Plasmonic gold nanostar-mediated photothermal immunotherapy for brain tumor ablation and immunologic memory

Abstract

Brain tumors present unique therapeutic challenges and they include glioblastoma (GBM) and metastases from cancers of other organs. Current treatment options are limited and include surgical resection, radiation therapy, laser interstitial thermal therapy and chemotherapy. Although much research has been done on the development of immune-based treatment platforms, only limited success has been demonstrated. Herein, we demonstrate a novel treatment of GBM through the use of plasmonic gold nanostars (GNS) as photothermal inducers for synergistic immuno photothermal nanotherapy (SYMPHONY), which combines treatments using gold nanostar and laser-induced photothermal therapy with checkpoint blockade immunotherapy. In the treatment of a murine flank tumor model with the CT-2A glioma cell line, SYMPHONY demonstrated the capability of producing long-term survivors that rejects rechallenge with cancer cells, heralding the successful emergence of immunologic memory. This study is the first to investigate the use of this novel therapy for the treatment of GBM in a murine model.

Cancers of the intracranial compartment present unique therapeutic challenges and, regardless of pathology, continue to offer dismal prognoses. Such cancers consist of primary malignant brain tumors, such as glioblastoma (GBM), as well as brain metastases from systemic cancers of lung, breast, skin or kidney. GBM represents the most common and aggressive primary malignant brain tumor with more than 10,000 newly diagnosed patients in the USA each year, and, to date, it remains universally lethal and the median survival is only 15 months after treatments including surgery, chemotherapy and radiation therapy [1]. Far more common, however, are brain metastases, which ironically continue to rise in prevalence as people survive longer with primary cancers due to treatment advances [2]. Of the 1.44 million newly diagnosed cancer patients in the USA per year, between 180,000 and 216,000 (nearly 15%) are diagnosed with brain metastases [3], with the most common primaries of lung, breast, melanoma and renal cell cancer [4]. The morbidity associated with intracranial tumor growth is substantial and arises not only from neurological deficits associated with direct brain compression or invasion but also from difficult-to-control seizures, peritumoral edema and steroid dependence [5]. These insults are compounded further by damage to normal brain tissue resulting from inherently nonspecific treatment modalities, such as chemotherapy and radiation [6]. While newer platforms like immunotherapy offer the promise of improved specificity, efficacy has been limited within the brain [7]. Radically new treatment approaches are urgently needed for effective intracranial cancer treatment.

Numerous studies suggest synergy between hyperthermia-inducing strategies and immune-based platforms, which share a common origin in the cancer vaccine work [8]. Anti-tumor immunity is heightened by several thermally induced mechanisms, including NK and T-cell ligand expression by tumor cells, increased antigen chaperoning by heat shock proteins, tumor cell exosome release, direct activation of infiltrating lymphocytes and improved vascular permeability [9]. Immunotherapies can thus benefit from targeted thermal therapies, especially with precise thermal ablation of cancer cells [10].

Among immunotherapies, immune checkpoint blockade is perhaps one of the most promising approaches, aiming to uncheck T-cell activity within the immunosuppressive tumor microenvironment [11]. Programmed death-ligand 1 (PD-L1), a protein overexpressed by many cancers (including brain tumors), binds to its receptor PD-1 on activated, infiltrating T cells, inhibiting their cytotoxic anti-tumor function and facilitating immune escape [12]. Immune checkpoint blockage has already been used for GBM treatment [13].

Nanoparticle-mediated thermal therapy is an emerging concept [14]. A special type of metallic nanoparticles, called ‘plasmonic’ nanoparticles, has received great interest because they have unique properties that allow them to amplify the optical properties of the excitation light and thus increase the effectiveness of light-based photothermal tumor ablation [15]. Plasmon refers to the oscillation of free electrons in the metallic nanostructure. With stimulation from external electromagnetic field, the oscillation of free electrons can be in resonance with external stimulation and the electromagnetic field on the metallic nanostructure is dramatically enhanced. This phenomenon is named as surface plasmon resonance. With the goal of developing biocompatible nanoparticles for in vivo applications, we have developed a novel method, which does not require toxic surfactant, to synthesize star-shaped gold nanoparticles (gold nanostars [GNS]) that exhibit strong plasmonic properties [16,17]. Due to tip-enhanced plasmonics, GNS with multiple sharp branches can exhibit enhanced electromagnetic properties, acting like ‘lightning rods’ to convert and amplify stereotactically-delivered laser light into heat efficiently, which makes GNS a superior photon-to-heat transducer [16]. Furthermore, tumors usually have leaky vasculature compared with healthy tissue and the GNS nanoparticles have capability to accumulate preferably in tumor due to the enhanced permeability and retention (EPR) effect. By selectively accumulating within the tumor and amplifying heat delivery of the laser across tiny distances, the GNS offer the ability to extend and ‘shape’ the laser heat field in a manner that conforms to tumor margins, which could improve tumor treatment specificity with reduced side effect to surrounding healthy tissue.

