Prussian blue nanoparticles (PBNPs) are a versatile platform for administering photothermal therapy (PTT) in cancer therapy applications. PBNPs combine biocompatibility, safety, and clinical translational potential with durable treatment outcomes in preclinical cancer models. In this perspective, we focus on aspects critical to the workflow of implementing PBNP-PTT in cancer treatment, drawing inspiration from adjacent scientific areas that have not been described in the context of PBNPs, but are important for improving the delivery of PBNP-PTT and its translation. Specifically, we will discuss machine learning approaches, multiomics analyses, and clinical strategies pertinent to PBNP-PTT. Machine learning approaches have the potential to enhance PBNP-PTT design, performance, and therapeutic outcomes. Complementing this, multiomics has the potential to describe the responses to PBNP-PTT, particularly its immune effects. By embedding these advances from nanoparticle engineering to therapy monitoring, PBNP-PTT can evolve from empirical tumor ablation toward a precision photothermal platform, enabling highly individualized cancer treatments with improved safety, efficacy, regulatory approval, and clinical predictability. We will also cover clinical strategies pertinent to the translation PBNP-PTT culminating with specific forward-looking perspectives. This convergence of nanotechnology, immunology, and data science positions PBNP-PTT at the forefront of next-generation cancer nanomedicine and immunotherapy.

Abstract Ovarian cancer (OC) is the most lethal gynecological malignancy with less than a 10% response rate to immune checkpoint blockade. Personalized vaccination with tumor lysate-pulsed dendritic cells amplifies T cell responses, including neoepitope-specific T cells, but is not curative. Thus, amplifying the pre-existing tumor-specific T cell response is insufficient. We developed a novel method to expand T cells ex vivo for OC specificity using Prussian Blue nanoparticle-photothermal therapy (PBNP-PTT), and verified in vivo efficacy using a syngeneic murine model. PBNP-PTT -generated ID8 OC cell lysate was used to stimulate bone marrow-derived dendritic cells (BMDCs) isolated from healthy female C57Bl6 mice. Autologous splenic CD3+ T cells were cocultured ex vivo with BMDCs and assessed in vitro for tumor-specific activation and killing using IFNy ELISpot and MTS cytotoxicity assays. Congenic mice bearing ID8B OC cells received either ex vivo expanded T cells or nonspecifically expanded T cells, and the accumulated ascites was profiled for cytokine composition. Adoptively transferred T cells were immunophenotyped with spectral flow cytometry.Ex vivo expansion of T cells with DCs primed using PBNP-PTT-generated OC cell lysate produced a pool of CD8+ T cells with enhanced IFNy and TNFa secretion in tumor coculture compared to nonspecifically expanded T cells. PBNP-PTT-expanded T cells exhibited greater tumor cell killing at increasing effector-to-target ratios and did not demonstrate off-target IFNy secretion. Tumor-bearing female C57Bl6 mice receiving OC-expanded T cells lived significantly longer and exhibited decreased tumor burden compared to mice receiving nonspecific T cells. Adoptively transferred T cells expanded with PBNP-PTT lysate also persisted longer in vivo with maintained activation in the ascites, and synergized with concurrent epigenetic therapy to further prolong survival. Ex vivo expansion methods using PBNP-PTT were also applied to healthy human blood donors to generate human T cells that demonstrate potent activation when challenged with human OC cell lines. In summary, we developed a novel method of engineering T cells ex vivo for specificity against ovarian cancer using nanoparticle photothermal therapy. This study is the first to harness PBNP-PTT ex vivo to demonstrate antitumor activity in vivo using an autologous mouse model of ovarian cancer. Citation Format: Abigail V. Lee, Erin Grundy, Jose Colina, Elizabeth Sweeney, Rohan Fernandes, Nethaji Muniraj, Russell Y. Cruz, Katherine B. Chiappinelli. Antigen-specific T cells engineered with nanoparticle photothermal therapy demonstrate antitumor efficacy in an immunocompetent murine model of ovarian cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2026; Part 1 (Regular Abstracts); 2026 Apr 17-22; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2026;86(7 Suppl):Abstract nr 5612.

