An Engineered Prussian Blue Nanoparticles-Based Nanoimmunotherapy Elicits Robust and Persistent Immunological Memory in a TH-MYCN Neuroblastoma Model

A combination therapy using Prussian blue nanoparticles (PBNP) as photothermal therapy (PTT) agents coated with CpG oligodeoxynucleotides, an immunologic adjuvant, as a nanoimmunotherapy (CpG-PBNP-PTT) for neuroblastoma (NB) is described. NB driven by MYCN ampli ﬁ cation confers high risk and correlates with a dismal prognosis, accounting for the majority of NB-related mortality. The ef ﬁ cacy of the CpG-PBNP-PTT nanoimmunotherapy in a clinically relevant, TH-MYCN murine NB model (9464D) overexpressing MYCN is tested. When administered to 9464D NB cells in vitro, CpG-PBNP-PTT triggers thermal dose-dependent immunogenic cell death and tumor cell priming for immune recognition in vitro, measured by the expression of speci ﬁ c costimulatory and antigen-presenting molecules. In vivo, intratumorally administered CpG-PBNP-PTT generates complete tumor regression and signi ﬁ cantly higher long-term survival compared to controls. Furthermore, CpG-PBNP-PTT-treated mice reject tumor rechallenge. Ex vivo studies con ﬁ rm these therapeutic responses result from the generation of robust T cell-mediated immunological performed.

optimize and maintain T cell activity [8,9] for better outcomes in high-risk NB patients.
Here, we expand upon our previous work using Prussian blue nanoparticles (PBNPs) as agents of photothermal therapy (PTT; PBNP-PTT) [10][11][12][13][14][15][16] by investigating the efficacy of PTT using PBNPs coated with unmethylated cytosine-phosphate-guanine oligodeoxynucleotides (CpG) in a syngeneic, murine model of NB. [17,18] PBNPs are ideal candidates for use as PTT agents, as they are biodegradable with limited toxicity, [13][14][15] and are FDA approved. [19] We have previously reported that PBNP-PTT elicits efficient conversion of near-infrared (NIR) wavelength light into heat by biocompatible PBNPs causing local heating of the tumor and stimulating immunogenic cell death (ICD), [10,[13][14][15][16] a favorable cell death phenotype that engages an antitumor immune response. [20,21] This triggers release of tumor-associated antigens as well as damage-related molecular patterns from the dying tumor cells which then stimulates the activation of immune cells to target nearby unirradiated residual cancer cells in a system known as the "abscopal effect." [22][23][24][25] We and others have previously reported that using PTT in combination with immunotherapy further improves the antitumor response of PTT compared with either therapy administered alone, by boosting immunity of the treated subjects and extending long-term durable protection against cancer recurrence. [13,15,16] CpG, the immunologic adjuvant that we have attached to PBNPs, is recognized by toll-like receptor 9 (TLR9) and stimulates a cascade of innate and adaptive immune responses, [26,27] and has recently been approved for clinical use in a hepatitis B vaccine by the FDA. [28] CpG has been previously studied in the context of NB. [13,15,16,29,30] Also, Molenkamp et al. demonstrated that intratumoral injection of CpG alone or in combination with radiotherapy increases T cell immune response in cancer patients. [31] However, CpG injected directly into a tumor is rapidly eliminated, thus restricting its immunostimulatory benefits. [32,33] By coating CpG onto the PBNPs (CpG-PBNPs), we prevent its rapid clearance, thus increasing its bioavailability in the local TME. We have previously reported that CpG-PBNPbased PTT (CpG-PBNP-PTT) stimulates antigen-presenting cells (APCs), such as dendritic cells (DCs), and helps to overcome the immunosuppressive TME by triggering a T cell response in the Neuro-2a model of NB. [15,16] However, most immunotherapeutic studies for NB, including our previous studies, use C1300-derived cell lines such as Neuro-2a, which lack MYCN amplification and the expression of the GD2 antigen, which is prominently expressed on the surface of high-risk human NB tumors. [18,34] Thus, to model high-risk NB patients, in the current study we explore the potential of locally administered CpG-PBNP-PTT therapy in 9464D TH-MYCN model. The 9464D TH-MYCN model (referred to as 9464D model hereafter) generated by Weiss et al. is a transgenic model in which human MYCN is overexpressed through a rat tyrosine hydroxylase (TH) promoter. [17,18,34] This model shows strong histological and genetic similarities with human NB including the expression of GD2, thus rendering it a more clinically relevant model. [35,36] Several nanoparticle-based platforms have been developed to administer treatments including photodynamic therapy, chemotherapy, or developmental photoactivated chemotherapy in combination with PTT, with encouraging results in diverse cancer models. [24,[37][38][39][40][41][42] The specific nanoparticle platform selected for treating a particular tumor indication depends, among other factors, on the biology of tumor being treated and the ease of manufacture of the nanoparticles under consideration. To address ease of manufacture, in the current study, we use a simple and scalable layer-by-layer coating scheme to generate stable PBNPs functionalized with CpG (CpG-PBNP) for use in PTT (CpG-PBNP-PTT) as a minimally invasive method to trigger a robust systemic antitumor immune response and potentially generate long-term antigen-specific T cell memory culminating in robust and persistent disease remission in the 9464D model of NB. The overall treatment scheme and hypothesized mechanism of action are shown in Figure 1. We first test whether CpG-PBNP-PTT "primes" 9464D cells for recognition by immune effector cells and elicits ICD as a function of thermal dose, in vitro. This thermal dose-dependent expression of costimulatory and antigen-presenting molecules has not been previously studied for PTT. Subsequently, we analyze the efficacy of CpG-PBNP-PTT to elicit complete tumor regression and robust long-term memory on single tumor-bearing mice in vivo. We then conduct ex vivo cocultures of the splenocytes from any long-term surviving, rechallenge mice with 9464D cells to confirm tumor-specific responses and immunological memory generation by the T cells. These ex vivo studies involving T cells also represent a novel aspect of our study for PTT. Finally, we assess the effects of our nanoimmunotherapy to generate a potent abscopal effect in a synchronous tumor model. Through these studies, we seek to offer CpG-PBNP-PTT as a potent anticancer nanoimmunotherapy leveraging the photothermal heating characteristics of the PBNPs along with the immunostimulatory properties of both PBNP-PTT and CpG in a clinically relevant 9464D NB model.