In a recent study, we merged GNS-enhanced photothermal treatment with checkpoint immunotherapy to demonstrate a novel platform, which we termed synergistic immuno photothermal nanotherapy (SYMPHONY), for the treatment of bladder cancer within a murine model [18]. In this study, we further demonstrate the utility of SYMPHONY for GBM treatment. In a murine GBM animal model, SYMPHONY proved uniquely capable of producing long-term survivors that reject rechallenge with cancer cells injection, heralding the successful emergence of immunologic memory. To the best of our knowledge, this study is the first to demonstrate the novel combination of GNS-mediated photothermal therapy and checkpoint immunotherapy for GBM treatment with a murine model.

Materials & methods

GNS synthesis

GNS were synthesized using the surfactant-free and seed-mediated method developed by our lab [16]. The GNS fabrication process started with the 12-nm gold nanosphere synthesis. HAuCl₄ was reduced by trisodium citrate under fast stirring in a boiled aqueous solution. The generated 12-nm gold nanospheres were then used as seeds and mixed with AgNO₃, ascorbic acid and HAuCl₄ quickly. Following GNS synthesis, thiolated polyethylene glycol (SH-mPEG, M.W. 6000) was used to functionalize the GNS surface in order to improve stability and in vivo circulation time. After functionalization with SH-mPEG (M.W. 6000), GNS nanoparticles were condensed to 20 mg/ml in phosphate-buffered saline (PBS) solution for IV injection. The gold concentration was measured with atomic absorption spectroscopy.

Animal model & photoimmunotherapy

The CT-2A murine glioma cell line was used in this study for animal model development. Chemical induction followed by implantation of tumor fragments using C57BL/6 mice were used to develop the CT-2A cell line [19]. The syngeneic CT-2A model is a robust phenotype for aggressive and poorly differentiated brain tumor. This brain tumor animal model with features of high-grade gliomas, malignant transformation, is highly vascularized, infiltrative and exhibit deficiency in the phosphatase and tensin homology protein [20,21]. The phosphatase and tensin homology mutation, which can result in tumor-induced immunosuppression, are clinically relevant because they are found in 40% of high-grade human gliomas and 70% of glioma cell lines [22,23]. In addition, CT-2A brain tumor cells and brain tumor stem cells are similar for being able to self-renew, express CD133 [24]. Importantly, compared with patient-derived xenograft GBM cell line, we can implant this syngeneic cell line into immunocompetent mice which allow us to assess immune dysfunction in this model. CT-2A is a syngeneic murine glioma cell line on C57BL/6 background that our group has the experience with. Its histology and immune-phenotypes are well characterized by our group. Compared with other established murine glioma cell lines including SMA-560 (on VM/Dk background) and GL261 (on C57BL/6 background), CT-2A cells are significantly more proliferative and invasive [25]. Our data suggest that immune dysfunction phenotypes seen in patients with GBM such as T-cell exhaustion and bone marrow T-cell sequestration are recapitulated in CT-2A tumor bearing mice [26]. According to the literature, the CT-2A model is considered to accurately represent several GBM characteristics including intra-tumoral heterogeneity, in vivo migratory patterns, radio-resistance, chemo-resistance and different modes of immune [20].

