Cell Therapy Research Articles

TMIC-42. CONQUERING SPP1+ MYELOID-DRIVEN RESISTANCE OF GLIOBLASTOMA TO IL13RA2-TARGETED CAR T CELL THERAPY

Abstract In the evolving landscape of glioblastoma (GBM) therapy, the recent proliferation of clinical trials exploring chimeric antigen receptor (CAR) T cell interventions has garnered attention, although their therapeutic efficacy remains limited. Despite recent progress in elucidating the role of the GBM microenvironment in immunotherapy resistance, particularly regarding intercellular networks and macrophages, significant ambiguities endure, impeding a comprehensive understanding of resistance mechanisms amidst the patient-specific heterogeneity of the tumor microenvironment (TME). In this study, we utilized single-cell RNA sequencing to analyze tumors from 41 glioma patients undergoing IL13Rα2-targeted CAR T cell therapy. Our findings unveiled heightened suppressive extracellular network signatures, particularly SPP-1, predominantly observed within myeloid cells from non-responsive patients. The clinical significance of both SPP1+ myeloid cell abundance and overall expression levels revealed associations with patient outcomes. Subsequently, transcriptomic insights were translated to myeloid cell functions in patients exhibiting high versus low SPP1 expression. SPP1-expressing macrophages displayed diminished antigen presentation, phagocytosis, and cell-mediated cytotoxicity, along with augmented extracellular matrix biogenesis and degradation, iron efflux, and suppressive interleukin pathways. Moreover, SPP1-high macrophages exhibited enhanced ligand-receptor interactions, implying a predisposition towards suppressive activities. Immunometabolism pathways, including lipid and ketone body biogenesis and regeneration, were upregulated in SPP1-expressing macrophages, indicating an increased reliance on non-glycolysis-mediated ATP production. To assess translational implications, the therapeutic efficacy of SPP1 blockade was evaluated in a syngeneic glioma model. Blockade of SPP1 with an anti-SPP1 antibody prior to CAR T cell infusion effectively mitigated the immunosuppressive milieu, reversing resistance in preclinical GBM models to murine IL13Rα2-targeted CAR T cell therapy. Subsequent profiling of tumors post-treatment provided insights into TME alterations following SPP1 blockade therapy. This investigation illuminates the intricate interplay between the TME and CAR T cell therapy resistance in GBM, underscoring SPP1 as a promising therapeutic target to surmount CAR T cell resistance and enhance treatment outcomes.

Charge-based immunoreceptor signalling in health and disease.

Immunoreceptors have crucial roles in sensing environmental signals and initiating immune responses to protect the host. Dysregulation of immunoreceptor signalling can therefore lead to a range of diseases, making immunoreceptor-based therapies a promising frontier in biomedicine. A common feature of various immunoreceptors is the basic-residue-rich sequence (BRS), which is a largely unexplored aspect of immunoreceptor signalling. The BRS is typically located in the cytoplasmic juxtamembrane region of immunoreceptors, where it forms dynamic interactions with neighbouring charged molecules to regulate signalling. Loss or gain of the basic residues in an immunoreceptor BRS has been linked to severe human diseases, such as immunodeficiency and autoimmunity. In this Perspective, we describe the role of BRSs in various immunoreceptors, elucidating their signalling mechanisms and biological functions. Furthermore, we highlight pathogenic mutations in immunoreceptor BRSs and discuss the potential of leveraging BRS signalling in engineered T cell-based therapies.

CTIM-20. IDENTIFYING CANDIDATE ANTIGEN TARGETS FOR PEDIATRIC BRAIN TUMOR IMMUNOTHERAPIES THROUGH COMPREHENSIVE IMMUNOGENOMIC ANALYSIS

Abstract BACKGROUND Identifying tumor rejection antigens continues to be a significant challenge in the development of effective immunotherapies and their application to pediatric brain tumors. METHODS To identify candidate antigen targets for Medulloblastoma (primary MB, n=170; recurrent MB, n=21), Diffuse Intrinsic Pontine Glioma (DIPG, n=10), and Pediatric High-Grade Astrocytoma (HGA, n=12) for adoptive cellular therapy, we conducted a comprehensive immunogenomic analysis of the transcription profiles of the most aggressive pediatric brain tumors and performed in silico antigen prediction across a wide range of antigen classes. IMPORTANCE OF THE STUDY Pediatric brain tumors are heterogeneous diseases, and current standard treatments do not adequately address this variability. Newer and safer patient-specific treatment approaches are needed for high-risk patients who do not respond to standard therapies. Cellular immunotherapy could be crucial for improving survival and reducing morbidity. For an effective immune response against these brain tumors, appropriate tumor antigens must be targeted. While subgroup-specific genetic mutations in medulloblastoma patients have been reported, the immunogenicity of these genetic alterations remains unknown. Using a custom antigen prediction pipeline, we identified potential tumor rejection antigens with significant implications for the development of cellular therapies for MB, DIPG, and HGA. RESULTS Antigen prediction analysis revealed that most patients express few candidate neoantigen targets that pass all filtering criteria. Overall, the proportion of predicted tumor-associated antigens (TAAs) was higher than that of neoantigens and gene fusions for all molecular subtypes, except for SHH, which exhibited a higher neoantigen burden. Notably, cancer-testis antigens and previously unrecognized neurodevelopmental antigens were found to be expressed in most patients across all medulloblastoma subgroups. Although the absolute immune cell content is predicted to be low, immune gene-signature analysis revealed subgroup-specific differences.

