy alone on survival in glioblastoma in a randomised Phase III study: 5-year analysis of the Eortc-Ncic trial Lancet Oncol. 10 5 2009 459 466 10.1016/s1470-2045(09)70025-7 Epub 2009/03/10 19269895
3 Chen P.Y. Hsieh H.Y. Huang C.Y. Lin C.Y. Wei K.C. Liu HL. Focused ultrasound-induced blood-brain barrier opening to enhance interleukin-12 delivery for brain tumor immunotherapy: a preclinical feasibility study J. Transl. Med. 13 2015 93 10.1186/s12967-015-0451-y Epub 2015/03/19 25784614
4 Grabowski M.M. Sankey E.W. Ryan K.J. Chongsathidkiet P. Lorrey S.J. Wilkinson D.S. Immune suppression in gliomas J. Neuro Oncol. 151 2021 3 12
5 Hugo W. Zaretsky J.M. Sun L. Song C. Moreno B.H. Hu-Lieskovan S. Genomic and transcriptomic features of response to anti-Pd-1 therapy in metastatic melanoma Cell 165 1 2016 35 44 10.1016/j.cell.2016.02.065 Epub 2016/03/22 26997480
6 Zhou D. Luo X. Zhou Z. Zeng X. Wan X. Tan C. Cost-effectiveness analysis of tislelizumab, nivolumab and docetaxel as second- and third-line for advanced or metastatic non-small cell lung cancer in China Front. Pharmacol. 13 2022 880280 10.3389/fphar.2022.880280 Epub 2022/09/13
7 Saba N.F. Steuer C.E. Ekpenyong A. McCook-Veal A. Magliocca K. Patel M. Pembrolizumab and cabozantinib in recurrent metastatic head and neck squamous cell carcinoma: a phase 2 trial Nat. Med. 29 4 2023 880 887 10.1038/s41591-023-02275-x Epub 2023/04/04 37012550
8 Reardon D. Omuro A. Brandes A. Rieger J. Wick A. Sepulveda J. Os10. 3 randomized phase 3 study evaluating the efficacy and safety of Nivolumab Vs Bevacizumab in patients with recurrent glioblastoma: checkmate 143 Neuro Oncol. 19 suppl_3 2017 iii21-iii
9 Caccese M. Indraccolo S. Zagonel V. Lombardi G. Pd-1/Pd-L1 immune-checkpoint inhibitors in glioblastoma: a concise review Crit. Rev. Oncol. Hematol. 135 2019 128 134 10.1016/j.critrevonc.2018.12.002 Epub 2019/03/02 30819441
10 Englander Z.K. Wei H.J. Pouliopoulos A.N. Bendau E. Upadhyayula P. Jan C.I. Focused ultrasound mediated blood-brain barrier opening is safe and feasible in a murine pontine glioma model Sci. Rep. 11 1 2021 6521 10.1038/s41598-021-85180-y Epub 2021/03/24 33753753
11 Anastasiadis P. Gandhi D. Guo Y. Ahmed A.K. Bentzen S.M. Arvanitis C. Localized blood-brain barrier opening in infiltrating gliomas with MRI-guided acoustic emissions-controlled focused ultrasound Proc. Natl. Acad. Sci. U. S. A. 118 37 2021 10.1073/pnas.2103280118 Epub 2021/09/11
12 Zhang Y. Liao C. Qu H. Huang S. Jiang H. Zhou H. Testing different combinations of acoustic pressure and doses of Quinolinic acid for induction of focal neuron loss in mice using transcranial low-intensity focused ultrasound Ultrasound Med. Biol. 45 1 2019 129 136 10.1016/j.ultrasmedbio.2018.08.023 Epub 2018/10/13 30309748
13 Zhao Y.Z. Lu C.T. Zhou Z.C. Jin Z. Zhang L. Sun C.Z. Enhancing chemotherapeutic drug inhibition on tumor growth by ultrasound: an in vivo experiment J. Drug Target. 19 2 2011 154 160 10.3109/10611861003801834 Epub 2010/05/01 20429773
14 Bekeredjian R. Kroll R.D. Fein E. Tinkov S. Coester C. Winter G. Ultrasound targeted microbubble destruction increases capillary permeability in hepatomas Ultrasound Med. Biol. 33 10 2007 1592 1598 10.1016/j.ultrasmedbio.2007.05.003 Epub 2007/07/10 17618040