Dulbecco's Modified Eagle Medium having 10% fetal bovine serum (Gemini Bio-Products, CA, USA), 2 mM 1-glutamine and 4.5 mg/ml glucose (Gibco, MD, USA) were used to culture CT-2A cells. Cells were collected for tumor development when they were in the logarithmic growth phase. C57BL/6 female mice were purchased from Charles River Laboratories (MA, USA) were used at 6–12 weeks of age. We have traditionally performed our tumor studies in female mice because the cell line authentication karyotyping confirmed that the CT-2A cell line that we possess is originally from a female mouse. For subcutaneous implantation, 5 × 10⁵ tumor cells were delivered in a total volume of 200 μl per mouse into the subcutaneous tissues of the right flank. For the photothermal treatment, 808 nm extracorporeal laser irradiation was delivered with a power density of 0.6 W/cm² for 10 min using a diode laser (Opto Engine LLC, UT, USA). Anti-PD-L1 (clone: B7-H1) was obtained from Bio-X-cell (NH, USA) and 200 μg per mouse was delivered intraperitoneally 30 min after laser treatment and then every 3 days. Humane end points include tumor size greater than 20 mm in one dimension, 2000 mm³ in total volume, or tumor ulceration or necrosis. Rechallenge was performed on cured mice where the treated tumors had disappeared completely. Animals were maintained under specific pathogen-free conditions at the Cancer Center Isolation Facility and the Vivarium of Duke University Medical Center. All mice were euthanized at the completion of the study. All experimental procedures were approved by the Institutional Animal Care and Use Committee.

Statistical analysis

Animal studies included female mice aged 6–12 weeks, without additional exclusion criteria employed. In the animal study protocol, all the mice were pooled and then sequentially assigned to each pertinent group. Survival comparisons were made using the Gehan–Breslow–Wilcoxon test. The figure legends indicate the specific statistical method employed for each data presentation. For the tumor volume change profile over time after initial treatment, one-way ANOVA statistical method was chosen for data analysis. For evaluating the tumor size change after rechallenge with cancer cells injection, two-tailed, unpaired Student's t-test was chosen to analyze results. The tumor volume result was shown as mean ± standard error of mean.

Results

SYMPHONY paradigm

Figure 1 shows the principle of SYMPHONY therapy with two treatment arms in this study. The first treatment arm is GNS-mediated photothermal therapy. After systemic administration, GNS nanoparticles accumulate preferentially in the tumor due to the EPR effect. Upon laser irradiation, GNS nanoparticles accumulated in tumors convert light to heat and kill cancer cells with increased temperature. The second treatment arm involves anti-PD-L1 antibody administration, which benefits both from improved access and T cell enabling in the setting of laser-induced hyperthermia to elicit synergistic and effective antitumor immunity. Figure 2 depicts the experiment design of SYMPHONY therapy on GBM photoimmunotherapy. Figure 3A shows a transmission electron microscopy image of the synthesized GNS with multiple sharp branches, providing tip-enhanced plasmonics. The photon-to-heat conversion efficiency of GNS was evaluated experimentally. As shown in Figure 3B, with 0.8 W/cm² 808 nm laser irradiation, 50 μg/ml (0.005%) GNS solution showed a temperature increase of 23.5°C, while pure water (0.8 ml) showed a temperature increase of only 2.6°C with the same laser irradiation.

Figure 1. . Synergistic immuno-photothermal nanotherapy (SYMPHONY).

By simultaneously ablating individual cancer cells with gold nanostar-enhanced photothermal therapy and enabling improved access and activity for anti-PD-L1 antibodies, the dual-modality can trigger a powerful thermally enhanced antitumor immune response to rapidly eradicate primary tumors and induce effective long-lasting immunity against recurrence.

PD-L1: Programmed death-ligand 1.

Figure 2. . Experiment design of synergistic immuno photothermal.

nanotherapy (SYMPHONY). After tumor development, the GNS was IV administrated and 1 day later the laser treatment was performed on the tumor with GNS accumulation. The anti-PD-L1 antibody was IP injected and rechallenge with glioblastoma cancer cells was performed on mice with disappeared tumor.

GNS: Gold nanostar; PD-L1: Programmed death-ligand 1.

Figure 3. . Gold nanostar characterization.

(A) Transmission electron microscopy image of the synthesized gold nanostar nanoparticles. (B) Temperature profile with 0.8 W/cm² laser irradiation. The solution temperature was monitored using a near-infrared camera.