IMMU-28. LOW-INTENSITY PULSED ULTRASOUND COMBINED WITH POLY-ICLC AND INTERLEUKIN-2 UNLOCKS THE IMMUNE PRIVILEGE OF THE BRAIN BY PROMOTING EPITOPE SPREADING AND ACTIVATION OF CNS-SPECIFIC NAÏVE T CELLS

Abstract Immunotherapies targeting glioma, such as chimeric antigen receptor (CAR) T cell therapies, offer new therapeutic avenues; however, their application has yielded limited success in clinical trials so far. Due to the antigenic heterogeneity of gliomas, targeting a few or several antigens still results in recurrence with “antigen-loss” tumors, indicating the need to engage the endogenous immune system to eliminate these therapy-resistant tumor cells. However, natural anatomical barriers and an immunosuppressive environment allow brain tumors to evade detection by the endogenous immune system. Low-intensity pulsed ultrasound combined with microbubbles (LIPU/MB) has recently been shown to transiently open the blood-brain barrier (BBB), enabling penetration of cells and drugs into the CNS. Using intravital two-photon imaging, two-photon microscopy, multiparametric spectral flow cytometry, and bone marrow chimera mice, alongside blood samples from glioblastoma patients treated with LIPU/MB, we observed that LIPU/MB promotes migration of CNS antigen-presenting cells from the perivascular space into the periphery. To assess the immunological relevance of LIPU/MB-mediated CNS antigen presentation to the peripheral immune system, we have developed new transgenic mice (GFAP-minigene mice) with an intact BBB expressing four immunogenic peptides under the CNS-specific GFAP promoter. In GFAP-minigene mice, LIPU/MB combined with toll-like receptor 3 agonist poly-ICLC and Interleukin-2 promoted priming, activation, and homing of naïve CNS-specific T cells into the brain parenchyma. Cytotoxic T cell activity in the brain was accompanied by MHC-I and MHC-II epitope spreading, mirrored by the occurrence of endogenous T cells in the brain, deep cervical lymph nodes, and spleen harboring other CNS-(minigene) antigen-specific T cell receptors. Collectively, our findings suggest that LIPU/MB combined with poly-ICLC can overcome inherent limitations of immunotherapy by enhancing CNS epitope spreading and activation of naïve CNS-antigen-specific T cells within the context of an intact blood-brain barrier.

FRAGMENTED THIGH INJURY: TREATMENT WITH PREBIOTICS, NANOPARTICLES, AND CELL THERAPY

The purpose of this study was to investigate the possibility of staged local treatment of an infected mine-explosive wound using a gel containing Bacillus spp., cerium oxide nanoparticles, and cell therapy. Object and Methods. The article presents a clinical case demonstrating the effective staged treatment of a 57-year-old patient with a fragmentary fracture of the right femur and a large purulent wound of the right thigh. The patient underwent Ex-fix rod-type femur-tibia stabilization and resection of a post-traumatic aneurysm of the right superficial femoral artery. In the first stage of treatment, two sessions of negative pressure wound therapy (NPWT) were applied, with pressures ranging from 75-115 mm Hg and a session duration of 4-5 days. In the second stage, local treatment involved a combination of antiseptics and probiotics (Bacillus spp.) alongside cerium oxide nanoparticles. By the 7th day, the eradication of pathogenic microflora was achieved, allowing for the third stage of treatment—application of autologous mesenchymal stem cells. Results: The treatment of combat trauma has become increasingly relevant in Ukraine and globally due to its complexity and the rising proportion of multiple and combined injuries, which account for 25-60% of cases. Infectious complications occur in 25% of combat trauma cases and are responsible for 70% of related deaths. Most structural and functional disorders associated with wound infections develop at the time of injury, intensify over time, and require immediate surgical intervention, improvements in local treatment methods, and new approaches for wound closure. Conclusions: The use of a combination of probiotics and NPWT therapy led to a rapid reduction of the inflammatory process. Clinical observations showed accelerated wound cleansing from fibrin and necrotic tissue. The combined treatment significantly reduced the size and depth of wound due to the formation of active pink granulation tissue (observed by days 8-9) and marginal epithelialization by days 10-13. During this period, no pathogenic flora were detected, allowing for the subsequent application of autologous mesenchymal stem cells. This treatment approach reduced the need for antibacterial drugs and prevented the need for additional surgical interventions. The inclusion of cerium oxide nanoparticles was an effective enhancement for cell transplantation, facilitating stem cell delivery and contributing to the treatment of large wounds.