Ovarian cancers (OCs) remain a lethal gynecological malignancy characterized by an immunosuppressive tumor microenvironment lacking effector lymphocytes. Thus, increasing tumor intrinsic immune signaling in OC remains a therapeutic goal for improving recruitment and activation of lymphocytes to the tumor microenvironment. A nanoparticle-based thermal treatment, Prussian blue nanoparticle photothermal therapy (PBNP-PTT), has shown strong antigen-specific lymphocyte recruitment to the tumor microenvironment through induction of immunogenic cell death in preclinical tumor models. Therefore, this study sought to determine the efficacy of PBNP-PTT in OC cell lines. We first demonstrate that PBNP-PTT induced tumor intrinsic immune signaling in OC cells and led to improved monocyte activation through tumor intrinsic proinflammatory cytokine release and presentation of damage-associated molecular patterns. Accompanying this intrinsic immunostimulatory effect, we report a novel epigenetic hallmark response to PBNP-PTT characterized by genome-wide H3K9 acetylation. Finally, increasing histone acetylation via histone deacetylase inhibition (Panobinostat) improved the tumor intrinsic immune signaling potential of PBNP-PTT. These findings indicate that PBNP-PTT activates the release of immunostimulatory factors in ovarian cancer cells alongside H3K9ac rewiring and suggests the potential of combining PBNP-PTT with HDACi to improve tumor intrinsic immune signaling in ovarian cancer.

Given the poor prognosis of Glioblastoma (GBM), there is an urgent need for therapies addressing GBM heterogeneity and achieving durable remission. In response, we have developed an autologous T cell product targeting multiple antigens using immunostimulatory photothermal Prussian blue nanoparticle-based photothermal therapy (PBNP-PTT). Our recent studies show that T cells expanded using PBNP-PTT are specific and cytolytic against GBM cell lines in vitro and in vivo. To expand autologous GBM-specific T cells, we subjected primary tumor cells, excised during surgery, to PBNP-PTT ex vivo. We then cultured these treated tumor cells with autologous DCs from the patient and stimulated T cells with the primed DCs. In proof-of-concept studies with tumor samples from GBM or metastatic brain tumors, we demonstrated that the PBNP-PTT platform expanded both CD4+ and CD8+ autologous T cells (n=5 patients). These T cells secreted IFN-ɣ specifically and dose-dependently (2.5-fold over controls) and exhibited specific cytolytic activity against target tumor cells (14.8-20.6% killing after 4 h co-culture). TCR sequencing revealed increased Simpson clonality (0.03 to 0.17) and maximum productive frequency (1.7 to 12.0) post-expansion, indicating selective expansion of TCR clones. Immunopeptidomics showed that DCs primed with PBNP-PTT-treated tumor cells present distinct antigens, facilitating personalized tumor-specific antigen presentation. These findings support using PBNP-PTT to stimulate and expand tumor-specific T cells for autologous ATCT in GBM.

Chimeric antigen receptor T cell (CAR T) therapy has demonstrated remarkable efficacy in hematologic malignancies but has struggled to achieve comparable success in solid tumors. A key obstacle is the extracellular matrix (ECM) in solid tumors, which significantly impedes CAR T cell infiltration. In clinical trials, neuroblastoma (NB) has shown responsiveness to GD2-directed CAR T therapy, however, the failure of GD2.CAR Ts to effectively clear bulky disease - characterized by dense ECM - highlights the critical challenge of infiltration. In this study, we demonstrate that GD2.CAR Ts exhibit a unique infiltration-restriction compared to other CAR Ts and endogenous T cells. A separate analysis of clinical datasets identified MMP7 and SPP1 (OPN) as candidate genes to improve the infiltration of GD2.CAR Ts as these were upregulated in tumor-infiltrating leukocytes. MMP-7 and OPN overexpression enhanced CAR T extravasation (p < .001) and interstitial movement (p < .05) in ECM-dense environments in vitro. Overexpression of either OPN (p < .0001) or MMP-7 (p < .001) improved tumor infiltration in a xenograft model of NB. This resulted in improved tumor control (94% reduction in tumor burden, p < .05) and a survival extension in OPN-GD2.CAR T treated mice compared to unmodified GD2.CAR Ts (median of 148 days, p < .05). OPN overexpression did not increase off-target infiltration into healthy tissues or promote tumor metastasis, highlighting its potential for safe therapeutic application. Our study provides a framework for further exploration of gene modifications to improve CAR T infiltration and efficacy in solid tumors and identifies SPP1 as a candidate gene to improve GD2.CAR T treatment of bulky tumors. ### Competing Interest Statement The authors have declared no competing interest.