CpG-PBNPs Function as Effective PTT Agents, Upregulate Molecules Implicated in Driving T Cell Responses, and Elicit ICD in 9464D Cells In Vitro
We used a layer-by-layer coating technique [43,44] to synthesize CpG-PBNPs and assessed the properties of the resultant CpG-PBNPs by measuring size distributions and zeta potentials using dynamic light scattering ( Figure S1A,B, Supporting Information). We analyzed the photothermal heating properties of the CpG-PBNPs in vitro which demonstrated a laser powerdependent increase (0.2-1.5 W) in temperature along with an increased thermal dose (expressed as cumulative equivalent minutes at 43 C; logΣCEM43). [45,46] We observed a maximum temperature of 77 C ( Figure S1C, Supporting Information) and the corresponding thermal dose of 9.58 at a concentration of 0.15 mg mL À1 CpG-PBNPs and laser power of 1.5 W ( Figure S1D, Supporting Information). We further observed that PBNP-PTT-and CpG-PBNP-PTT-treated 9464D cells exhibited a laser power-dependent thermal injury, as assessed by inverted phase contrast microscope using 10Â magnification 24 h posttreatment ( Figure S1E, Supporting Information).
We also analyzed a cohort of 137 pediatric patients with NB via RNA-seq data from the 2018 NCI TARGET study. We confirmed that high MYCN expression is restricted to patients with high risk tumors and observed an inverse correlation with the expression of MYCN and markers of immunogenicity, T cell infiltration and function, immune suppression, and toll-like receptors ( Figure S2A,B, Supporting Information). Thus, to understand the correlation between MYCN and immune signatures, we leveraged the 9464D NB model which overexpresses MYCN to determine the baseline immunogenicity of these tumors, and assess the effect of CpG-PBNP-PTT on these immune signatures.
We first measured the induction of MHC molecules which are critical for antigen presentation to T cells. [47,48] PBNP-PTT and CpG-PBNP-PTT at 1.5 W significantly enhanced MHC I expression on 9464D cells as compared with vehicle (Figure 2A,B; Figure S3A, Supporting Information). MHC II expression was also significantly higher on 9464D cells treated with PBNP-PTT at 1.5 W, or CpG-PBNP-PTT at 0.5 and 1.5 W, as compared with vehicle ( Figure 2C,D; Figure S3B, Supporting Information). Next, we examined the effect of CpG-PBNP-PTT on the expression of costimulatory molecules CD80 and CD86, which play a pivotal role in T cell activation and proliferation, and their absence can result in T cell anergy or apoptosis. [49][50][51][52] We observed a significant increase in the expression of CD80 and CD86 post 1.5 W PBNP-PTT, or 1.5 W CpG-PBNP-PTT as compared with vehicle ( Figure 2E-H; Figure S3C,D, Supporting Information).