SYMPHONY for improved tumor ablation & immunologic memory

Figure 4 demonstrates the effectiveness of the SYMPHONY platform. CT-2A murine glioma cells (5 × 10⁵) were implanted within the right flank of C57BL/6 mice on day 0. When the average tumor size reached 5–6 mm, mice were randomly divided into six groups for various combinations of treatments involving laser irradiation, GNS injection and anti-PD-L1 immunotherapy. Six groups were included in this study with each group having ten mice: Anti-PD-L1 + GNS + Laser (SYMPHONY); Anti-PD-L1 alone; GNS alone; Laser alone; GNS + Laser; Untreated control. Following randomization, 2 mg GNS were IV injected via tail vein. One day following the GNS injection, extracorporeal laser application (808 nm, 10 min) was performed on tumors. The first administration of IP anti-PD-L1 was performed 30 min following laser treatment. Anti-PD-L1 antibody was administrated every 3 days until the end of the experiment, and tumor volumes were assessed every 3 days as well. Figure 4A shows the increase of tumor volumes of the different mouse groups over time. The SYMPHONY group reflects the most effective therapeutic effect as it exhibited the greatest restriction to tumor growth. It is noteworthy that the therapeutic response for GNS + laser groups resulted in smaller tumor growth as compared with anti-PD-L1 therapy alone. This result demonstrates the effectiveness of GNS-mediated photothermal therapy in treating primary tumors. Figure 4B depicts the tumor volume of mice in different groups 37 days after treatment. The SYMPHONY group and the Laser + GNS group produced the two most effective results showing the smallest tumor growth. Furthermore, only these two groups (SYMPHONY group and Laser + GNS group; indicated in the blue color in Figure 4B) resulted in long-term tumor-free survival in subsets of mice.

Figure 4. . Therapeutic response after treatment.

(A) Plot of tumor volumes over time. Only the GNS + Laser (five out of ten mice) and GNS + Laser + anti-PD-L1 (SYMPHONY) (three out of ten mice) groups produced tumor-free long-term survival. (B) Tumor volume of mice in different groups 37 days after treatment. The SYMPHONY group and the Laser + GNS group produced the two most effective results showing the smallest tumor growth and resulted in long-term tumor-free survival in subsets of mice. Tumor volume data are shown as mean ± standard error of mean. Group with tumor-free survival mice are shown in the blue color and with a triangle symbol (Δ). Reproduced with permission from [18] Creative Commons Attribution 4.0 International License © Nature. (2017). GNS: Gold nanostar; PD-L1: Programmed death-ligand 1; SYMPHONY: Synergistic immuno photothermal nanotherapy.

Figure 5 shows the plots of the tumor volumes of tumor-free survivors from the photothermal therapy (GNS + Laser; n = 5) and the SYMPHONY (combined photothermal therapy and anti-PD-L1 immunotherapy; n = 3) groups that were rechallenged in the contralateral flank with 5 × 10⁵ CT-2A glioma cells on Day 50. Two out of three mice (67%) from the group receiving SYMPHONY rejected the rechallenge while this was true for two out of five mice (40%) from the group receiving GNS-mediated photothermal therapy. However, While effective in treating tumors (Figure 4), the GNS-mediated photothermal therapy was not as sufficient as SYMPHONY to prevent the recurrence from the tumor rechallenge (Figure 5). We have traditionally performed our tumor studies in female mice because the cell line authentication karyotyping confirmed that the CT-2A cell line that we possess is originally from a female mouse. The numbers for rechallenge are indeed low: n = 5 versus n = 3 mice treated with GNS+Laser ‘only’ versus SYMPHONY treatments, but experimental results are statistically significant in our studies. This result demonstrates that SYMPHONY therapy can produce long-term survivors that reject rechallenge with tumor, underlining the successful emergence of effective long-lasting anticancer immunologic memory, or ‘Cancer Vaccine’ effect.

Figure 5. . Rechallenge study.

Tumor-free survivors from the GNS + Laser (n = 5) and GNS + Laser + anti-PD-L1 (SYMPHONY; n = 3) groups in (A) were rechallenged in the contralateral flank with CT-2A glioma cells on Day 50. The group receiving SYMPHONY was more effective in rejecting the rechallenge when compared with the GNS + Laser group. Tumor volume data are shown as mean ± standard error of mean and are compared using two-tailed, unpaired Student's t-test. p = 0.0112.

GNS: Gold nanostar; PD-L1: Programmed death-ligand 1; SYMPHONY: Synergistic immuno photothermal nanotherapy.