Tumor-associated macrophages (TAMs) exhibit hybrid phenotypes ranging between M1-like (anti-tumor) and M2-like (pro-tumor) macrophages. In most tumor types, a higher M1/M2 ratio favors antitumor immunity. Therefore, strategies to enhance the M1/M2 ratio can potentially alter the tumor microenvironment (TME) towards antitumor immunity. These strategies include reprogramming resident TAMs, altering the fate of tumor-infiltrating monocytes towards M1-like macrophages, and finally, adoptive cell therapy (ACT) with M1-like macrophages. A significant challenge with the above strategies is to retain the antitumor properties of M1-like macrophages in an overwhelmingly immunosuppressive TME. In this study, we demonstrate a macrophage-based ACT where treatment with HDAC6 inhibitors (HDA6is) retains the M1-like characteristics while significantly suppressing the tumor growth in syngeneic SM1 murine melanoma and with radiation therapy (RT). We reprogrammed GFP-expressing antitumor M1 macrophages ex-vivo with HDAC6 inhibitors to lock into the M1 phenotype and administered intratumorally as ACT in the syngeneic SM1 murine melanoma model and humanized NSG-SGM3 melanoma model. We performed histological analysis of tumors for macrophage markers, immune phenotyping of infiltrated immune cells, single-cell secretome analysis of tumor macrophages, and single-cell RNA-seq analysis of CD45+ immune cell populations. Initially, tail vein injected (i.v) administration of luciferase-expressing monocytes in combination with HDAC6i reached the tumors and diminished SM1 tumor growth compared to the control group. On the contrary, luciferase-expressing M1 macrophages failed to reach the tumor as visualized by IVIS imaging, suggesting that i.v is not an effective route for macrophage therapy. Whereas, intratumor HDAC6i treated M1 macrophage ACT resulted in diminished tumor growth. Single-cell (Sc) RNA-seq analysis of CD45+ sorted immune cells revealed a significant increase in the M1/M2 macrophage ratio in concurrence with flow cytometry data. NicheNet cell-cell interaction analysis indicated that M1-like TAMs activated infiltrating T-cells and monocytes through ligand-receptor interactions. Cell fate analysis of infiltrated monocytes indicated differentiation toward M1-like macrophages in ACT tumors. Flow cytometry analysis indicated an increase in CD8 effector T-cells. Sc-secretome analysis of F4/80 sorted tumor macrophages by the Isoplexis platform revealed polyfunctionality of HDAC6-treated M1 macrophages. Histological examination of tumor sections for macrophage phenotypic markers suggested that transplanted macrophages retained the M1 phenotype post-ACT in both SM1 murine and NSG-SGM3 melanoma tumor models. Our study comprehensively demonstrated the potential of reprogramming macrophages with HDAC6 inhibitors as a viable macrophage cell therapy for treating solid tumors. Satish Kumar Reddy Noonepalle, Maria Gracia Hernandez, Nima Aghdam, Nithya Gajendran, Manasa Suresh, Xintang Li, Abishek Sehgal, David Quiceno, Michael Berrigen, Tessa Knox, Karen Tan, Eduardo Sotomayor, Katherine Chiappinelli, Duncan Wardrop, Anelia Horvath, Anatoly Dritschilo, Rohan Fernandes, Karthik Musunuri, Alejandro Villagra. HDAC6 inhibitors reprogram macrophages in antitumor-adoptive cell therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2253.

Activation and exhaustion signatures are not dependent on MMP-7 or OPN expression.

OPN or MMP-7 expression enhances primary T cell movement through ECM.

Summary of gene sets used for candidate gene identification.