Next, we assessed the potential of CpG-PBNP-PTT to elicit ICD, which is important to trigger tumor immunogenicity and strengthen therapeutic outcomes. [15,16] We first investigated the status of the biochemical correlates indicating ICD, which include cell surface expression of calreticulin, along with extracellular release of ATP and high mobility group box 1 (HMGB1). [14,20,21] We observed a significant increase in surface calreticulin after PBNP-PTT at 1.5 W or CpG-PBNP-PTT at 0.5 and 1.5 W relative to the vehicle (Figure 2I,J; Figure S3E, Supporting Information). Furthermore, a decrease in intracellular HMGB1 levels (suggestive of HMGB1 release) was observed on treatment with PBNP-PTT at 1.5 W or CpG-PBNP-PTT at 1.5 W as compared with the vehicle (Figure 2K,L; Figure S3F, Supporting Information). Next, we observed a significant decrease in intracellular ATP levels in cells treated with PBNP-PTT at 0.2, 0.5, and 1.5 W or CpG-PBNP-PTT at 0.2, 0.5, and 1.5 W as compared with vehicle, as well as PBNPs-or CpG-PBNPs-treated groups, suggesting significant ATP release ( Figure 2M; Table 1). Finally, we observed that 9464D cells exhibited significantly enhanced cell death in a laser power-dependent manner with PBNP-PTT at 0.5 or 1.5 W or CpG-PBNP-PTT at 0.5 and 1.5 W ( Figure 2N,O). Our in vitro studies demonstrate that CpG-PBNP-PTT nanoimmunotherapy is potent in increasing expression of molecules involved in antigen presentation, costimulatory markers and triggering ICD in 9464D cells when administered at a specific thermal dose (laser power of 1.5 W).   Encouraged by the induction of immune signaling markers and ICD in vitro culminating in tumor cell death, we next investigated the potential of CpG-PBNP-PTT to treat 9464D tumors in vivo. To study the effect of CpG-PBNP-PTT on the TME, we first established 9464D tumors in C57BL/6J mice and evaluated the phenotype of the resulting tumors for human NB-associated genes ( Figure S4A, Supporting Information). Most importantly, for the correlation to human tumors, we  www.advancedsciencenews.com www.advnanobiomedres.com observed the expression of GD2 synthase (one of the key enzymes in the production of the disialoganglioside GD2) in 9464D cells, which is not expressed in other NB mouse models. [18] Interestingly, our in vitro studies also showed significantly upregulated expression of the GD2 antigen in 9464D cells treated with PBNP-PTT (1.5 W) or CpG-PBNP-PTT (0.5 and 1.5 W) ( Figure S4B-D, Supporting Information). Next, we tested the therapeutic efficacy of our nanoimmunotherapy. For these studies, 9464D cells were inoculated in the mice (day 0). Tumor-bearing mice (%5 mm tumors; day 18) were divided into three treatment groups (n ¼ 5/group); 1) vehicle, 2) PBNP-PTT, and 3) CpG-PBNP-PTT, and the treatments were conducted accordingly ( Figure 3A). The maximum temperature maintained during PTT was %120 C as measured by a thermal imaging camera ( Figure 3B). The corresponding thermal dose administered was %23.5 (log (ΣCEM43)) for both the PBNP-PTT-and CpG-PBNP-PTT-treated groups ( Figure 3C). Importantly, 100% of mice (5/5) treated with CpG-PBNP-PTT exhibited complete tumor regression and survival on day 80   www.advancedsciencenews.com www.advnanobiomedres.com posttumor inoculation compared with 60% (3/5) PBNP-PTTtreated mice ( Figure 3D). However 100% of vehicle-treated mice (5/5) succumbed to high tumor burden by day 65 (median survival 38 days) ( Figure 3D,E).
To elucidate whether CpG-PBNP-PTT generated immunological memory and conferred protection against tumor recurrence, we rechallenged the surviving mice from PBNP-PTT or CpG-PBNP-PTT groups with 1 million 9464D cells on day 80. We observed that two of three surviving mice in the PBNP-PTT-treated group survived tumor rechallenge ( Figure 3F). Remarkably, four of five CpG-PBNP-PTT-treated mice exhibited protection against the tumor rechallenge and continued to survive tumor-free for an additional 45 days (day 125 in the study) at which point they were sacrificed for downstream analysis ( Figure 3G). Thus, long-term survival after tumor inoculation and rechallenge at day 125 was 80% for tumor-bearing mice treated with CpG-PBNP-PTT, twofold higher than that observed in mice treated with PBNP-PTT (40%). Taken together, our findings suggest CpG-PBNP-PTT is a robust therapy capable of inducing tumor regression, long-term survival, and protection against tumor rechallenge in the 9464D NB model.

T Cells Isolated from Long-Term Surviving, PTT-Treated Mice Exhibit Long-Term Memory and a Tumor-Specific T Cell Response Ex Vivo
To elucidate the antitumor effects triggered by CpG-PBNP-PTT, we analyzed the levels of T cells in the spleens of the long-term surviving, rechallenged mice on day 125. In this study, as a treatment-naive control group (control), we used age-matched mice that were inoculated with 1 million 9464D cells. When tumors in these animals reached %15 mm, we sacrificed mice from treatment naive, control group (n ¼ 4), PBNP-PTT-treated (n ¼ 2), and CpG-PBNP-PTT-treated (n ¼ 4) groups on the same day (day 125 for the rechallenged mice) for downstream analysis.
We observed a significant increase in CD3þ lymphocytes in the spleens of PBNP-PTT and CpG-PBNP-PTT rechallenged mice, compared with the spleens of mice in the control group ( Figure 4A,B). However, there were no statistically significant differences in the numbers of CD4þ or CD8 þ T cells between treatment groups ( Figure 4C-E). Development of long-term, antigen-specific memory is a hallmark of the adaptive immune system. [53,54] Expression of the adhesion molecule CD44 is observed on memory cells distinguishing it from the naive T cells. [55] Furthermore, CD69 is a key marker found on the tissue-resident memory T cells, and is prominently expressed on memory T cells both in humans and mice at various sites, including lymph nodes, liver, skin, intestines, and lungs. [56] We observed a significant increase in the CD3 þ CD44þ memory cells in the spleens of PBNP-PTT and CpG-PBNP-PTT rechallenged mice as compared with mice in the control group ( Figure 4F,G). We also observed a significant increase in the CD69 expressing CD4 þ T cells in PBNP-PTT and CpG-PBNP-PTT rechallenged mice as compared with control mice ( Figure 4H,I).