IMMU-01. DE NOVO-DESIGNED BINDER-BASED CAR T CELLS ENABLE EFFECTIVE TARGETING OF GLIOBLASTOMA

Abstract Glioblastoma is a devastating disease with poor responses to all current therapies. Chimeric antigen receptor (CAR) T cell therapy has achieved impressive success against hematological malignancies but produces responses in only a limited number of glioblastoma patients. Here, we developed a novel platform for CAR constructs using de novo designed protein binders to target tumor antigens. We firstly designed a binder CAR targeting epidermal growth factor receptor (EGFR), which is highly expressed in glioblastoma, to validate the feasibility of our platform. Our EGFR binder CAR design promoted CAR T cell proliferation, cytotoxic cytokine secretion and exhaustion resistance, leading to better antitumor performance both in vitro and in vivo. Bulk and single-cell transcriptional profiling of binder CAR T cells revealed an underlying mechanism involving enhanced effector activity and reduced exhaustion responses. Mechanismly, the binder CARs exhibited higher surface expression and greater resistance to degradation triggered by tumor antigens. Moreover, we designed and engineered a novel CD276 binder CAR T cell product and verified its antitumor function, further demonstrating the general applicability of our binder-based CAR platform. Overall, our study provides an alternative avenue for the design of a CAR antigen-binding domain to potentiate CAR T cell antitumor efficacy.

EXTH-63. FOCUSED ULTRASOUND HYPERTHERMIA MEDIATED CONTROL OF THERMAL RESPONSIVE CAR T CELL ACTIVITY IN BREAST CANCER BRAIN METASTASIS

Abstract HER2-targeted therapies are promising treatment options for metastatic breast cancer and have improved the median overall survival. However, breast cancer brain metastasis (BCBM), observed in up to 50% of HER2-positive breast cancer patients, remains a clinical challenge. While Chimeric Antigen Receptor (CAR) T cell therapy is promising therapeutic strategy to address the unmet need in treating BCBM, improving its effectiveness and safety is critical for clinical translation. We hypothesized that spatially and temporally controlled activity of CAR-T cells with a thermal gene switch (TS) under MRI-guided focused ultrasound (MRgFUS) would potentiate anti-tumor responses in difficult-to-treat BCBM. To test our hypothesis, we developed a closed-loop controlled MR-thermometry (MRTI) based FUS system (MRgFUS) and αHER2 CAR-T cells engineered with TS expressing the reporter gene firefly luciferase (TS.Fluc) or bi-specific engager on NKG2D ligands (TS.BTE) upon MRgFUS-hyperthermia (41.5°C). In vivo quantification of TS activity from intratumorally delivered TS.Fluc αHER2 CAR-T cells in HER2+ BCBM mice model (HER2+ MDA-MB-468 in NSG mice with primary human CAR-T cells) followed by pulsed MRgFUS-hyperthermia resulted in a ~10-fold increase in luminescent activity compared to unheated mice. To assess longitudinal activation, we quantified TS activity with a second sonication 4 days post the initial FUS treatment. Prior to the second sonication, TS.Fluc expression had returned to baseline levels, while following it robust TS.Fluc expression was observed again, demonstrating that MRgFUS can activate engineered T cells with TS in brain tumors with high spatiotemporal control. Subsequently we investigated the therapeutic efficacy of intratumorally delivered TS.BTE αHER2 CAR T cells in mixed tumor model of heterogeneous HER2 MDA-MB-468 (75% HER2+) and observed significant tumor regression of HER2- tumor cells in the treated group compared to controls, demonstrating the potential of the proposed strategy to noninvasively drive the local production of key transgenes and potentiate anti-tumor responses in difficult-to-treat BCBM.

IMMU-10. IDENTIFICATION OF A NEW THERAPEUTIC ANTIGEN FOR GLIOBLASTOMA USING A MONOCLONAL ANTIBODY LIBRARY FROM PATIENT-DERIVED TUMOR SPHERES