We describe the use of ultrasound image guidance to improve treatment outcomes when administering interstitial photothermal therapy (I-PTT), an experimental cancer treatment modality. I-PTT is a promising thermal therapy for tumors using intratumorally injected nanoparticle-based photothermal agents activated by an interstitially placed laser diffuser. We hypothesized that ultrasound-based image guidance yields improved tumor treatment outcomes in terms of tumor regression and survival by improving the accuracy of the placement of the laser fiber and nanoparticles within a tumor and facilitating more precise PTT delivery. To test this hypothesis, we assessed the effect of ultrasound-guided I-PTT (US I-PTT) on neuroblastoma, an aggressive solid tumor of childhood, using the 9464D syngeneic model in C57BL/6 mice. US I-PTT using Prussian blue nanoparticles activated by an interstitial cylindrical laser diffuser generated an equivalent in vivo thermal dose as blinded, non-image-guided I-PTT (B I-PTT). However, US I-PTT resulted in significantly higher treatment accuracy compared to B I-PTT, attributable to the image guidance. Importantly, this improved accuracy translated to improved treatment outcomes wherein mice treated with US I-PTT exhibited significantly improved tumor regression, tumor-free survival, and long-term survival compared to mice treated with B I-PTT. Further, histological analyses of the tumors post-PTT confirmed the advantages conferred by US I-PTT over B I-PTT for tumor control. These proof-of-concept results demonstrate the value of using ultrasound guidance for I-PTT treatment and the translational implications of this approach to provide a more accurate and effective treatment for neuroblastoma.

Chimeric antigen receptor (CAR) T-cell therapy has demonstrated remarkable efficacy against hematologic malignancies but has struggled to achieve comparable success in solid tumors. A key obstacle in solid tumors is the extracellular matrix (ECM), which impedes CAR T-cell infiltration. In clinical trials, neuroblastoma has shown responsiveness to GD2-directed CAR T-cell therapy; however, the failure of GD2.CAR T cells to effectively clear bulky disease-characterized by dense ECM-highlights the critical challenge of infiltration. In this study, we demonstrate that GD2.CAR T cells exhibit a unique infiltration restriction compared with other CAR T cells and endogenous T cells. A separate analysis of clinical datasets identified MMP7 and SPP1 [which encodes osteopontin (OPN)] as candidate genes to improve the infiltration of GD2.CAR T cells as these were upregulated in tumor-infiltrating leukocytes. MMP-7 and OPN overexpression enhanced CAR T-cell extravasation and interstitial movement in ECM-dense environments in vitro. Overexpression of either OPN or MMP-7 significantly improved tumor infiltration in a xenograft model of neuroblastoma. This resulted in improved tumor control and a survival extension in OPN-GD2.CAR T cell-treated mice compared with unmodified GD2.CAR T cells. OPN overexpression did not increase off-target infiltration into healthy tissues or promote tumor metastasis, highlighting its potential for safe therapeutic application. Our study provides a framework for further exploration of gene modifications to improve CAR T-cell infiltration in solid tumors and identifies OPN as a candidate to explore in this regard. See related Spotlight by Gasparetto and Chiarle, p. 1698.