Memory T cells can be further classified into central memory T cells (T CM ) and effector memory T cells (T EM ) depending on their proliferative capacity, effector function, and migration potential. While protective memory is mediated by T EM that display immediate effector function, T CM cells home to secondary lymphoid organs and provide reactive memory by readily proliferating and differentiating to effector cells upon antigenic stimulation. [37,57] Therefore, to understand the T cell responses driving the rejection of 9464D tumor rechallenge, we measured the proportions of both T CM (CD3 þ CD62L þ CD44þ) and T EM (CD3 þ CD62L-CD44þ) cells for the CD4þ and CD8þ populations in the spleens of the various treatment groups. We observed that the percentage of CD4 þ T CM and CD4 þ T EM cells was significantly higher in PBNP-PTT or CpG-PBNP-PTT rechallenged mice as compared with mice in the control group ( Figure 5A-C). We observed no significant difference in the percentage of CD8 þ T CM cells; however, the percentage of CD8 þ T EM cells was significantly increased in the PBNP-PTT-or CpG-PBNP-PTT-treated rechallenged groups compared with control ( Figure 5D-F), indicating that a robust immunologic memory is elicited by PTT-based treatments.
Building on these observations, we performed an ex vivo study using isolated splenic T cells from the PBNP-PTT (n ¼ 2) or CpG-PBNP-PTT (n ¼ 4) rechallenged or treatment-naive control mice (n ¼ 4; same animals/treatment groups used in Figure 4) and cocultured them with 9464D cells to confirm the establishment of a 9464D cell-specific immunological memory postrechallenge. T cells isolated from the spleens of control, PBNP-PTT, or CpG-PBNP-PTT rechallenged mice were cultured for 4 days in the presence of IL-2, anti-CD3, and anti-CD28 ( Figure S5A, Supporting Information), following which we cocultured them with 9464D cells for 2 days. Importantly, we observed a significant decrease in the percentage of CD4 þ T CM cells and a significant increase in the percentage of both CD4þ and CD8 þ T EM cells in the PBNP-PTT and CpG-PBNP-PTT rechallenged mice as compared with the control mice ( Figure 5G-L). These data support the idea that tumor-specific memory T cells can become reactivated upon reexposure to 9464D cells.
We further analyzed the tumor-specific memory developed by our nanoimmunotherapy by evaluating the percentage of live GD2 þ 9464D cells in the coculture ( Figure S5B, Supporting Information). We observed a significant decrease in live GD2 þ 9464D cell population in the PBNP-PTT or CpG-PBNP-PTT rechallenged mice compared with control mice ( Figure 5M,N). These data suggest that our nanoimmunotherapy generated a potent tumor-specific cytotoxicity, as decreased GD2þ staining in the coculture is suggestive of T cell-mediated 9464D cell killing. Important to note here that although we do not observe a statistically significant difference in the magnitude of responses (e.g., %CD3 þ CD44þ or %CD4þ effector memory cells) between the PBNP-PTT and CpG-PBNP-PTT treatments for the assays/analyses in Figure 4 and 5, which may suggest that the CpG coating does not provide any benefit over PBNP-PTT. Importantly, we must consider that the PBNP-PTT group composed of two out of five mice that were able to survive the rechallange whereas three of the five mice had already succumbed to the tumor burden. However, CpG-PBNP-PTT treatment significant improved the therapeutic outcomes and composed of four out of five mice (twofold increase in long-term survival; Figure 3), suggesting the importance of the CpG coating for treatment outcomes.  Figure 6). For these studies, the tumor that reached a size of 5 mm (%60 mm 3 ) first (i.e., the larger tumor) was designated as the "primary" tumor (4.5-5 mm) and treated with the treatments in groups 1-5. The contralateral tumors in these groups (designated "secondary" tumor), which were generally slightly smaller (4-4.7 mm), were left untreated to evaluate the abscopal effect of the treatments. A maximum tumor temperature of %120 C was maintained attained during PTT (groups 4 and 5), which was measured using a thermal imaging camera ( Figure 6C) and the corresponding thermal doses were %24.4 log (ΣCEM43) ( Figure 6D). Both PBNP-PTT and CpG-PBNP-PTT generated  Figure 6. Effect of CpG-PBNP-PTT on tumor regression and long-term survival in a synchronous, 9464D tumor-bearing mice. Schematic of the treatments. A) Mice bearing two contralateral %5 mm diameter 9464D tumors were treated on one tumor (designated "primary" tumor) with either vehicle (PBS), PBNP, CpG-PBNP, PBNP-PTT, or CpG-PBNP-PTT and the contralateral tumor (designated "secondary" tumor) was left untreated. B) The vehicle group received 50 μL of PBS i.t., the PBNP-treated group received 50 μL of 1 mg mL À1 PBNPs i.t, the CpG-PBNP-treated group received 50 μL of 1 mg mL À1 CpG-PBNPs i.t, the PTT-treated groups received 50 μL of 1 mg mL À1 PBNPs or CpG-PBNPs i.t., and were irradiated with an 808 nm laser for 10 min at a temperature maintained at 120 C. In addition, the CpG-PBNP and CpG-PBNP-PTT groups received two boosters with CpG-PBNP on days 2 and 5. The dosage of CpG for these two groups was 2 μg of CpG conjugated onto PBNP on days 0, 2, and 5 i.t. C) Temperature versus time profiles and D) thermal doses administered expressed in cumulative equivalent minutes at 43 C (log(ΣCEM43)) of 9464D tumor-bearing mice treated i.t. with 50 μL of 1 mg mL À1 CpG-PBNPs or PBNPs and irradiated with a NIR laser at 120 C for 10 min (0.15 mg mL À1 ). E) Tumor growth curves of "primary" tumors of www.advancedsciencenews.com www.advnanobiomedres.com complete regression of primary tumors in 100% of mice, compared with 0% of vehicle-, PBNP-, and CpG-PBNP-treated mice ( Figure 6E(a-e); Figure 6G; Table 2). In addition, mice treated with either PBNP-PTT or CpG-PBNP-PTT exhibited significantly delayed tumor progression of secondary tumors, compared with mice in the vehicle-and PBNP-treated groups ( Figure 6F(a-e); Figure 