Abstract Glioblastoma (GBM) has a poor prognosis despite intensive treatment by surgery, radiation, and chemotherapy, thus new treatment strategies are urgently needed. Various innovative cancer therapies targeting cancer-specific cell surface antigens, such as chimeric antigen receptor T cell therapy, are developed and more cell surface antigens that is highly specific for GBM cells are needed. Although extensive transcriptome analyses or proteomic analysis of GBM cells were performed, few transcripts specific for GBM cells have been identified. However, GBM cell-specific antigen epitopes formed by post-translational modifications of proteins may have been missed by transcriptome or proteomic analysis, and could still be discovered by thoroughly searching for cancer-specific monoclonal antibodies. In this study, we aimed to identify GBM-specific antigens using a monoclonal antibody library from patient-derived tumor spheres, create CAR-T cells against the antigen, and check antitumor efficiency of the CAR-T cells. Approximately 25,000 monoclonal antibody-producing hybridomas were establishment using BALB/c mice and patient derived tumor spheres. Among them, a antibodies that bind to plural glioblastomas and not to plural normal brain cells were selected and we identified the antigen as Prostaglandin F2 receptor negative regulator (PTGFRN) by the expression cloning method. PTGFRN witch is expressed in several cancers regulate migration, angiogenesis, and cell polarity and decreases the radiosensitivity of GBM cells. We created CAR-T cells against the antigen and co-cultured it with GBM cells, which significantly increased the production of IL-2 and INF-γ. Furthermore, the CAR-T cells injected intracranial in an orthotopic xenograft model with patient-derived GBM cells, significantly reduced tumor growth. PTGFRN CAR-T-cell therapy may be a potential novel therapeutic target for GBM.

TMIC-22. MYELOID CELLS INDUCE IMMUNOSUPPRESSION AND HYPORESPONSIVENESS OF CHIMERIC ANTIGEN RECEPTOR T CELLS IN THE NEURONAL MICROENVIRONMENT OF GLIOBLASTOMA

Abstract Chimeric antigen receptor T (CART)-cell therapy has shown little success in glioblastoma and other solid tumors, unlike in hematologic malignancies. The immunosuppressive milieu and intratumoral heterogeneity are some obstacles limiting the effectiveness of CART-cell therapy in glioblastoma. Our goal in this study is to investigate how CART-cell transcriptional adaptations and cellular interactions in the glioblastoma microenvironment drive T-cell hyporesponsiveness. Using a human neocortical slice model, we performed physically interacting cell sequencing (PIC-seq) to investigate the transcriptional diversity of NKG2D CAR-T cells and control T-cells in the glioblastoma ecosystem. Through the integration of spatially resolved T-cell receptors sequencing and PIC-seq, we investigated the cellular and receptor-ligand interactions in CART-treated glioblastoma cells. Gene regulatory networks were reconstructed to identify key transcriptional regulators of CD8 CART-cell dysfunction. We used in-silico perturbation to understand transcriptional drivers that could potentially enhance the cytotoxic response of CAR-Ts. Primary patient-derived glioma cells were inoculated into human neocortical slices from surgical access tissue specimens. After 3 days of tumor growth, slices were treated with NKG2D CARTs or control T-cells. We observed that after 2 days of anti-tumor T-cell response in the CART group, tumor growth did not differ between conditions. After 7 days of treatment, PIC-seq was used to profile CARTs (tomato+) and tumor cells (GFP+) as well as physically connected cells (+/+). We identified a transcriptional shift towards T-cell exhaustion in CART cells compared to T-cells lacking the NKG2D construct. This transcriptional phenotype was driven by myeloid-CART and myeloid-tumor interaction. We identified that tumor-associated macrophages with enhanced phagocytic function in proximity to mesenchymal-like tumor cells located within hypoxic niches are the main drivers of T-cell dysfunction. Gene regulatory network analysis demonstrated that MAF and BACH2 are key transcriptional regulators of CART-cell function. Myeloid-tumor and myeloid-T-cell interactions promote an immunosuppressive microenvironment and hyporesponsive T-cells in glioblastoma. Our data has the potential to catalyze the re-engineering and optimization of CART-cell therapies with sustained cytotoxic effect against glioblastoma.

Advances in CAR NK Cell Therapy for Targeting and Eradicating Circulating Tumor Cells: Challenges and Solutions for Aging Patients Mini Review

Chimeric Antigen Receptor (CAR) Natural Killer (NK) cells represent a promising advancement in cancer immunotherapy, particularly for targeting circulating tumor cells (CTCs) and preventing metastasis. This review examines the latest developments in CAR NK cell therapy, including diverse NK cell sources, genetic engineering techniques, and dual mechanisms of action. Targeting CTCs with CAR NK cells shows significant potential in aggressive cancers like triple-negative breast cancer (TNBC) and pancreatic cancer. The impact of aging on NK cell function, especially regarding cytotoxicity, cytokine secretion, and persistence, poses challenges for elderly patients, but strategies such as interleukin-15 and metabolic interventions offer potential solutions. The review also addresses current limitations, such as poor persistence in immunosuppressive microenvironments and low solid tumor infiltration, while proposing combination therapies to enhance effectiveness. Although still in earlier clinical stages compared to CAR T cells, CAR NK cells’ safety profile and MHC-independent recognition mechanisms make them a versatile therapeutic option. Future directions include optimizing NK cell persistence, improving infiltration, and developing age-adapted therapies for elderly patients. _________________________________________________________________________________________ Keywords: CAR NK cells, CAR T cells, Circulating tumor cells (CTCs), Cell Therapy, Aging

TRBC1-CAR T cell therapy in peripheral T cell lymphoma: a phase 1/2 trial.