Abstract Background: CAR-T therapy has been largely ineffective in solid tumors because the dense, rigid, extracellular matrices (ECMs) and basement membranes (BMs) that encapsulate these tumors exclude T cells. Tumor-specific ECMs and BMs, made of type I and type IV collagen respectively, are heavily cross-linked and characterized by small pores. T cell infiltration into tumors relies upon the enzymatic degradation of these matrix fibers, and adhesion molecule-mediated movement. Gene expression analysis of tumor infiltrating leukocytes from patient samples showed that MMP-7, an enzyme that degrades type IV collagen, and osteopontin (OPN), a molecule that interacts with adhesion molecules to mediate movement, were highly upregulated in infiltrating cells compared to those that were tumor adjacent, suggesting they may be important in navigating tumor-specific environments. We hypothesized that overexpressing MMP-7 and/or OPN in CAR-T cells will enhance their infiltration into solid tumors. Methods: We overexpressed MMP-7 and OPN in second-generation CAR-Ts (GD2-28Z and HER2-28Z) and evaluated their infiltration in vitro using Halo invasion assays. T cell movement out of type IV collagen and into tumor droplets, composed of tumor targets polymerized in type I collagen, was visualized over time to characterize T cell movement through the tumor-specific BM and ECM, respectively. The expansion, phenotype, and cytotoxicity of OPN and MMP-7-modified GD2-28Z CAR-Ts was compared to unmodified GD2 CAR-Ts by counting, flow cytometry and chromium release, respectively. We then used a xenograft model of neuroblastoma to determine if increases in GD2 CAR-T infiltration led to reduced tumor burden. Results: In vitro, MMP-7 overexpression increased GD2-28Z (2.05-fold, p<0.0001) and HER2-28Z (3.16-fold, p<0.0001) CAR-T infiltration, and OPN overexpression increased GD2-28Z (1.20-fold, p=0.0496) and HER2-28Z (1.49-fold, p=0.002) CAR-T infiltration. In vivo, 52 days after CAR-T infusions, mice treated with OPN-modified GD2-28Z CAR-Ts had reduced tumor burden (0.206 cm, ±0.21) compared to GD2-28Z CAR-Ts (0.903 cm, ±0.33, p=0.0449). Conclusions: The overexpression of MMP-7 or OPN in second-generation CAR-Ts enhances CAR-T infiltration in vitro, and the expression of these genes does not impair CAR-T expansion, phenotype or cytotoxicity. The overexpression of OPN in GD2-28Z CAR-T enhances tumor clearance in a xenograft model of neuroblastoma, and may increase overall survival. Citation Format: Stacey N. Van Pelt, Bilal Omer, Lindsay Talbot, Cliona Rooney, Rohan Fernandes, Candise Tat, Mark White. Tunneling CARs: Gene modifications to enhance CAR-T infiltration into solid tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 42.

Background & AimGlioblastoma (GBM) is a deadly disease with a relative five-year survival rate of 7.5%, due in part to extensive intratumoral heterogeneity. Because of the poor prognosis for patients with GBM, there is an urgent need for therapies that address GBM heterogeneity and generate durable and persistent remission. In response to this need, we have generated an autologous T cell product targeting multiple antigens using immunostimulatory photothermal Prussian blue nanoparticle-based photothermal therapy (PBNP-PTT). In recently published studies, we demonstrated that T cells expanded using the PBNP-PTT-based method are specific and cytolytic against GBM cell lines both in vitro and in vivo. Acknowledging the limitations of adoptive T cell therapies (ATCTs) for solid tumors and the constraint of chimeric antigen receptor (CAR) T cells to target limited antigens, here we aim to develop autologous and personalized GBM-specific T cells for ATCT using primary tumor cells and peripheral blood sourced from patients with GBM.Methods, Results & ConclusionTo facilitate the expansion of autologous GBM-specific T cells, we subjected primary tumor cells excised during surgery to PBNP-PTT ex vivo. Next, we cultured the PBNP-PTT-treated tumor cells with autologous DCs isolated from the patient, and stimulated autologous T cells with the primed DCs. In proof-of-concept studies using tumor samples from patients with GBM or metastatic brain tumors, we demonstrated that the PBNP-PTT-based platform facilitated the expansion of both CD4+ and CD8+ autologous T cells (n=5 patients). Upon co-culture with the target tumor cells, these T cells secreted IFN-γ in a specific and dose-dependent manner (2.5-fold over controls). Further, T cells expanded using PBNP-PTT elicited specific cytolytic activity against target tumor cells (14.8-20.6% killing after 4 h co-culture). TCR sequencing analysis revealed increased Simpson clonality (0.03 to 0.17) and increased maximum productive frequency (1.7 to 12.0) before and after expansion, suggesting that the PBNP-PTT platform selectively expands a subset of TCR clones. Further, immunopeptidomics analysis demonstrated that DCs primed with PBNP-PTT-treated tumor cells present distinct antigens, suggesting that this method facilitates the presentation of personalized tumor-specific antigens for T cell engagement. These findings provide proof-of-concept data supporting the use of PBNP-PTT to stimulate and expand tumor-specific T cells for use as an autologous ATCT approach for GBM.