6H; Table 2). The tumor progression in secondary tumors of the PBNP-PTT-or CpG-PBNP-PTT-treated mice at day 50 was significantly delayed with respect to CpG-PBNP-treated mice, demonstrating the abscopal effect elicited by our PTT-based nanoimmunotherapy ( Figure S6, Supporting Information; Table 2). In line with these findings, we observed that unirradiated PBNPs or CpG-PBNPs were unable to extend survival benefits to the tumor-bearing mice relative to vehicle-treated mice. However, all animals irradiated with PTT (PBNP-PTT or CpG-PBNP-PTT) exhibited significantly increased survival relative to vehicle-and PBNP-treated controls ( Figure 6H; Table 3). There was also a significant increase in the survival of the CpG-PBNP-PTT-treated mice relative to the CpG-PBNPtreated mice (p < 0.05). Although there was no statistically significant difference in survival between the CpG-PBNP-PTTor PBNP-PTT-treated groups, the median survival was higher for the CpG-PBNP-PTT-treated group (62 days) versus the PBNP-PTT-treated group (59 days), which can likely be attributed to the presence of the CpG coating on the CpG-PBNPs used for PTT. Based on these findings, we conclude that this nanoimmunotherapy potently eradicated the primary tumors and generated an abscopal effect on contralateral tumors as evident by the significantly delayed tumor progression, and increased survival relative to vehicle-and PBNP-treated groups for PBNP-PTT, and all others groups for CpG-PBNP-PTT, except CpG-PBNP-PTT versus PBNP-PTT where there was a slight increase in median survival.

Discussion
Here, we illustrated a CpG-PBNP-PTT-mediated nanoimmunotherapy that triggered systemic antitumor immune responses, leading to long-term antigen-specific T cell memory, providing robust and persistent tumor remission in a clinically relevant syngeneic, 9464D model of NB. Our previous studies in Neuro-2a NB suggest that PBNP-PTT triggers ICD. [14,15] Furthermore, CpG-PBNPs were also observed to activate DCs which then culminates in T cell activation. [15,16] To mechanistically define the role of the T cell response of our novel nanoimmunotherapy in a clinically relevant MYCN overexpressing NB model, we first focused on T cell activation. T cell activation relies on the MHC I/MHC II and T cell receptor (TCR) interaction. While MHC I molecules are known to be expressed by most nucleated cells, MHC II molecules were initially believed to be expressed only by professional APCs. However, recent reports suggest the expression of MHC II and components related to its pathway on a variety of human tumor cells, including many solid tumors such as melanoma, breast cancer, prostate cancer, glioma, and colorectal cancer, where they play a pivotal role in T cell activation. [48,[58][59][60][61][62][63][64][65] Further studies in multiple tumor types have reported an association between MHC II expression on tumor cells and a favorable prognosis. [44] Our in vitro studies revealed that PTT mediates increases in both MHC I and MHC II on 9464D cells at a specific thermal dose (Figure 2), suggestive of engaging the first step in T cell activation (antigen presentation). The MHC:TCR cross-linking is the initial step in T cell activation, followed by the cross-linking of costimulatory molecules Table 2. List of p-values for effect of the CpG-PBNP-PTT on tumor progression in a synchronous, 9464D TH-MYCN murine model of NB (Figure 6; Supporting Information Figure 6).  with this complex that initiates T cell proliferation and survival. [51] Thus, we next investigated the expression of costimulatory molecules CD80 and CD86, which are known to interact with CD28 to activate both naive and memory T cells. [48,51,66] Furthermore, the paucity of these costimulatory molecules is linked to T cell anergy, [50] and their overexpression on tumor cells enhances antitumor immune response, which has been explored in several cancer immunotherapy clinical trials. [49,51,67] Our findings revealed significantly higher expression of CD80 and CD86 in high thermal dose PTT-treated groups as compared with controls ( Figure 2H-K), suggesting that PTT primes these cells for immune recognition at this thermal dose. Nonimmunogenic "cold" tumor cells, such as NB, have typically downregulated or lost their antigen-presenting capabilities along with low expression of costimulatory molecules. [50,68,69] Here, we have shown that PBNP-PTT or CpG-PBNP-PTT can reverse this downregulation and elicit expression of both costimulatory molecules and markers involved in antigen presentation. Thus, our in vitro findings illustrate that our nanoimmunotherapy has the potential to prime the tumor cells for immune cell recognition by upregulating MHC I, MHC II, CD80, and CD86. It can further trigger the immunogenic responses by causing ICD ( Figure 2L-R), findings consistent with earlier studies by our group that show PBNP-PTT leading to cytotoxicity and ICD generation in another NB model. [14,15] Interestingly, we also observed significantly higher GD2 expression on 9464D cells after treatment with 1.5 W PBNP-PTT or CpG-PBNP-PTT. The current standard treatment for high-risk NB involves antidisialoganglioside GD2 monoclonal antibody that targets GD2 on neuroblasts. [70,71] Therefore, the PTT-mediated enhanced GD2 expression observed here may offer an improved response to this current treatment modality ( Figure S4B,C, Supporting Information). Another critical feature of a robust T cell response is the generation of long-term immunological memory. To investigate whether PBNP-PTT or CpG-PBNP-PTT could confer immunological protection against relapse, we rechallenged the long-term surviving PBNP-PTT-or CpG-PBNP-PTT-treated mice with 1 million 9464D cells on day 80. Eighty percent of mice in the CpG-PBNP-PTT treatment group rejected rechallenge, compared with 40% in the PBNP-PTT treatment group, indicating the development of a robust antitumor immunological memory generated by CpG-PBNP-PTT-based nanoimmunotherapy (Figure 3).