Relapsed/refractory peripheral T cell lymphomas (PTCLs) are aggressive tumors with a poor prognosis. Unlike B cell lymphomas, treatment of PTCL has not benefited from advances in immunotherapy. This is largely due to a lack of suitable target antigens that discriminate malignant from normal T cells, thus avoiding severe immunosuppression consequent to depletion of the entire T cell compartment. We recently described a targeting strategy based on the mutually exclusive expression of T cell antigen receptor beta-chain constant domain (TRBC) 1 and 2. Selective targeting of the T cell antigen receptor beta-chain expressed by the (clonal) malignancy spares normal T cells expressing the other chain. The LibraT1 study is an ongoing, multicenter, international, single-arm phase 1/2 study of TRBC1-directed autologous chimeric antigen receptor (CAR) T cells (AUTO4) in relapsed/refractory TRBC1-positive PTCL. Primary objectives were assessment of safety and tolerability of AUTO4 infusion. Key secondary endpoints included efficacy, CAR T cell expansion and persistence. Here we describe the findings from dose escalation in LibraT1 in the first ten patients, in a non-prespecified interim analysis. AUTO4 resulted in low frequency of severe immunotoxicity, with one of ten patients developing grade 3 cytokine release syndrome. Complete metabolic response was observed in four of ten evaluable patients, with remissions being durable beyond 1 year in two patients. While an absence of circulating CAR T cells was observed, CAR T cells were readily detected in lymph node biopsy samples from sites of original disease suggesting homing to tumor sites. These results support the continuing exploration of TRBC1 targeting in PTCL. ClinicalTrials.gov registration: NCT03590574 .

CTIM-21. FIRST-IN-HUMAN PHASE I CLINICAL TRIAL USING 8R-70CAR T CELLS FOR NEWLY DIAGNOSED ADULT GLIOBLASTOMA

Abstract BACKGROUND To address the challenges of chimeric antigen receptor (CAR) T cell therapy in GBM, we utilized IL-8 release from tumor cells as a ‘GPS homing signal’ to enhance T-cell trafficking with a novel CD70-specific CAR design (8R-70CAR) that targets GBM. The 8R-70CAR offers several advantages: 1) Improved CAR T-cell trafficking, enhancing antitumor efficacy; 2) Reduced CD70, reshaping the tumor microenvironment (TME) and boosting endogenous immunity; 3) Utilized CD27 as the CAR, a co-stimulatory molecule that enhances T-cell functions; 4) Enabled CAR T cells to act as a sink to neutralize or remove IL-8, a pro-cancer chemokine, from the TME; and 5) Required only 120 ml of whole blood (not leukapheresis) for CAR T cell production. Preclinically, 8R-70CAR significantly enhanced CAR-T cell tumor migration and persistence, leading to complete tumor regression and sustained immune memory in a chemo/radiation/PD-1-blockade-resistant GBM model. These findings led to a phase-I trial (IMPACT trial: FDA-IND#23881, NCT05353530) using 8R-70CAR T cells for CD70 -positive newly diagnosed adult GBM. METHODS We aim to recruit 18 adult patients (3 + 3 dose-escalation design) with CD70-positive (>20%) tumors. The CAR T cells will be given intravenously after completing standard-of-care (SOC) therapy. Primary endpoints: Evaluate safety, tolerability, and dose-limiting toxicities (DLTs) to determine the maximum tolerated dose. Secondary endpoints: Assess preliminary efficacy. Immune monitoring and correlative studies will also be conducted. RESULTS To date, four patients have been enrolled in the IMPACT trial, with 2 eligible patients participating in treatment at dose level 1 (1x10^6/kg). One patient has completed treatment with 8R-70CAR T cells, experienced no DLTs, and is currently in follow-up. The remaining patient is under SOC therapy before receiving CAR T cell treatment. CONCLUSIONS This study aims to address current therapeutic gaps and offers options for implementing a novel CAR design for the development of a more effective therapeutic modality for GBM.