15 Stieger S.M. Caskey C.F. Adamson R.H. Qin S. Curry F.R. Wisner E.R. Enhancement of vascular permeability with low-frequency contrast-enhanced ultrasound in the chorioallantoic membrane model Radiology 243 1 2007 112 121 10.1148/radiol.2431060167 Epub 2007/03/30 17392250
16 Huang X. Yuan F. Liang M. Lo H.W. Shinohara M.L. Robertson C. M-HIFU inhibits tumor growth, suppresses Stat3 activity and enhances tumor specific immunity in a transplant tumor model of prostate cancer PLoS One 7 7 2012 e41632 10.1371/journal.pone.0041632 Epub 2012/08/23 22911830
17 Liu F. Hu Z. Qiu L. Hui C. Li C. Zhong P. Boosting high-intensity focused ultrasound-induced anti-tumor immunity using a sparse-scan strategy that can more effectively promote dendritic cell maturation J. Transl. Med. 8 2010 7 10.1186/1479-5876-8-7 Epub 2010/01/29 20105334
18 Zhou Q. Zhu X.Q. Zhang J. Xu Z.L. Lu P. Wu F. Changes in circulating immunosuppressive cytokine levels of cancer patients after high intensity focused ultrasound treatment Ultrasound Med. Biol. 34 1 2008 81 87 10.1016/j.ultrasmedbio.2007.07.013 Epub 2007/09/15 17854983
19 Zhang Y. Sriramaneni R.N. Clark P.A. Jagodinsky J.C. Ye M. Jin W. Multifunctional nanoparticle potentiates the in situ vaccination effect of radiation therapy and enhances response to immune checkpoint blockade Nat. Commun. 13 1 2022 4948 10.1038/s41467-022-32645-x Epub 2022/08/24 35999216
20 Duan Q. Zhang H. Zheng J. Zhang L. Turning cold into hot: firing up the tumor microenvironment Trends Cancer 6 7 2020 605 618 10.1016/j.trecan.2020.02.022 Epub 2020/07/02 32610070
21 Majc B. Novak M. Kopitar-Jerala N. Jewett A. Breznik B. Immunotherapy of glioblastoma: current strategies and challenges in tumor model development Cells 10 2 2021 10.3390/cells10020265 Epub 2021/02/13
22 Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy Nat. Rev. Cancer 12 4 2012 252 264 10.1038/nrc3239 Epub 2012/03/23 22437870
23 Topalian S.L. Hodi F.S. Brahmer J.R. Gettinger S.N. Smith D.C. McDermott D.F. Safety, activity, and immune correlates of anti-Pd-1 antibody in cancer N. Engl. J. Med. 366 26 2012 2443 2454 10.1056/NEJMoa1200690 Epub 2012/06/05 22658127
24 Chauhan P. Lokensgard JR. Glial Cell Expression of Pd-L1 Int. J. Mol. Sci. 20 7 2019 10.3390/ijms20071677 Epub 2019/04/17
25 Chamberlain M.C. Kim BT. Nivolumab for patients with recurrent glioblastoma progressing on bevacizumab: a retrospective case series J. Neurooncol. 133 3 2017 561 569 10.1007/s11060-017-2466-0 Epub 2017/05/14 28500559
26 Sanders S. Debinski W. Challenges to successful implementation of the immune checkpoint inhibitors for treatment of glioblastoma Int. J. Mol. Sci. 21 8 2020 10.3390/ijms21082759 Epub 2020/04/23
27 Lim M. Xia Y. Bettegowda C. Weller M. Current state of immunotherapy for glioblastoma Nat. Rev. Clin. Oncol. 15 7 2018 422 442 10.1038/s41571-018-0003-5 Epub 2018/04/13 29643471
28 Mo F. Pellerino A. Soffietti R. Rudà R. Blood-brain barrier in brain tumors: biology and clinical relevance Int. J. Mol. Sci. 22 23 2021 10.3390/ijms222312654 Epub 2021/12/11
29 Sweeney M.D. Sagare A.P. Zlokovic BV. Blood-brain barrier breakdown in Alzheimer disease and other neurodegenerative disorders Nat. Rev. Neurol. 14 3 2018 133 150 10.1038/nrneurol.2017.188 Epub 2018/01/30 29377008