To further illustrate a robust T cell memory suggested by the rechallenge studies, we first analyzed the infiltration of T cells in the spleens of rechallenged mice. T cells play a critical role in eliciting immune responses and influencing the clinical outcome against various diseases, including autoimmune diseases, infection, allergic diseases, and cancer. [52,72] We found significantly higher infiltration of CD3þ cells ( Figure 4A,B) along with a significant increase in CD3 þ CD44þ memory cells in the spleens of PBNP-PTT-and CpG-PBNP-PTT-treated mice that survived rechallenge, compared with treatment-naive control mice ( Figure 4F,G). Furthermore, there was a significant increase in the CD69 expression on CD4 þ T cells, which is a key marker for tissue-resident memory T cells both in humans and mice [56] ( Figure 4H,I).
We also found an increase in CD4þ central and effector memory along with CD8þ effector memory T cells in the spleens of the surviving CpG-PBNP-PTT and PBNP-PTT rechallenged mice compared with the treatment-naive control mice ( Figure 5A-F). Central memory T cells are responsible for long-term memory and usually reside in the T cell niche of secondary lymphoid organs; however, on antigenic stimulation they readily proliferate and differentiate into effector memory T cells which then elicit immediate effector function. [73,74] Building upon our in vivo study, we validated the generation of a long-term antigen-specific T cell memory response in the ex vivo coculture analysis. Importantly, we observed a decrease in the CD4þ central memory and an increase in the CD4þ and CD8þ effector memory upon coculturing the T cells with 9464D cells, suggesting the generation of antigen-specific memory in the T cells, critical to the rejection of tumor rechallenge ( Figure 5G-L). Furthermore, we observed a T cell-mediated 9464D tumor cells killing upon coculture, suggestive of tumor-specific memory ( Figure 5M,N), a key criteria for therapeutic success.
Finally, we evaluated the potential of CpG-PBNP-PTT to elicit an abscopal effect in a synchronous tumor model ( Figure 6). This aspect is particularly important as %70% of NB cases present with metastatic disease at initial diagnosis. [75] Thus, an effective robust treatment should be able to extend its effects to more distant sites of tumor dissemination. Induction of abscopal effect is shown to involve activation of a systemic antitumor immune response against tumor antigens. [24,25] Our results indicated complete tumor regression on the PTT-treated flank ( Figure 6E(a-e)) and a significantly slower tumor progression on the untreated flank of PBNP-PTT-and CpG-PBNP-PTTtreated mice compared with vehicle-treated nice ( Figure 6F(a-e)), with significantly enhanced long-term survival in the PBNP-PTT-and CpG-PBNP-PTT-treated mice compared with vehicle treated mice, thus extending its abscopal effect to the secondary tumor site. Although we were unable to observe a statistically significant difference in survival between PBNP-PTT and CpG-PBNP-PTT in the synchronous tumor model with CpG-PBNP-PTT yielding only a modest increase in median survival compared to PBNP-PTT, we are investigating combinations of CpG-PBNP-PTT with other immunotherapies checkpoint inhibitors or immune effector cell therapies to better exploit any therapeutic advantage offered by the CpG coating (as observed in the single tumor model).
Overall, we observed that CpG-PBNP-PTT nanoimmunotherapy potently triggered strong antitumor immune responses with abscopal effects driven by T cell activation and long-term robust tumor-specific T cell memory. We elucidated that the antitumor effects CpG-PBNP-PTT are mediated by direct killing of cancer cells by PTT, along with generation of ICD and upregulation of immunostimulatory molecules, which ultimately leads to T cell activation and generation of tumor-specific T cell memory that prevents tumor relapse. As we tested our nanoimmunotherapy in a syngeneic preclinical NB model, there is potential to build upon these promising results for clinical translation.