EXTH-55. ANTI-TUMOR ACTIVITY OF A NEW ONCOLYTIC ADENOVIRUS DELTA-24-RGDOX-IL15 CO-EXPRESSING OX40L AND IL-15

Abstract Accumulating evidence show that the interaction between oncolytic viruses (OVs) and host immune system plays an essential role in cancer virotherapy. Compared to lytic potency of OVs, enhancement of OV-mediated anti-tumor immunity has become a more attractive strategy to improve OV efficacy. To this end, we have reported that the third-generation oncolytic adenovirus (OA), Delta-24-RGDOX, which expresses the immune co-stimulator OX40 ligand (OX40L), is more potent to elicit anti-tumor immunity than the second-generation OA Delta-24-RGD. Since IL-15 activates T and NK cells and promotes persistence of CD8+ memory T cells, we hypothesize that co-expression of OX40L and IL-15 by OAs will further potentiate their efficacy. For this purpose, we constructed the next generation OA Delta-24-RGDOX-IL15. In cultured human and murine tumor cell lines, this new virus effectively expressed the two molecules and displayed comparable potency in viral replication and oncolysis as OAs expressing either of the two (Delta-24-RGDOX, Delta-24-RGD-IL15). Then, we tested the anti-tumor activity of Delta-24-RGDOX-IL15 (two doses, intratumorally, one week interval) in two syngeneic tumor models in C57BL/6 mice: 1) intracranial (i.c.) gliomas from GL261-5 cells; 2) subcutaneous (s.c.)/i.c. melanomas from B16 cells (virus injected into s.c. tumors only). We found Delta-24-RGDOX-IL15 was more efficacious than the other two OAs to inhibit tumor growth of both treated tumor and the untreated i.c. tumors in the second model, leading to long-term survivors (p < 0.05). Different from adoptive cell therapies with cells engineered to express IL-15, we haven’t observed any obvious toxicity with this new virus when it was injected intratumorally into either the i.c. or s.c. tumors. Further studies are in progress to analyze the modifications of the tumor microenvironment induced by the addition of IL-15 to Delta-24-RGDOX. We expect this virus to be a good candidate to be combined with CAR T cell therapies.

DDDR-29. MATRIX STIFFNESS INDUCED PHENOTYPE IN BRAIN METASTATIC BREAST CANCER CELLS DETERMINES RESPONSE TO THERAPY

Abstract Breast cancer is the most prevalent form of cancer, representing 12.5% of all cancer cases diagnosed worldwide. Breast cancer cells can metastasize to distant organs (i.e., brain) and remain dormant for extended periods of time. This aspect makes it very difficult to treat these cells as they exhibit reduced growth as well as resistance to therapy. Despite advances in our understanding of breast cancer metastasis, the specific mechanisms that enable dormant cancer cells to resist therapeutics, particularly in brain metastatic breast cancer (BMBC), remain unclear. Herein, we employed brain tissue mimetic hyaluronic acid (HA) hydrogels of varying stiffness (i.e., ~0.4 kPa vs. ~4.5 kPa) to investigate the response to chemo or targeted therapy in dormant versus proliferative BMBC cells. It was revealed that BMBC cells grown on soft HA hydrogels (~0.4 kPa) displayed a non-proliferative (i.e., dormant) phenotype and exhibited resistance to Paclitaxel (PTX) or Lapatinib (LAP). However, BMBC cells grown on stiff HA hydrogels (~4.5 kPa) displayed a proliferative phenotype and sensitivity to PTX or LAP. Furthermore, we found that resistance to therapy was due to the enhanced expression of the serum/glucocorticoid regulated kinase 1 (SGK1) gene, mediated, in part, via the p38 mitogen-activated protein kinase (MAPK) pathway. Consequently, SGK1 inhibition using a SGK inhibitor in BMBC cells cultured on soft HA hydrogels resulted in a dormant-to-proliferative switch as well as response to PTX or LAP. In sum, our study demonstrated that matrix stiffness plays a major role in the dormancy-associated therapy response, mediated, in part, via the p38/SGK1 pathway.

Polyphenol-Nanoengineered Monocyte Biohybrids for Targeted Cardiac Repair and Immunomodulation.

Myocardial infarction is one of the leading cause of cardiovascular death worldwide. Invasive interventional procedures and medications are applied to attenuate the attacks associated with ischemic heart disease by reestablishing blood flow and restoring oxygen supply. However, the overactivation of inflammatory responses and unsatisfactory drug delivery efficiency in the infarcted regions prohibit functional improvement. Here, a nanoengineered monocyte (MO)-based biohybrid system, referred to as CTAs @MOs, for the heart-targeted delivery of combinational therapeutic agents (CTAs) containing anti-inflammatory IL-10 and cardiomyogenic miR-19a to overcome the limitation of malperfusion within the infarcted myocardium through a polyphenol-mediated interfacial assembly, is reported. Systemic administration of CTAs@MOs bypasses extensive thoracotomy and intramyocardial administration risks, leading to infarcted heart-specific accumulation and sustained release of therapeutic agents, enabling immunomodulation of the proinflammatory microenvironment and promoting cardiomyocyte proliferation in sequence. Moreover, CTAs@MOs, which serve as a cellular biohybrid-based therapy, significantly improve cardiac function as evidenced by enhanced ejection fractions, increased fractional shortening, and diminished infarct sizes. This polyphenol nanoengineered biohybrid system represents a general and potent platform for the efficient treatment of cardiovascular disorders.