Discussion

Traditional hyperthermia modalities, such as microwave, or radiofrequency, and focused ultrasound have been used to control macroscopic heating around the tumor region, but cannot target or ablate cancer cells at the microprecision scale. Nanoparticle-mediated thermal therapy has recently received increasing interest. GNS, which accumulate in and around cancer cells, can be triggered with light to rapidly achieve high ablative intratumoral temperatures (>55°C) and can also induce milder fever range (41–43°C) hyperthermia in the tumor microenvironment [16,17,27]. GNS therefore can act as exceptional light ‘nano-enhancers’ and nano-sources of heat that can ablate tumor cells in their microenvironment (i.e., from the inside) with high spatial accuracy. Cancer photothermal treatment precision is improved with this feature, leading to the use of reduced laser energy in order to destroy cancer cells. This advantage of GNS-mediated photothermal therapy has promise to achieve the same anticancer therapeutic efficiency with reduced laser energy as well as improve thermal treatment specificity, which is superior over traditional thermal therapies including microwave, radiofrequency and focused ultrasound. The photon-to-heat conversion capability of GNS is calculated to be 160,000-times higher than water given the same mass, which demonstrates that GNS are a superior photon-to-heat conversion transducer. Theoretical calculations have shown that GNS have the highest absorption-to-scattering ratio of the commonly used plasmonic gold nanoparticles [28], which is consistent with the very high conversion efficiency we have measured in this study. We have shown that gold nanoparticles accumulate preferentially within tumors following intravenous injection due to the effects on tumor vasculature allowing for EPR [27]. Nanoparticles can accumulate selectively in tumors after escaping the circulation via the EPR effect, which originates from the tumor vasculature leakiness. Furthermore, the inefficient tumor lymphatic system also enhances the nanoparticles’ retention in tumors. To take full advantage of the EPR effect, nanoparticles must be designed to have a narrow size range between 10 and 100 nm. GNS are therefore synthesized to have hydrodynamic sizes well within this range. Combining their selective accumulation in tumor with plasmon-enhanced light absorption and photon-to-heat conversion, GNS are uniquely capable of selectively ablating tumor regions, while maintaining surrounding normal tissues at safe and nonablative temperatures. This feature allows us to achieve rapid and precise hyperthermia throughout the tumor without harming tissue beyond its margins, which makes GNS an ideal agent for precise cancer photothermal therapy at the cellular level. We have investigated the temperature profile in a previously published paper. The tumor temperature with GNS injection is significantly higher than that without GNS injection [27]. We have investigated the long-term toxicity and biodistribution of GNS after systematical administration in previous studies and the results showed that GNS can accumulate in tumors through the EPR effect and there is no observed toxicity associated with GNS infusion up to 6 months [27,29].

SYMPHONY is a novel treatment approach that combines plasmon-activated GNS, laser-induced photothermal therapy and checkpoint blockade immunotherapy. The synergistic effect between nanoparticle-mediated photothermal therapy and immune checkpoint blockade has also been found in previous studies. It has been reported that a combination of photothermal ablation of primary tumor with single-walled carbon nanotubes and anti-cytotoxic CTLA 4 antibody immunotherapy can trigger immune response to treat not only primary tumor but also cancer metastasis [30]. The efficacy of combined GNS-mediated photothermal therapy and anti-PD-L1 immunotherapy most likely relies on several synergistic processes [18]. Specific ablation using GNS-enhanced photothermal treatment can kill tumor cells and release tumor-associated antigens as well as damage associated molecular pattern molecules, such as heat shock proteins. After cell death, the released intracellular damage associated molecular patterns can get involved in the immune response by interacting with antigen-presenting cells, which can process and present tumor-associated antigens to T cells. After combination with anti-PD-L1 treatment, tumor antigen presentation can trigger immune response and generate tumor-specific T cells, which circulate around body and kill both primary and metastatic cancer cells. Our studies have demonstrated the effectiveness of SYMPHONY in laser light enhancing, selective targeting of tumor cells, photothermal treatment and activating the immune response for cancer therapy. Synergistic combinatorial approaches such as SYMPHONY have great potential to reverse tumor-associated immunosuppression in order to improve cancer immunotherapy outcomes. Of great importance is the possibility that such an approach can induce long-term immunological memory that can provide protection against cancer recurrence long after treatment of the initial tumors. T-cell activation and tissue inflammation can be enhanced by using drugs that block the interaction between PD-1 and PD-L1. But drugs based on checkpoint inhibition have decreased efficiency with time and can only be effective for a small portion of patients. Nanoparticle-mediated thermal therapy has promise to broaden and stabilize the therapeutic effect of PD-L1/PD-1 inhibitors. In our study, there is no statistically significant difference in tumor size between GNS+Laser+anti-PD-L1 and GNS+Laser treatment in Figure 4A & B. However, there is significant difference in tumor size after rechallenge as shown in Figure 5. The mice with GNS+Laser+anti-PD-L1 treatment successfully rejected the rechallenge, indicating a generation of memorized antitumor immune response. Our results have indicated that by using a combination of immune-checkpoint inhibition and GNS-mediated photothermal therapy, it is possible to achieve eradication of primary treated tumors and, more importantly, subsequent rejection of ‘rechallenge’ cancer cells on cured mice, indicating that the combined treatment induced effective long-lasting immunity. In other words, SYMPHONY treatment functions as both a primary ablative treatment and a vaccine, simultaneously. SYMPHONY may be particularly well suited to tumors of the intracranial compartment, as laser ablation is becoming a mainstay of surgical treatment for these lesions, and SYMPHONY specifically addresses weaknesses of the current technologies and therapies in use. This study demonstrates the therapeutic efficacy of SYMPHONY treatment against lethal GBM cancer in a murine flank tumor model. In future studies, we plan to use an orthotopic GBM model to further investigate the detailed mechanism of SYMPHONY therapy against intracranial gliomas and will increase the size of investigated groups to achieve better statistical significance.