Pediatric NB Analyses: The results published here are based upon data generated by the Therapeutically Applicable Research to Generate Effective Treatments (TARGET, https://ocg.cancer.gov/programs/target) initiative (NB, phs000467). The data used for this analysis are available at https://portal.gdc.cancer.gov/projects. Data from the NB (TARGET, 2018) study were obtained through Pediatric cBioPortal, downloaded in June 2020. [73][74][75] MYCN expression in NB risk groups was compared using one-way ANOVA. The heat map was generated through Pediatric cBioPortal. A total of 139 patients annotated with RNA-seq data from TARGET 2018 study were chosen for this analysis, but patients TARGET-30-PASSWW and TARGET-30-PARGKK were excluded as their transcriptomes were not complete.
Nanoparticle Synthesis and Characterization: PBNPs were synthesized using a one-pot synthesis system as previously described, [15] where an aqueous solution of 1.0 mM FeCl 3 ·6H 2 O and 0.5 mM citric acid in 20 mL of DI water was mixed with 20 mL aqueous solution containing 1 mM K 4 Fe(CN) 6 ·3H 2 O and 0.5 mM citric acid with vigorous stirring. The resulting precipitate containing PBNPs was isolated by adding equal volumes of acetone and centrifuging at (10 400 Â g for 10 min) at room temperature (RT). The collected PBNPs were rinsed three times and resuspended in Milli-Q water by sonication for 5 s using a Q500 sonicator (QSonica LLC, Newton, CT, USA) at high power (amplitude ¼ 40%).
CpG-PBNPs were synthesized using a layer-by-layer coating approach, [15] where PBNPs (3 mg mL À1 ) were contacted with equal volumes of PEI (12 mg mL À1 ) in acetate buffer (pH 5.2) at RT for 1 h on an orbital shaker. PEI-coated PBNPs (PEI-PBNPs) were washed four times in 50 mL ethanol and centrifuged at 10 400 Â g for 10 min and resuspended by sonication in Milli-Q water. We further coated 500 μL PEI-PBNPs (2 mg mL À1 ) with 300 μL of an aqueous solution of CpG (containing 100 μg CpG in endotoxin-free water) under stirring at RT for 15 min. The resultant CpG-coated PEI-PBNPs (CpG-PBNPs) were collected by centrifuging the mixture at 21 000 Â g for 15 min. Dynamic light scattering on a Zetasizer Nano ZS (Malvern Instruments, Malvern, UK) was used to measure the stability of the nanoparticles by measuring the size (hydrodynamic diameter) and charge distributions (zeta potential) of the PBNPs, PEI-PBNPs, and CpG-PBNPs.
In Vitro PTT Experiments: Five million 9464D cells were exposed to varying degrees of laser power (0.2-1.5 W cm À2 ) for 10 min with a fixed concentration (0.15 mg mL À1 ) of PBNPs or CpG-PBNPs using an 808 nm NIR continuous wave, collimated diode laser (Laserglow Technologies, Toronto, ON, Canada) in 1.5 mL tube. A power meter (Thorlabs, Newton, NJ) was used to confirm the laser power administered in each study. Time-based temperature measurements were captured using an i7 thermal imaging camera (FLIR, Arlington, VA, USA) at 1 min interval for 10 min. 9464D cells were plated in 6-well plate and incubated at 37 C under 5% CO 2 posttreatment, and visualized under the inverted phase contrast microscope (Leica DMi1 Inverted Microscope, Leica Microsystems) post 24 h for morphological changes using 10Â magnification. The cells were further analyzed for viability using the CellTiter-Glo assay (Promega Corporation, Madison, WI) according to manufacturer's instruction. Briefly, CellTiter-Glo reagent was added to the wells (50 and 50 μL media) and incubated at RT for 10 min in the dark and then the luminescence was recorded using SpectraMax i3x (Molecular Devices, LLC, San Jose, CA). Blank wells (media alone) were measured for luminescence and subtracted from the values in experimental wells. Results were expressed as a percentage of vehicle-treated control cells. In Vivo Studies: All animal studies were approved by the Institutional Animal Care and Use Committee (IACUC) at the George Washington University, Washington, DC, USA (Protocol # A396). Humane care of the animals was ensured in accord with IACUC guidelines. For all in vivo studies, 5-week-old female C57BL/6J mice purchased from Jackson Laboratory (Bar Harbor, ME, USA) and acclimated for a week. The tumor sizes were measured twice a week using a digital caliper, and the tumor volume was estimated by calculation as volume (mm 3 ) ¼ (length Â width 2 )/2. Animals were euthanized through cervical dislocation after CO 2 narcosis when tumor sizes reached 15 mm diameter in any dimension or ulcerations occurred. If the animals displayed any signs of distress, they were immediately euthanized.
In Vivo NB Model: The 9464D model was established by subcutaneously inoculating 1 million 9464D cells in 100 μL PBS in 5-6-week-old female C57BL/6J mice (The Jackson Laboratory, Bar Harbor, ME), as described by Kroesen et al. [18] Mice were treated when tumors reached a diameter of at least 5 mm (%60 mm 3 ). For the synchronous tumor model, 1 million 9464D cells were inoculated in both flanks of mice simultaneously. The tumor that reached a size of 5 mm (%60 mm 3 ) first (i.e., the larger tumor) was designated as the "primary" tumor and treated. The contralateral tumors (generally slightly smaller; designated as the "secondary" tumor) were left untreated to evaluate the abscopal effect of the treatments.