INTRODUCTION: Adoptive T cell therapy (ATCT) has successfully treated hematological malignancies and is currently under investigation for solid tumor therapy. In contrast to existing chimeric antigen receptor (CAR) T cell and/or antigen-specific T cell approaches, which target defined antigens, our PBNP-based approach can target a broad repertoire of antigens in GBM in an antigen-agnostic manner. METHODS: We treated GBM cells with PBNP-based photothermal therapy (PBNP-PTT) prior to culturing with dendritic cells (DCs), and subsequent stimulation of T cells. RESULTS: When PBNP-PTT was administered to U87 cells at an immunogenic thermal dose, we effectively expanded U87-specific T cells. DCs cultured ex vivo with PBNP-PTT-treated U87 cells enabled 15- 30-fold expansion of CD4+ and CD8+ T cells. Upon co-culture with target U87 cells, these T cells secreted IFN-G in a specific and dose-dependent manner (up to 647-fold over controls). T cells manufactured using PBNP-PTT elicited specific cytolytic activity against target U87 cells (33-93% killing at an E:T ratio of 20:1), while sparing normal human astrocytes. Similarly, PBNP-PTT on SNB19 cells resulted in a 7-39-fold expansion of T cells, which elicited 25-66% killing of SNB19 cells at an E:T ratio of 20:1. U87-luc cells were then orthotopically inoculated into NSG mice to establish xenograft GBM, and U87-specific T cells developed via PBNP-PTT were intracranially injected. These T cells eliminated U87 tumors in vivo and significantly improved mouse survival compared to mice treated with PBS (p = 0.0047) or non-specific T cells (p = 0.0123) from the same donor. CONCLUSIONS: These findings provide proof-of-concept data supporting the use of PBNP-PTT to stimulate and expand tumor-specific T cells ex vivo for potential use as an ATCT approach for patients with GBM. We are currently using patient-derived tumor samples to generate IND-enabling data.

Abstract The interplay between tumor cells and immune cells within the tumor microenvironment (TME) dictates protumor or antitumor immune responses. Tumor-associated macrophages (TAMs) respond to signals of the TME and exhibit a spectrum of phenotypes ranging between M1 (antitumor) and M2 (protumor) macrophages. In most tumor types, including melanoma, the balance between M1 and M2 macrophages is critical with a higher M1/M2 ratio favoring antitumor immunity. Therefore, strategies enhancing the M1/M2 ratio can significantly alter the TME towards antitumor immunity. In this study, we administered luciferase and GFP-expressing M1 macrophages as an adoptive cell therapy (ACT) through intravenous (i.v) and intratumor routes respectively, into mice bearing SM1 murine melanoma tumors to determine the effective treatment modality. We reprogrammed antitumor M1 macrophages ex-vivo with HDAC6 inhibitors to lock into the M1 phenotype and administered intratumorally as ACT in the syngeneic SM1 murine melanoma and humanized NSG-SGM3 melanoma models. We performed histological analysis of tumors for macrophage markers, immune phenotyping of infiltrated immune cells, single-cell secretome analysis of tumor macrophages, and single-cell RNA-seq analysis of CD45+ immune cell populations to demonstrate the benefit of macrophage ACT. Tail vein injected, luciferase-expressing M1 macrophages localized in the lungs and spleen, failed to reach the tumor as visualized by IVIS imaging, suggesting that i.v administration is ineffective for macrophage therapy. On the contrary, intratumor M1 macrophage ACT resulted in diminished tumor growth. Single-cell RNA-seq analysis of the CD45+ sorted tumor-associated immune cell population revealed distinct macrophage subsets and a significant M1/M2 macrophage ratio increase. NicheNet cell-cell interaction analysis indicated that M1-like TAMs activated infiltrating T-cells and monocytes through ligand-receptor interactions. Trajectory analysis of infiltrated monocytes indicated differentiation toward inflammatory macrophages in ACT tumors. Flow cytometry analysis corroborated that ACT increased the M1/M2 macrophage ratio and an increase in CD8 effector T-cells. Furthermore, HDAC6i-treated macrophages increased antigen cross-presentation. Single-cell secretome analysis of F4/80+ TAMs by the Isoplexis platform revealed polyfunctionality of HDAC6-treated M1 macrophages secreting inflammatory cytokine Tnfa and T-cell recruiting chemokine Cxcl10. Histological examination of tumor sections for macrophage phenotypic markers suggested that transplanted macrophages retained the M1 phenotype post-ACT in both SM1 murine and NSG-SGM3 melanoma tumor models. We demonstrated the potential of reprogramming macrophages ex vivo with HDAC6 inhibitors as a feasible macrophage cell therapy to treat solid tumors. Citation Format: Satish Kumar Reddy Noonepalle, Nithya Gajendran, Manasa Suresh, Xintang Li, Maria D. Hernandez, Christian Zevallos Delgado, Nima Aghdam, Michael Berrigen, Tessa Knox, Karen Tan, Marie Durr, Eduardo Sotomayor, Katherine B. Chiappinelli, Duncan Wardrop, Anelia Horvath, Brett A. Shook, Norman H. Lee, Anatoly Dritschilo, Rohan Fernandes, Karthik Musunuri, Maho Shibata, Alejandro Villagra. Reprogramming tumor microenvironment with intratumor macrophage adoptive cell therapy in melanoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 5247.