30 Pardridge WM. The blood-brain barrier: bottleneck in brain drug development NeuroRx 2 1 2005 3 14 10.1602/neurorx.2.1.3 the journal of the American Society for Experimental NeuroTherapeuticsEpub 2005/02/18 15717053
31 Bunevicius A. McDannold N.J. Golby AJ. Focused ultrasound strategies for brain tumor therapy Oper. Neurosurg. 19 1 2020 9 18 10.1093/ons/opz374 (Hagerstown, Md)Epub 2019/12/20
32 Chen D. Varanasi S.K. Hara T. Traina K. Sun M. McDonald B. Ctla-4 blockade induces a microglia-Th1 cell partnership that stimulates microglia phagocytosis and anti-tumor function in glioblastoma Immunity 56 9 2023 2086 2104 10.1016/j.immuni.2023.07.015 e8Epub 2023/08/13 37572655
33 Ausejo-Mauleon I. Labiano S. de la Nava D. Laspidea V. Zalacain M. Marrodán L. Tim-3 blockade in diffuse intrinsic pontine glioma models promotes tumor regression and antitumor immune memory Cancer Cell 41 11 2023 1911 1926 10.1016/j.ccell.2023.09.001 e8Epub 2023/10/07 37802053
34 See A.P. Parker J.J. Waziri A. The role of regulatory T cells and microglia in glioblastoma-associated immunosuppression J. Neurooncol. 123 3 2015 405 412 10.1007/s11060-015-1849-3 Epub 2015/07/01 26123363
35 Fecci P.E. Mitchell D.A. Whitesides J.F. Xie W. Friedman A.H. Archer G.E. Increased regulatory T-cell fraction amidst a diminished cd4 compartment explains cellular immune defects in patients with malignant glioma Cancer Res. 66 6 2006 3294 3302 10.1158/0008-5472.Can-05-3773 Epub 2006/03/17 16540683
36 Bou-Gharios J. Noël G. Burckel H. Preclinical and clinical advances to overcome hypoxia in glioblastoma multiforme Cell Death Dis. 15 7 2024 503 10.1038/s41419-024-06904-2 Epub 2024/07/14 39003252
37 Feldman L. Hypoxia within the glioblastoma tumor microenvironment: a master saboteur of novel treatments Front. Immunol. 15 2024 1384249 10.3389/fimmu.2024.1384249 Epub 2024/07/12
38 Verheggen I.C.M. de Jong J.J.A. van Boxtel M.P.J. Gronenschild E. Palm W.M. Postma A.A. Increase in blood-brain barrier leakage in healthy, older adults Geroscience 42 4 2020 1183 1193 10.1007/s11357-020-00211-2 Epub 2020/07/01 32601792
39 Zhao Y.L. Xia X. Liang C.E. Li XM. Pharmacokinetic changes induced by focused ultrasound in Glioma-bearing rats using dynamic contrast-enhanced MRI Measurement J. Biol. Regul. Homeost. Agents 32 3 2018 655 661 Epub 2018/06/21 29921395
40 Grasso G. Torregrossa F. Noto M. Bruno E. Feraco P. Buscemi F. MR-guided focused ultrasound-induced blood-brain barrier opening for brain metastasis: a review Neurosurg. Focus 55 2 2023 E11 10.3171/2023.5.Focus23227 Epub 2023/08/02
41 Batchelor T.T. Gerstner E.R. Emblem K.E. Duda D.G. Kalpathy-Cramer J. Snuderl M. Improved tumor oxygenation and survival in glioblastoma patients who show increased blood perfusion after Cediranib and Chemoradiation Proc. Natl. Acad. Sci. U. S. A. 110 47 2013 19059 19064 10.1073/pnas.1318022110 Epub 2013/11/06 24190997
42 Jordan B.F. Sonveaux P. Targeting tumor perfusion and oxygenation to improve the outcome of anticancer therapy Front. Pharmacol. 3 2012 94 10.3389/fphar.2012.00094 Epub 2012/06/05 22661950
43 De Vleeschouwer S., Bergers G. Glioblastoma: to target the tumor cell or the microenvironment? In: De Vleeschouwer S., Eds. Glioblastoma. Brisbane (AU): Codon Publications.