TMIC-09. DENDRITIC CELL DYSFUNCTION RESTRICTS TUMOR ANTIGEN SPREADING IN ADOPTIVE CELLULAR TRANSFER THERAPY-ESCAPED GLIOMAS: IMPLICATIONS FOR TUMOR IMMUNE EVASION

Abstract Dendritic cells (DCs) play a pivotal role in initiating anti-tumor immune responses. Our group has developed an adoptive cellular transfer therapy (ACT) that significantly increases the infiltration of DCs and T cells into tumors, markedly enhancing survival in murine brain tumor models. However, tumors develop mechanisms to restrict the immune surveillance and the efficacy of immunotherapy, leading to tumor escape. In this study, we identified a mechanism involving DC dysfunction in ACT-escaped brain tumors. The finding that DCs are enriched after ACT treatment prompts an investigation into whether DC function is impaired in ACT-escaped tumors. Our results showed that DCs from both primary and ACT-escaped tumors were dysfunctional in their ability to activate T cells when co-cultured with tumor-reactive T cells or OT1 T cells. RNA-seq data revealed that, compared to primary tumor DCs, ACT-escaped tumor DCs exhibited a more pronounced reduction in conventional DC markers and antigen-presenting genes, alongside increased tolerance markers. Gene set enrichment analysis indicated that epithelial-mesenchymal transition (EMT) and hypoxia pathways were significantly enriched in ACT-escaped tumor DCs, suggesting divergent DC dysfunction mechanisms in primary versus escaped tumors. The anti-tumor role of DCs is primarily exerted through the activation of tumor-reactive T cells. Therefore, we assessed the cytotoxicity and exhaustion status of T cells in tumors. Surprisingly, we found that T cells from ACT-escaped tumors exhibited significantly higher levels of cytotoxicity and lower levels of T cell exhaustion compared to primary tumors. This suggests a T cell exhaustion-independent mechanism in ACT-escaped tumors. We further demonstrated that T cells in ACT-escaped tumor are mainly sourced from the adoptive transferred T cells, which recognize primary tumor antigens but not escaped tumor antigens. These results suggest that a combined mechanism of tumor antigen shifting and impaired DC function leads to a failure of antigen spreading and tumor eradication.

Integrating transcriptomic data and digital pathology for NRG-based prediction of prognosis and therapy response in gastric cancer

Background Cancer is characterized by its ability to resist cell death, and emerging evidence suggests a potential correlation between non-apoptotic regulated cell death (RCD), tumor progression, and therapy response. However, the prognostic significance of non-apoptotic RCD-related genes (NRGs) and their relationships with immune response in gastric cancer (GC) remain unclear. Methods In this study, RNA-seq gene expression and clinical information of GC patients were acquired from The Cancer Genome Atlas and the Gene Expression Omnibus databases. Cox and LASSO regression analyses were used to construct the NRG signature. Moreover, we developed a deep learning model based on ResNet50 to predict the NRG signature from digital pathology slides. The expression of signature hub genes was validated using real-time quantitative PCR and single-cell RNA sequencing data. Results We identified 13 NRGs as signature genes for predicting the prognosis of patients with GC. The high-risk group, characterized by higher NRG scores, demonstrated a shorter overall survival rate, increased immunosuppressive cell infiltration, and immune dysfunction. Moreover, associations were observed between the NRG signature and chemotherapeutic drug responsiveness, as well as immunotherapy effectiveness in GC patients. Furthermore, the deep learning model effectively stratified GC patients based on the NRG signature by leveraging morphological variances, showing promising results for the classification of GC patients. Validation experiments demonstrated that the expression level of SERPINE1 was significantly upregulated in GC, while the expression levels of GPX3 and APOD were significantly downregulated. Conclusion The NRG signature and its deep learning model have significant clinical implications, highlighting the importance of individualized treatment strategies based on GC subtyping. These findings provide valuable insights for guiding clinical decision-making and treatment approaches for GC.

Advanced Non-Operative Interventions for Anterior Knee Pain.

This review presents evidence for advanced non-operative interventions, including extracorporeal shockwave therapy (ESWT), prolotherapy, platelet-rich plasma (PRP), adipose tissue-derived cells, bone marrow aspirate concentrate, various additional non-corticosteroid injectates, and needle-based interventions for common causes of anterior knee pain in the adult population. These etiologies include osteoarthritis of the knee, patellofemoral pain syndrome, chondromalacia patella, Hoffa fat pad impingement syndrome, patellar/quadriceps tendinopathy, and prepatellar bursitis. This review discusses patient care options using a case-based understanding of interventions by condition while recognizing strength of evidence. ESWT and PRP are the most robustly studied and have greatest evidence for treating tibiofemoral osteoarthritis and for long-term benefit in treating patellar tendinopathy. PRP may have evidence for treatment of chondromalacia and prolotherapy for management of tibiofemoral arthritis; both have limited evidence. Botulinum neurotoxin type A has strong evidence to support use in treating patellofemoral pain syndrome. There is limited evidence to support the use of viscosupplementation, percutaneous needle tenotomy, and medicinal signaling cell-based therapies beyond platelet-rich plasma for anterior knee pain. There is limited research on the management of quadriceps tendinopathy, prepatellar bursitis, patellofemoral osteoarthritis, and Hoffa's fat pad impingement syndrome. Further research and standardization of protocols are necessary to fully assess these treatments' efficacy. ESWT, cell-based, and needle-based interventions, may serve as effective treatment options for patients with anterior knee pain. Selection of each intervention requires understanding the evidence, level of risk, and appropriate application based on a patient's level of activity to enable clinicians to enhance patient outcomes and quality of life.