To confirm the establishment of the model, total RNA was extracted from the mice tumor cells using the RNeasy Mini Kit (Cat# 74104) following the manufacturer's instructions (Qiagen, Hilden, Germany) and quantified using ND-1000 NanoDrop Spectrophotometer (NanoDrop Technologies, Inc., Wilmington, DE). The cDNA was synthesized using iScript cDNA synthesis kit (Bio-Rad, 1708891), and mRNA was quantified using MyIQ single-color real-time PCR detection system (Bio-Rad, Hercules, CA) and iQ SYBR green Supermix (Bio-Rad, 1708882). Primers ( Table 4) were customized from Invitrogen (Waltham, MA, USA) and checked for correct amplification and dissociation of the PCR products. mRNA expression was determined relative to PBGD expression using the method described by Kroesen et al. [18] In Vivo PTT: For single tumor studies, mice were randomly divided into three groups when tumors reached a diameter of at %5 mm (%60 mm 3 ) (n ¼ 5/group): 1) Vehicle (intratumoral [i.t.] injection of 50 μL PBS on day 0), 2) PBNP-PTT (i.t. injection of 50 μL of 1 mg mL À1 PBNPs and irradiated with NIR laser to temperature of 120 C for 10 min), and 3) CpG-PBNP-PTT nanoimmunotherapy (i.t. injection of 50 μL of 1 mg mL À1 CpG-PBNP with 2 μg bound CpG, irradiated with NIR laser to reach a maximum temperature of 120 C for 10 min, 50 μL CpG-PBNP boosts were administered at the initial site of tumor [without PTT] on days 2 and 5). Surviving mice were rechallenged with 1 million 9464D cells %80 days posttumor-free survival to assess the immunological memory.
Synchronous tumor-bearing mice were randomly divided into five groups (n PBNPs and irradiated with NIR laser to temperature of 120 C for 10 min), and 5) CpG-PBNP-PTT (i.t. injection of 50 μL of 1 mg mL À1 CpG-PBNPs with 2 μg bound CpG, irradiated with NIR laser to temperature of 120 C for 10 min, CpG-PBNPs boosts were administered at the initial site of tumor [without PTT] on days 2 and 5). The tumor that reached a size of 5 mm (%60 mm 3 ) first (i.e., the larger tumor) was designated as the "primary" tumor and treated. The contralateral tumors (generally slightly smaller; designated as the "secondary" tumor) were left untreated to evaluate the abscopal effect of the treatments. Mice were anesthetized during the procedure using 2-5% isoflurane. Temperatures attained during PTT treatment were monitored using the i7 FLIR camera. Tumor growth of the mice was monitored following inoculation and treatments by routine caliper measurements.
Ex Vivo T Cell Studies: T cells were isolated from the spleens of either treatment-naive control group or PBNP-PTT-/CpG-PBNP-PTT-treated mice (125 days posttumor inoculation and rechallenge for these PTTtreated groups) using a Pan T Cell Isolation Kit II (Miltenyi Biotec). For the treatment-naive control group, we used age-matched mice that were inoculated with 1 million 9464D cells and euthanized when their tumors reached %15 mm diameter. This was scheduled so that we were able to acquire spleens of mice from the treatment-naive control group and PBNP-PTT-and CpG-PBNP-PTT-treated groups on the same day for downstream analysis. T cells were expanded in vitro for 4 days using TexMACS medium supplemented with 100 U mouse Recombinant IL-2 (STEMCELL Technologies) in the presence of anti-CD3 and anti-CD28 antibodies. Subsequently, 9464D cells were cocultured with the T cells for 2 days, after which the cells were blocked using TruStain FcX (anti-mouse CD16/32) antibody and then stained with fluorescent antibodies APC anti-CD3, Brilliant Violet 421 anti-CD4, Alexa Fluor 488 anti-CD8, APC/Cy7 anti-CD44, Brilliant Violet 650 anti-CD62L, and PE/Cy7 anti-GD2 and Zombie Violet Fixable Viability Dye; all antibodies were used at 1:100 dilution. Stained cells were visualized on the BD Celesta Cell Analyzer (BD Biosciences Franklin Lakes, NJ). Flow cytometry results were analyzed using FlowJo (Ashland, OR) software and only live single cells were gated for analysis.
Statistical Analysis: 1) Preprocessing of data: normalization was used in this study to represent flow cytometry count data (normalized to mode) and the expression of intracellular ATP levels in the various treatment groups (as a percentage of the vehicle-treated group). All animal studies were performed in a nonblinded fashion, and mice that attained similar average volumes of the primary tumors were randomized into the various treatment groups. All other data acquired in this study were presented as acquired without any transformation or exclusion of outliers. 2) Data presentation: results obtained in this study are expressed as mean AE standard deviation. 3) Sample size (n) for each statistical analysis: all in vitro studies had a sample size of n ¼ 3. All in vivo studies had a sample size of n ¼ 5.

Supporting Information
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