Aim: We investigate combining Prussian Blue nanoparticles (PBNPs), as photothermal therapy (PTT) agents, with agonistic CD137 antibodies (alpha CD137) on a single nanoparticle platform to deliver non-toxic, anti-tumor efficacy in SM1 murine melanoma. Methods: We electrostatically coated PBNPs with alpha CD137 (alpha CD137-PBNPs) and quantified their physicochemical characteristics, photothermal and co-stimulatory capabilities. Next, we tested the efficacy and hepatotoxicity of PTT using alpha CD137-PBNPs (alpha CD137-PBNP-PTT) in SM1 tumor-bearing mice. Results: The alpha CD137-PBNPs retained both the photothermal and agonistic properties of the PBNPs and alpha CD137, respectively. In vivo, SM1 tumor-bearing mice treated with alpha CD137-PBNP-PTT exhibited a significantly higher survival rate (50%) without hepatotoxicity, compared with control treatments. Conclusion: These data suggest the potential utility of co-localizing PBNP-PTT with alpha CD137-based agonism as a novel combination nanomedicine.

Abstract The immunosuppressive nature of ovarian cancer (OC) allows these tumors to evade the immune system, contributing to late-stage diagnoses and poor clinical outcomes for patients. However, a higher number of tumor-infiltrating lymphocytes is associated with improved clinical outcomes in OC. One type of lymphocyte, the T cell, can traffic to the tumor and kill tumor cells. We have shown that treating tumor cells with Prussian Blue nanoparticles and photothermal therapy (PBNP-PTT) results in immunogenic cell death, a form of cell death that is visible to the immune system, including T cells. PBNP-PTT-treated tumor cells were then used to expand tumor-specific T cells from partially genetically matched healthy donors against breast and brain cancer cell lines. These expanded T cells were better able to recognize and kill the target tumor cell lines than bulk unexpanded T cells from the same donors. Hence, we hypothesized that using PBNP-PTT-treated OC cells would allow us to expand a polyclonal tumor-specific T cell product that is cytolytic against OC cells in vitro and in vivo.We first evaluated whether multi-antigen OC-specific T cells could be expanded from partially genetically matched healthy donors and confirmed that PBNP-PTT elicited immunogenic cell death in the HLA-A*02:01 human OC cell lines Hey and TykNu. Using our established in vitro T cell expansion protocol, we then successfully expanded T cells from multiple HLA-A*02:01 healthy donors (n=3) against these cell lines. Functional analyses—including interferon-gamma ELISPOT, multi-color flow cytometry, and cytotoxicity assays—demonstrated that the expanded T cells had enhanced anti-tumor responses against OC cell lines compared to bulk unexpanded T cells derived from the same donors. Current work is expanding this approach to a syngeneic murine setting in C57BL/6J mice. Moreover, we will evaluate HLA-matched T cells and tumor cells from patients with OC ex vivo. Collectively, these efforts serve as a novel approach towards a curative immunotherapy for OC. Citation Format: Erin E. Grundy, Abigail Lee, Samantha Chin, Melissa Hadley, Danielle Schmidt, Loretta Wang, Olivia Cox, Khadra Omar, Catherine M. Bollard, Rohan Fernandes, Katherine B. Chiappinelli. Generation of multi-antigen specific T cells for ovarian cancer using Prussian Blue nanoparticles and photothermal therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2628.