EXTH-79. BREAKING THE BARRIER: ADOPTIVE CELLULAR THERAPY HALTS GLIOMA IMMUNOSUPPRESSION BY TARGETING MYELOID-DERIVED SUPPRESSOR CELL MIGRATION AND CELL CYCLE MECHANISMS

Abstract INTRODUCTION Our group previously demonstrated that adoptive cellular therapy (ACT) significantly increased overall survival in mice across several brain cancer models. ACT remodels the tumor microenvironment (TME) in a pleiotropic manner, and specifically depletes immunosuppressive cells like myeloid-derived suppressor cells (MDSCs) which are a significant barrier to efficacious immunotherapy responses. This led us to hypothesize that ACT depletes MDSCs from the glioma microenvironment by inhibiting MDSC proliferation and migration, thereby overcoming glioma immunosuppression. METHODS We quantified MDSC cell cycle, apoptosis, and localization in the TME and peripheral lymphoid tissues using flow cytometry as well as chemokines in the TME from glioma-bearing mice treated with ACT. C57BL/6J mice intracranially received KR158b-luciferase glioma and were treated with ACT. After treatment, MDSC cell cycle was quantified using bromodeoxyuridine and 7-aminoactinomycin to define cell cycle phases. MDSC apoptosis and cell death states were quantified by measuring annexin-V and propidium iodide. Anatomic localization of MDSCs was measured by comparing host (CD45.2) and transferred HSC-derived cell markers (CD45.1) in the TME and spleen. A multiplex array was performed using tumor lysates to quantify cytokine and chemokine levels within the TME. RESULTS We observed that MDSCs in the TME were significantly less proliferative and exhibited significantly increased levels of apoptosis and cell death after ACT. HSC-derived MDSCs were significantly enriched in the spleen of ACT-treated mice, but both transferred HSC-derived MDSCs and host MDSCs were depleted in the TME. Finally, ACT significantly decreased several chemokines in the TME, including interleukin-16 and monocyte chemoattractant protein-5. CONCLUSION These results indicate that ACT shifts MDSC cell cycle states away from proliferation towards cell death, and inhibits peripheral recruitment to the TME. Our investigation revealed novel, myeloproliferative chemokines that are decreased by ACT, representing a novel mechanism underlying disrupted MDSC migration to the glioma microenvironment and ultimately to overcoming glioma-mediated immunosuppression.

IMMU-34. PREDICTION OF TUMOR-ASSOCIATED ANTIGENS USING RADIOGENOMICS

Abstract INTRODUCTION Glioblastoma remains a disease with dismal prognosis. Immunotherapy demonstrated promising results against some refractory cancers. However, comparative results have not been accomplished in glioblastoma therapy due to lack of suitable tumor targets and considerable clonal heterogeneity. OBJECTIVE We propose leveraging radiogenomics using machine learning based on Pre-Op MRI to predict tumor antigen expression profiles displaying specific targets for immunotherapy. METHODS We collected RNAseq data and Pre-Op Axial T2-FLAIR images in 29 patients from IVY Glioblastoma Atlas Project. MRIs were segmented using LIFEx software as follows: 1) tumor + edema (T+E) and 2) whole brain (WB). The intended prediction was for tumor-associated antigens. H2O.ai software was used for prediction using an automated supervised machine learning algorithm. Antigens that achieved >55% area under the curve (i.e., prediction) were reanalyzed adjusting for reproducibility, time of analysis and maximum precision. RESULTS An average of 53.2 T2-FLAIR axial slices per patient were used for 3D reconstruction. We selected the following genes (ERBB2, TP53, IL10RA, CD163, EGFR and MGMT) for downstream analysis given their clinical, immunologic, and prognostic relevance. Regarding tumor-associated antigen prediction for ERBB2, TP53, IL10RA, CD163, EGFR and MGMT predicted 52%, 56%, 66%, 66%, 56% and 0%, on T+E segmentation respectively; The values were 49%, 93%, 87%, 47%, 37% and 0% for the respective WB group. Reanalysis revealed 100% prediction accuracy for the presence of mutant TP53 and IL10RA in both segmentation groups (T+E, WB). CD163 and EGFR prediction was 54% and 61% for the T+E group and 48% for both antigens in the WB group. CONCLUSION We successfully predicted the presence of two out of six antigens (i.e., mutant TP53 and IL10RA) in this patient cohort. Our approach can provide expeditious antigen identification to enable cellular therapy manufacturing for patients with unresectable newly diagnosed tumorsor those which relapsed geographically. Furthermore, this MRI-based methodology might provide a precision medicine approach to places that cannot afford costly testing (i.e., RNAseq).