Conclusion

In this study, we demonstrated that the SYMPHONY therapy combining GNS-mediated photothermal therapy and checkpoint immunotherapy can improve the therapeutic effect against aggressive GBM cancer in a murine animal model. The tumor-bearing mice that were cured by the SYMPHONY treatment successfully rejected rechallenge with memorized anticancer immunoresponse, like an ‘anticancer vaccine’ effect. The results of this pilot study indicate that the novel SYMPHONY therapy has great potential as a new treatment option for aggressive GBM cancer.

Future perspective

GBM is one of most deadly cancer disease and less than 5% of patients survives for more than 3 years. Current therapeutic options including surgery, radiation therapy and chemotherapy have limited effect on GBM treatment. It has been a long goal to activate immune system to treat cancer. We have demonstrated that our novel SYMPHONY therapy, a combination of plasmonic GNS-mediated photothermal therapy and anti-PD-L1 immunotherapy can trigger memorized immunoresponse and reject rechallenge with cancer cells injection using a murine GBM animal model. The proposed SYMPHONY approach could provide an effective treatment when aggressive tumors cannot be surgically removed. Of special importance is the possibility that such an approach can induce long-term immunological memory that can provide protection against tumor recurrence long after treatment of the initial tumors. In other words, SYMPHONY works like both a primary ablative treatment and a vaccine simultaneously. This effective integration of the latest advances in nanotechnology and immunotherapy shows great promise to treat not only unresectable primary tumors, but also distant cancer metastasis by enhancing the systemic activity of specific and adaptive immune responses. Further fundamental studies will achieve better understanding and optimal exploitation of the mechanisms underlying these novel synergistic treatment modalities in order to enhance and broaden the effect of immune-checkpoint inhibitors for successful eradication of metastatic cancer. For future studies, detailed immunoresponse mechanism and how to optimize SYMPHONY treatment protocol should be carefully investigated. In addition, questions related with GNS nanoparticles, such as long-term toxicity, large-scale synthesis, as well as product quality control should be well studied before this novel therapy could be considered for future clinical translation to treat deadly GBM cancer. The approval for use in humans by the US FDA for CTLA-4 and PD-L1-based immunotherapies will make the drugs readily available for combination clinical trials. Similarly, as gold nanoparticles are considered biologically inert (nontoxic) and FDA-approved lasers are currently available for clinical use, the path to prove the safety of synergistic therapies for use in humans could be conceivable in a reasonable future. This strategy could lead to an entirely new treatment paradigm that challenges traditional surgical resection approaches not only for GBM but also for many cancers and metastases.

Summary points.

• We have successfully applied synergistic immuno photothermal nanotherapy (SYMPHONY) therapy for glioblastoma treatment with murine animal model.

• SYMPHONY therapy shows improved therapeutic effect than photothermal therapy or checkpoint immunotherapy alone.

• Memorized anticancer immunoresponse was observed more effective for tumor-bearing mice with SYMPHONY treatment than that for mice with photothermal therapy or checkpoint immunotherapy alone.

• SYMPHONY therapy has promise to be applied for aggressive glioblastoma treatment in future preclinical and clinical applications.